cst of phenol water system experiment

[ps2id id=’background’ target=”/]

When small quantity of phenol is mixed with water and the mixture is shaken, phenol dissolves, forming a single layer. On adding larger quantities of phenol, however, two kinds of compositions (layers) of liquid are formed. The lower layer consists of small amounts of water dissolved in phenol (phenol saturated with water) and upper layer of phenol dissolved in water (water saturated with phenol). 1

Aim:  To determine the critical solution temperature or upper consolute temperature of phenol-water system.

REQUIREMENTS

Apparatus:        Hard glass tube

Weigh accurately 4 gms (w1 gm) of phenol in a glass tube. (Do not touch phenol, it is corrosive). Introduce glass rod and thermometer (with 0.1◦C accuracy) into it. Fix the tube in a vertical position in a water bath (beaker containing water). Set up the apparatus as shown in the figure. Add 2 ml of distilled water to phenol and heat the test tube in a water bath, whose temperature is initially raised to about 30◦C on low flame with constant stirring. Note the temperature (T1) of solution at which a clear and transparent solution is obtained. It is the solution of water in phenol. Remove the tube outside the water bath and with constant stirring note the transition temperature t◦ C at which the turbidity reappears (this is because water separates out as a different layer.). Add 2 ml more of distilled water and record the transition temperature. Continue the addition of 2 ml distilled water and record the transition temperature till the temperature goes through a maximum value (CST) and comes down to about 40 ◦ C.

Density of water d = 1.0 gm/ml

P = % Phenol = 100W1

[ps2id id=’conclusion’ target=”/]

The critical solution temperature for phenol water system is ___ ◦ C at___% by weight of phenol.

1. More HN, Hajare AA. Practical Physical Pharmacy, Career publications; 2010: 86-87.

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What exactly happens at the critical solution temperature of a phenol water system?

What makes phenol that was turbid go clear beyond the critical solution temperature? We say beyond the critical solution temperature the constituents become soluble. What is the reaction or the exact change involved in the phenol water system (say with water in excess) beyond the critical solution temperature.

• physical-chemistry

• 2 $\begingroup$ There is no reaction. As for the exact change... well, it's like you said: the components become soluble. I mean, they were soluble to some extent even before that point, but here they become soluble infinitely . $\endgroup$ –  Ivan Neretin Commented Oct 26, 2015 at 15:32
• $\begingroup$ Critical Solution Temperature (CST) $\endgroup$ –  MaxW Commented Oct 26, 2015 at 16:48
• $\begingroup$ In chemistry a system a two liquid system is said to have two phases. The system is turbid below the CST because one phase is droplets suspended in the other phase. $\endgroup$ –  MaxW Commented Oct 26, 2015 at 16:56

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Water-Phenol Miscibility Diagram

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Journal of the Iranian Chemical Society

Anmol Mathur

Phenolic compounds are ubiquitous in today's modern pharmaceutics with immense uses in the aviation to paint and to the cosmetics industries. Phenol's properties have been studied for centuries, particularly its partial miscibility with water. Generally, partially miscible liquids become more soluble with the increase in temperature and at a certain temperature they are completely miscible. This temperature is known as the critical solution temperature (CST) or consolute temperature. The temperature above which the phases of a system are completely miscible is known as the upper consulate temperature (UCT) and it gets affected by the addition of impurities. To find the miscibility temperature, the mixture was heated in a boiling tube until the turbidity disappeared and the final temperature was noted. Then, the mixture was cooled down and the temperature noted when the turbidity reappeared. Solutions of impurities of different concentrations were formed and their effect on the UCT of the ternary system created by the impurities, Phenol, and water was analyzed. It was found that the ionic compounds, which get hydrated with water, show lesser increase in CST as they decrease the miscibility to a lesser extent. Further, at all concentrations, CST of phenol-water system containing Camphor as impurity is maximum and it increases with increasing concentration. Thus, Camphor is the most suitable substance to be added to increase the UCT of phenol-water system.

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Oxidative cleavage of β-O-4 bonds in lignin model compounds with polymer-supported Ni–Salen catalysts †

First published on 19th September 2024

Transition metal-catalyzed lignin oxidative cleavage reactions have attracted considerable attention. In this work, polymerized ionic liquid-tagged Salen ligands have been initially synthesized, followed by anion exchange, and then coordination with Ni( II ) via a –N 2 O 2 – tetradentate structure. Finally, the as-obtained Ni–Salen complexes were polymerized to give a Ni–Salen polymer catalyst (poly-Ni-[Salen-Vim][OAc] 2 ). The resulting catalyst showed 99% conversion and 88% selectivity to oxidative cleavage products for the oxidative cleavage of a lignin model compound (2-phenoxy-1-phenylethanone) without any base additive at 110 °C. The polymeric ionic liquid-tagged Salen(Ni) catalysts can be separated easily by centrifugation after the reaction and recycled for five runs with a slight loss of activity. Additionally, studies on birch lignin depolymerization indicated that polymer-supported Ni Salen catalysts were able to cleave β-O-4 linkages to produce dimeric products. Further investigation suggests that the oxidative cleavage reaction was proceeded via a radical pathway.

1 Introduction

Different catalytic systems have been studied for lignin oxidation. 17–19 It has been found that inexpensive commercially available copper salts (CuCl, CuCl 2 , and Cu(OAc) 2 ) and bases (NaOH) are efficient catalysts for the selective cleavage of C–C bonds of β-O-4 lignin model substrates under mild conditions. 17 It has been demonstrated that the addition of a base has a facilitating effect on the breaking of the β-O-4 bond, in which the base is able to elongate the C α –H bond of the lignin model substrate so that hydrogen can be easily dissociated. For instance, the covalent triazine skeletons, a metal-free catalyst, showed good catalytic activity for the C–C bond cleavage of PP-one due to the presence of strongly basic sites, which helped to activate the C β –H bond. 20 Besides, it was found that potassium tert -butoxide with strong basicity exhibited high catalytic activity for the oxidative cleavage of PP-ol. 9 Moreover, a catalytic amount of solid base (K 7 HNb 6 O 39 ), coupled with the Cu/C 3 N 4 catalyst, showed enhanced catalytic performance for the oxidative cleavage of PP-one. 21 However, the above reaction system could require severe reaction conditions or addition of a base, which might cause corrosion and difficulties in separation and recycling. In view of these considerations, our group has developed protonated ionic liquid [Bim][Pic]-stabilizing vanadium–oxygen clusters, which exhibited high catalytic activity for selective aerobic oxidation to cleave β-O-4 linkages into phenols, esters and acids. It was found that the highly reversible interconversion of V 4+ and V 5+ species in vanadium oxo-clusters allowed the coexistence of mixed valence vanadium species, which was responsible for oxidizing PP-ol to PP-one. 22 However, it is still difficult to separate the catalyst from the reaction system.

Salen complexes have the advantages of being inexpensive, easy to synthesize and relatively stable. 23 In addition, it has been found that the catalytic activity of metal-Salen catalysts can be tuned by modulating the moieties of Salen ligands. 24 V, Cu, Co, and Mn Salen based catalysts have been reported to catalyze the oxidation of lignin and model compounds. 25–27 For example, Co Salen catalysts have been most frequently investigated in the catalytic oxidation of lignin and model compounds because of their compatibility with aqueous reaction media. Exposure of Co complexes to oxygen has been reported to form a Co III -superoxide adduct and Co III -hydroperoxide adduct. 28,29 Although homogeneous metal-Salen complexes have shown high catalytic properties, their cost-effectiveness, poor stability and separation difficulties would still hinder their further application. There have been more studies on the design and application of heterogeneous Salen complex catalysts, 30 such as covalent grafting of a Salen complex on graphene, 31 covalent grafting on silica 32 and constant potential condition deposition methods, 33 which might improve stability and recyclability. Especially, the entrainment of am ionic liquid (IL) moiety on a polymeric Salen complex catalyst can facilitate the isolation of products from the catalyst, and also the counter anions of the IL have the ability to regulate the oxidative activity of the catalyst. 34–36 Furthermore, divinylbenzene has been widely used as a cross-linker in polymeric catalysts since the copolymerization between divinylbenzene and 1-vinylimidazolium can form highly thermally stable and porous polymers, being favorable for catalytic performance. 37–39

Inspired by other and our group's previous studies on oxidative cleavage of β-O-4 bonds in lignin model compounds, in this work we reported polymeric ionic liquid-tagged Salen ligands, coordinating with a Ni( II ) via the –N 2 O 2 – tetradentate structure to give a polymeric Ni–Salen catalyst (poly-Ni-[Salen-Vim][OAc] 2 ), and the polymeric ionic liquid-tagged Salen(Ni) catalysts acted as highly efficient catalysts for oxidative cleavage of β-O-4 lignin model compounds under mild and non-alkaline/acidic reaction conditions.

2 Experimental section

2.1 chemicals and materials.

Birch lignin was derived from birch sawdust according to a reported method. 4 Briefly, 10 g of wood chips and 100 mL of methanol containing 3% (w/w) hydrogen chloride were carefully added to a 500 mL round-bottomed flask with a condenser. The mixture was refluxed under vigorous stirring for 70 h. The reaction was stopped and the reaction mixture was cooled to room temperature, and then the mixture was filtered to remove residual solids and washed several times with a small amount of methanol. The collected filtrate was concentrated by rotary evaporation to less than 50 mL and transferred to a 250 mL beaker containing ice water with vigorous stirring. Finally, the birch lignin was collected by filtration and dried under vacuum at 60 °C.

2.2 Synthesis and characterization of lignin model compounds and lignin preparation

2.3 catalyst preparation, 2.4 typical reaction procedure.

For oxidative degradation of birch lignin, after the oxidative degradation reaction, the autoclave was cooled to room temperature and depressurized. Then the reaction mixture was centrifuged, and the insoluble fraction was washed with methanol. The total solution was combined and transferred to a round-bottom flask and the methanol was removed under vacuum. The resulting oil was dissolved in CH 3 CN and was analyzed by HPLC-MS (Q Exactive Orbitrap LC-MS/MS system, Thermo Fisher Scientific) equipped with a chromatographic column (Thermo Hypersil GOLD C18, 3 μm, 2.1 mm × 100 mm) in secondary mass spectrometry mode (HCD) and an electrospray ion source (ESI). The small molecular weight substances derived from birch lignin oxidative degradation were determined quantitatively by the external standard method according to previous reports. 43,44 The dimers were analyzed by using dimeric β-O-4 lignin model compounds as external standards.

2.5 Catalyst characterization

3 results and discussion, 3.1 synthesis and characterization of the polymeric ni–salen catalyst.

The porous structure of the catalyst was determined from nitrogen adsorption–desorption isotherms. As shown in Fig. S10, † the catalyst had a rapidly increasing nitrogen adsorption capacity at low relative pressure ( P / P 0 < 0.001), and the N 2 adsorption–desorption isotherm was a type IV isotherm, with H4 hysteresis loops at higher pressures, which corresponded to the presence of mesoporous and microporous structures in the polymer framework. The pore size distribution confirmed the presence of micropores and abundant mesopores between 1 and 6 nm (Fig. S10b † ). Besides, the poly-Ni-[Salen-Vim][OAc] 2 catalyst has a high BET surface area of 211 m 2 g −1 , a mesoporous pore volume of 0.3 cm 3 g −1 and an average pore size of 6.5 nm. The high BET surface and porous structures might be conducive to mass transfer and facilitate catalytic reactions.

SEM images showed that the catalyst had a large flaky irregular shape, as seen in Fig. 3a . The voids generated between neighbored sheets correspond to the mesoporous detected from nitrogen sorption isotherms (Fig. S10 † ). The SEM image confirmed the cross-linked porous structure, indicating that the thin-layer nanostructures could be formed due to the steric effect of long carbon chains. The flaky structure and hierarchical pores are favorable to the mass transfer of reactants as well as full exposure of active sites for efficient oxidative cleavage of PP-one. EDS elemental mapping images showed that the C, N, O and Ni elements were evenly distributed in the polymer framework of poly-Ni-[Salen-Vim][OAc] 2 .

Thermogravimetric analysis (TGA) was performed to investigate the thermal stability of the catalyst from 40 °C to 800 °C under a N 2 atmosphere. As shown in Fig. S11, † the decrease in mass (6%) was related to the removal of water molecules absorbed by the catalyst below 200 °C, and the main reason for the 52% decrease in mass in the temperature range of 200–500 °C was related to the removal of the ionic liquid groups from the catalyst. 52 The further weight loss at higher temperature (>500 °C) could be attributed to the decomposition of the polymeric backbone. 37 The above results indicated that the catalyst is thermally stable at the reaction temperature.

3.2 Catalytic performance

As shown in Table 1 , it was observed that both polymeric Co and Ni Salen catalysts showed high conversion, but the poly-Ni-[Salen-Vim] [OAc] 2 catalyst afforded higher selectivity towards oxidative products than the poly-Co-[Salen-Vim] [OAc] 2 catalyst (entries 5–7). The low selectivity of the oxidative cleavage products could be attributed to the occurrence of a condensation reaction. Especially, the conversion of PP-one was very poor and the condensation products were formed under the conditions of a N 2 atmosphere, which revealed that molecular oxygen plays an essential role in the oxidative cleavage reaction ( Table 1 , entry 8). Nevertheless, it can be seen that poly-Ni-[Salen-Vim][Br] 2 showed a much lower conversion and product yield than the Ni-[Salen-Vim][OAc] 2 catalyst ( Table 1 , entry 9), reflecting that the OAc − anions in the ionic porous polymer exerted a positive impact on the oxidative cleavage likely due to their basicity. 53,54 In view of the high catalytic performance of the poly-Ni-[Salen-Vim] [OAc] 2 catalyst, further investigation was carried out to gain deeper insight into the role of the poly-Ni-[Salen-Vim][OAc] 2 catalyst in the oxidative cleavage of PP-one.

First, the effect of the catalyst dosage on the catalytic performance was studied. As shown in Fig. 4a , the conversion of PP-one and the yields of products obviously increased with the dosage but the catalytic activity remained almost unchangeable as the dosage reached as high as 50 mg. This indicated that polymeric Salen ligand-coordinated Ni( II ) sites were actually catalytic sites and more active sites were favorable for the oxidation reaction. Furthermore, the conversion of PP-one increased gradually with the reaction temperature ranging from 80 to 110 °C, but the conversion leveled off at a slightly higher reaction temperature ( Fig. 4b ). Meanwhile, the yields towards phenol and BA can reach a maximum around 110 °C. Especially, as the temperature was further increased to 130 °C, the yield of MP was slightly reduced along with an increase of the yield of BA, strongly suggesting that MP might be the intermediate product of the reaction. As shown in Fig. 4c , the conversion of PP-one reached 99%, and the highest yields of phenol and MB were 80% and 81% as the time was increased to 12 h. Moreover, if the reaction time was further prolonged to 14 h or 16 h, the conversion and yields of phenol and BA remained almost constant, reflecting that the catalyst can maintain the high selectivity towards oxidative cleavage of β-O-4 bonds even at a longer reaction time. Interestingly, MP reached its maximum yield at 8 h but reduced with reaction time, along with an obvious rise of MB yield, indicating that MP was indeed the intermediate product of the oxidative cleavage reaction in line with that shown in Fig. 4b .

The reusability of the poly-Ni-[Salen-Vim] [OAc] 2 catalyst was investigated, as catalyst reusability is one of the most valuable characteristics of the catalyst. After reaction, the poly-Ni-[Salen-Vim][OAc] 2 catalyst can be easily separated by simple centrifugation and washed with ethanol. As shown in Fig. 5a , after five recycles, the conversion of PP-one showed a decrease, but the selectivity of oxidative cleavage products maintained a high level (around 90%). Especially, after the five cycles, the spent catalyst showed basically the same characteristic peaks of FT-IR spectra as the fresh catalyst ( Fig. 5b ), indicating that there was no significant change in the main functional group of the poly-Ni-[Salen-Vim][OAc] 2 catalyst. However, the loss of catalyst mass was observed after five cycles, indicating that the polymeric catalyst may undergo degradation under the reaction conditions. 55,56 Besides, it was demonstrated by hot filtration experiments (Fig. S12 † ) that the conversion of PP-one increased by 10% when the catalyst was removed from the reaction system and the reaction time continued to extend from 4 h to 12 h, indicating a slight leaching of Ni species into the effluent.

The β-O-4 lignin model compounds contain methoxy groups on the aromatic ring, which are structurally similar to natural lignin. Therefore, the suitability of the poly-Ni-[Salen-Vim][OAc] 2 catalyst for the oxidative cleavage of a range of methoxy-containing β-O-4 lignin model compounds other than PP-one was also examined. As shown in Table 2 , the catalytic cleavage of the β-O-4 lignin model compounds with the methoxy group can be effectively converted to phenols, aromatic acids and corresponding aromatic esters under the same reaction conditions. These results confirmed clearly that the poly-Ni-[Salen-Vim][OAc] 2 exhibits good catalytic performance in the oxidative cleavage of β-O-4 lignin model compounds with the methoxy group.

Based on the above results, the present poly-Ni-[Salen-Vim][OAc] 2 catalyst was compared with reported catalysts for the oxidative cleavage of PP-one using O 2 as the oxidant. As shown in Table S1, † the conventional C–O and C–C bond breaking systems for β-O-4 lignin model compounds still require the addition of bases, which are not friendly from the green development point of view or require more severe reaction temperatures to achieve PP-one oxidative cleavage (Table S2, † entries 1–7). Comparatively, the present poly-Ni-[Salen-Vim][OAc] 2 catalyst can be used for oxidative cleavage of lignin model compounds under much mild reaction conditions, and furthermore it acted as a heterogeneous catalyst without the addition of a base, facilitating the separation of the catalyst. Moreover, its catalytic activity is comparable with or better than those of reported systems (Table S2, † entry 8).

Finally, to evaluate the applicability of the catalytic system, the oxidative depolymerization of birch lignin was carried out under the same reaction conditions. After the reaction, the conversion of birch lignin was ca . 74%. The liquid fraction was then analyzed by HPLC-MS and HPLC. It was observed that the liquid fraction contained three dimeric products, which are shown in Fig. S13 and S14, † respectively. The results indicated that the present catalyst indeed exhibited activity for the oxidative depolymerization of birch lignin, producing some small molecular products as well as methanol-soluble low-molecular products. After the reaction, only a small amount of birch lignin (around 20%) cannot be dissolved in methanol, indicating that most of the birch lignin has been depolymerized into low-molecular products. However, the corresponding monomers were not detected under the present conditions likely due to the relatively low reaction temperature in methanol media.

3.3 Reaction pathway

In order to further explore the reaction pathways of the oxidative cleavage process, controlled experiments were carried out. The depolymerization of the β-O-4 lignin model compounds can be achieved by either C α –C β bond or C β –O bond breakage. C–C bond breakage of PP-one produces BA and phenyl formate, which is subsequently decarboxylated to produce phenol, while C–O bond breakage produces phenol, BA and phenylglyoxylic acid, which is further converted to MP, BA or MB. In order to get deeper insight into the main pathways, the reaction was carried out under the same reaction conditions using possible intermediates as substrates. As shown in Scheme 2 (eqn (4)), when phenylglyoxylic acid, an intermediate product of C–O bond cleavage, was used as a substrate, the conversion was 65%, and the yields to BA, MP, and MB were 52%, 9%, and 1%, respectively, while BA can be converted to MB with a yield of 10% under identical reaction conditions (eqn (5)). Furthermore, if phenyl formate, the intermediate product of C–C bond cleavage, was used as the substrate, it was almost fully converted to phenol (eqn (6)). However, 2-phenylacetophenone gave poor conversion (36%) via the C–C cleavage route (eqn (7)), which indicated that the catalyst is indeed less active for C–C bond cleavage. Actually, the reaction of PP-one catalyzed by the poly-Ni-[Salen-Vim][OAc] 2 catalyst produced a small amount of MP ( Fig. 5c ), which would be produced via the C–O cleavage pathway. As a result, the β-O-4 lignin model compound PP-one is mainly oxidatively cleaved via the C–O cleavage pathway.

Based on the results of radical inhibition experiments, control experiments and previous investigation, 58 a plausible catalytic reaction pathway is proposed in Scheme 3 . Firstly, molecular oxygen reversibly binds to Ni( II ) to generate Ni( III )-superoxide complexes, 48,59 and PP-one binds to the Ni( III )-superoxide complexes to form active intermediates. Next, there are two possible paths for the catalytic oxidative cleavage reaction. Path A generates phenol and phenylglyoxylic acid by cleavage of the C–O bond of the intermediate, and phenylglyoxylic acid can be further converted to BA, MB, and MP with the participation of molecular oxygen and methanol by the action of Ni sites. Path B generates phenyl formate and BA via C–C bond cleavage, wherein phenyl formate is easily converted to phenol in the presence of methanol and polymeric Salen Ni catalysts. According to our present investigation as shown in Scheme 2 and Fig. 4 , path A should be dominant for the oxidative cleavage reaction.

4 Conclusions

Data availability, conflicts of interest, acknowledgements.

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• Published: 27 September 2024

Prohibitin 2 orchestrates long noncoding RNA and gene transcription to accelerate tumorigenesis

• Tianyi Ding 1 , 2 , 3   na1 ,
• Haowen Xu 1 , 2 , 3   na1 ,
• Xiaoyu Zhang 1 , 2 , 3   na1 ,
• Fan Yang 1 , 2 , 3 ,
• Jixing Zhang 1 , 2 , 3 ,
• Yibing Shi 1 , 2 , 3 ,
• Yiran Bai 1 , 2 , 3 ,
• Jiaqi Yang 1 , 2 , 3 ,
• Chaoqun Chen 1 , 2 , 3 ,
• Chengbo Zhu 1 , 2 , 3 &
• He Zhang   ORCID: orcid.org/0000-0001-9954-4451 1 , 2 , 3  

Nature Communications volume  15 , Article number:  8385 ( 2024 ) Cite this article

Metrics details

• Cancer epigenetics
• Epigenetics

The spatial co-presence of aberrant long non-coding RNAs (lncRNAs) and abnormal coding genes contributes to malignancy development in various tumors. However, precise coordinated mechanisms underlying this phenomenon in tumorigenesis remains incompletely understood. Here, we show that Prohibitin 2 (PHB2) orchestrates the transcription of an oncogenic CASC15-New-Isoform 2 ( CANT2 ) lncRNA and the coding tumor-suppressor gene CCBE1 , thereby accelerating melanoma tumorigenesis. In melanoma cells, PHB2 initially accesses the open chromatin sites at the CANT2 promoter, recruiting MLL2 to augment H3K4 trimethylation and activate CANT2 transcription. Intriguingly, PHB2 further binds the activated CANT2 transcript, targeting the promoter of the tumor-suppressor gene CCBE1 . This interaction recruits histone deacetylase HDAC1 to decrease H3K27 acetylation at the CCBE1 promoter and inhibit its transcription, significantly promoting tumor cell growth and metastasis both in vitro and in vivo. Our study elucidates a PHB2-mediated mechanism that orchestrates the aberrant transcription of lncRNAs and coding genes, providing an intriguing epigenetic regulatory model in tumorigenesis.

Introduction

Tumor cell heterogeneity results in the coexistence of aberrant lncRNAs and abnormal coding genes within the same or different genomic loci, giving rise to diverse malignancies with various pathogenic causes. For instance, in the human genome, abnormal coding genes MYC is generated from the chr8q24 locus, a well-known genomic region implicated in the progression of various malignancies, and lncRNAs generated from this locus are recognized as contributing factors in the tumorigenesis of difference cancer types 1 , 2 , 3 , 4 , 5 , 6 . However, many critical lncRNAs and coding genes originating from distinct loci coordinately result in tumorigenesis through unique mechanisms. For example, abnormal p53 has been reported to lead to genetic instability and uncontrolled cell proliferation in gastric cancer 7 . Meanwhile, lncRNA GCLET increased FOXP2 expression and remarkably impacted gastric cancer phenotypes 8 . Additionally, our previous studies have identified that the coding gene NTS and the lncRNA ROR , derived from different loci, exerted distinct influences on the progression of uveal melanoma 9 , 10 . Therefore, it is intriguing to explore how these coding genes and lncRNAs from the same or various loci coordinately contribute to the progression of malignancies.

The eukaryotic genome is well organized and evolutionarily conserved, yet exhibits spatial plasticity with cell-specific characteristics 11 , 12 , 13 . Recent studies demonstrate that the intricate transcription of coding genes and lncRNAs are influenced by genetic and epigenetic mechanisms across various dimensions from one to three dimensional levels 14 , 15 , 16 , 17 , 18 , 19 . Plenty of elementary chromatin features, including DNA sequence variations, nucleosome density and position, histone methylation modification and transcription factor binding, serving as the cornerstone of transcription at coding genes and lncRNAs 20 , 21 , 22 , 23 . The activity of regulatory factors and their functional network could partially explain cell-specific regulatory mechanisms during carcinogenesis 24 . For instance, the ETS family members of transcription factors have clearly been shown to be a driving event in prostate tumorigenesis via chromosomal translocation events 25 . Furthermore, the open or closed status of specific genomic loci is closely linked to active and inactive transcription, respectively, and determines the chromatin accessibility of regulatory factors 26 , 27 , 28 . For example, the chromatin regulator BRD8, conjugating with H2AZ, sustained a repressive chromatin state in the p53 locus, preventing the activation of p53 and promoting the development of glioblastoma 29 . Typically, these multifaceted regulatory mechanisms establish an environment that permits the operation of key regulatory factors in the process of transcription.

Prohibitin-2 (PHB2) and its homologs are widely expressed scaffold proteins involved in numerous signaling pathways that regulate metabolism, mitochondrial autophagy, and cell migration and proliferation, thereby influencing diseases such as cancer and inflammation 30 , 31 . As a precursor of melanoma, melanocytes are involved in melanin biosynthesis, which is related to mitochondrial function. Previous studies have shown that the PHB family plays a crucial role in maintaining mitochondrial integrity, participating in melanin production and carcinogenic pathways, and potentially functioning as regulators of melanin production signals 32 .This suggests the potential of developing new drug candidates for the treatment of melanoma and other types of cancer. Furthermore, PHB2 acts as a transcriptional co-regulatory factor that can bind to histone-modifying enzymes, mediating the transcriptional regulation of tumor-related genes and thus affecting tumorigenesis 33 , 34 . Given these facts, it is plausible that unknown shuttle factors like PHB2 could coordinately orchestrate the transcription of coding genes and lncRNAs, thereby influencing tumorigenesis.

In this study, we identify that transcriptional regulator PHB2 serves as a shuttle factor which coordinately orchestrates the transcription of the oncogenic CANT2 lncRNA derived from chromosome 6p22.3 and the coding tumor-suppressor gene CCBE1 derived from chromosome 18q21.32 for accelerating tumorigenesis of melanoma. Our study provides a PHB2-mediating mechanism orchestrating aberrant transcription of lncRNA and coding gene, thus proposing an epigenetic regulatory model of tumorigenesis.

Open chromatin recruits PHB2 to activate the transcription of the CANT2 lncRNA at chr6p22.3 locus

To validate our hypothesis that a shuttle factor could bridge the transcriptional connection between noncoding RNAs and coding genes in melanoma, we focused on the canonical cancer susceptibility locus chr6p22.3. This locus is known to be the origin of various noncoding transcripts, including the CASC15 lncRNA, which is implicated in the metastasis melanoma cells 35 , 36 . We initially performed rapid amplification of cDNA ends (RACE) assay at this locus to elucidate the precise transcripts present in A375 and A875 melanoma cells. A lncRNA with 1939 bp spanning 9 exons was identified at the CASC15 locus in melanoma cells (Fig.  S1A, B ). Specifically, exons 2–5 and 8 were consistent with the predicted exons (Fig.  1A , white box). However, exon 1 exhibited an additional 72-bp fragment at the 5’ terminus, while exon 6–7 and 9 were unique (Fig.  1A , blue box). To further strengthen the validation of this identified isoform, we employed three series of RNA-seq and TT-seq data conducted on A375 melanoma cells, available at the public GEO database (GSE223887 37 , GSE223888 37 , and GSE232375 38 ), to examine gene density at the CASC15 locus (Fig.  S2A, B ). Utilizing these comprehensive datasets as a unified reference, we aligned all nine exons of the isoform with the read peaks, and confirmed that their expressions in A375 cells were consistent with our RACE findings (Fig.  S2B ). We then confirmed the absence of coding evidence for this transcript with both CPAT 39 (Fig.  S3A ) and CPC2.0 ( http://cpc2.gao-lab.org/index.php ) 40 (Fig.  S3B ) using the human genome as reference. Collectively, these data showed that this isoform of the CASC15 lncRNA is a non-coding transcript identified in cutaneous melanoma, and we therefore named it CASC15-New-Isoform 2 ( CANT2 ) lncRNA (GenBank number: OR811110).

A Genomic structure of CANT2 and a schematic of the core promoter fragment deletion by CRISPR-Cas9. The rectangles indicate the exons of CANT2 . The black line indicates introns. The red line indicates the core promoter of CANT2 . B Chromatin accessibility status at the chr6p22.3 locus with ATAC-seq and Omni-ATAC datasets obtained from the public GEO database under accession codes GSE188398 42 , GSE134432 43 , and GSE241445 44 . C Real-time PCR of luciferase activity of the DNA fragments of CANT2 promoter. Ctrl, control group without any Firefly reporter plasmids; Mock, group with wild-type Firefly reporter plasmids; 1–5, groups with different 200 bp DNA fragment of CANT2 promoter contained in Firefly reporter plasmids. All groups had Renilla reporter plasmids. All data were calculated the ratio of firefly to Renilla luciferase activity (Fluc/Rluc) in dual luciferase reporter system. For comparison, the ratio of Fluc/Rluc of the mock was arbitrarily set as 1 in the calculation. All the experiments were performed in triplicate and data are presented as the mean ± SD using an unpaired two-tailed t test; * P  < 0.05; ** P  < 0.01. D Schematic diagram of variant primer sets in FAIRE assay. a , b , c : primers in SOX4 , CANT2 , PRL promoter regions, respectively. E PCR of FAIRE assay at CANT2 core promoter region. Representative images from three independent experiments. F Quantification of FAIRE assay at Chr6p22.3 in melanoma cells (A375 and A875) and normal cells (PIG1). Input DNA was used as a positive control. All the experiments were performed in triplicate and data are presented as the mean ± SD using a Dunnett’s multiple comparisons test; **** P  < 0.0001. G Real-time of CANT2 expression at RNA level in melanoma cells lines. All the experiments were performed in triplicate and data are presented as the mean ± SD using an unpaired two-tailed t test; *** P  < 0.001; **** P  < 0.0001. H Subcellular localization of CANT2 in melanoma cells. GAPDH and U6 RNA served as positive controls for the cytoplasmic (black) and nuclear (orange) fractions, respectively. All the experiments were performed in triplicate. I Survival curves of SKCM patients with a high or low PHB2 expression (cutoff = 0.5, P  < 0.05) was analyzed in GEPIA using a Log-rank (Mantel-Cox) test. J ,  K ChIP analysis of PHB2 ( J ), H3K4me3 ( K ) and MLL2 ( K ) at the CANT2 core promoter. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. All the experiments were performed in triplicate and data are presented as the mean ± SD using a Dunnett’s multiple comparisons test; * P  < 0.05, ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001. L , M  PCR of ChIP analysis of H3K4me3 ( L ) and MLL2 ( M ) at the CANT2 core promoter in melanoma and normal cells. Representative blots from three independent experiments. N Co-IP assay was performed to show the interaction between PHB2 and MLL2 in normal and melanoma cells. IgG was used as a negative control. Representative blots from three independent experiments.

Complex mechanisms such as epigenetics may play a crucial role in regulating the transcription of various isoforms at chr6p22.3 locus. Components of epigenetics, including 3D genome organization, chromatin remodeling, and histone modification, typically exert significant influence in this context. With this understanding, we investigated the chromatin topology landscape at chr6p22.3 by analyzing Hi-C data of melanoma with the 3D Genome Browser (accessible at http://3dgenome.fsm.northwestern.edu/view.php ) 41 . However, our analysis of the Hi-C maps did not unveil any significant differences in higher-order chromatin structures such as topologically associating domain (TAD) organization between melanoma samples and normal samples (Figure S4A ). Alternatively, we shifted our focus to chromatin accessibility dynamics using ATAC-seq and omni-ATAC datasets (GSE188398 42 , GSE134432 43 , and GSE241445 44 ) performed on malignant melanoma cells and patient-derived melanoma cultures (MM lines). Comparative analysis with normal human keratinocyte cells revealed prominent peaks in the promoter region of CANT2 lncRNA, located at chr6:21,885,810–21,886,009, in melanoma cells such as COLO-823 and WM-266-4 (Fig.  1B ). Similarly, significant ATAC-seq peaks were observed in the CANT2 promoter region in A375 melanoma cells used in our study (Fig.  1B ). Moreover, patient-derived melanoma cultures, such as MM087 and MM099, which has undergone phenotype switching to a dedifferentiated, mesenchymal-like and therapy-resistant cell state 43 , exhibited higher peaks compared to the melanoma culture MM001 with a melanocyte state (Fig.  1B ). To determine how CANT2 lncRNA could alter tumor behavior, the 200-bp core promoter (Fig.  1A , fragment IV) of CANT2 was identified via the dual luciferase reporter assay (Figs.  S4B and  1C ). We further employed formaldehyde-assisted isolation of regulatory elements (FAIRE) to examine the chromatin accessibility of CANT2 locus in melanoma (Fig.  1D ), and found that an open chromatin status was exhibited in melanoma cells (Fig.  1E , lanes 2–3) compared with normal cells (Fig.  1E , lane 1). Similarly, we further confirmed the open chromatin status of CANT2 locus by using FAIRE-qPCR assay (Fig.  1F , middle). Likewise, the adjacent SOX4 and PRL genes remained in a closed chromatin status both in tumor and normal cells (Fig.  1F , left and right). Subsequently, we assessed the expression of CANT2 lncRNA in tumor cells. As anticipated, our findings revealed that CANT2 was highly expressed in both A875 and A375 cells (Fig.  1G ). Additionally, we examined the expression of previous reported noncoding RNA transcripts including CANT1 , CASC15 and CASC15-S within this locus. Interestingly, we observed that CANT1 , CASC15 and CASC15-S were not expressed in melanoma cells used in our study (Figure  S5A ). Moreover, a cytoplasmic and nuclear RNA isolation assay indicated that CANT2 lncRNA predominantly localized in the nucleus (Fig.  1H ). These findings unveiled that CANT2 is a nuclear lncRNA in human cutaneous melanoma cells.

We next aimed to explore the potential epigenetic regulation of CANT2 lncRNA. Intriguingly, our investigation into the gene expression profile of skin cutaneous melanoma (SKCM) revealed that PHB2, a known transcriptional co-regulator capable of binding with histone modification enzymes 33 , 34 , was highly expressed in SKCM (Fig.  S5B ). Additionally, high PHB2 expression correlated with inferior overall survival (Fig.  1I ). We also identified higher PHB2 expression in melanoma cells compared to normal control cells (Fig.  S5C ). Subcellular localization studies revealed that PHB2 was presented in both the cytosol and nucleus of melanoma cells (Fig.  S5D ). Chromatin immunoprecipitation (ChIP) qPCR showed that PHB2 could bind to this open chromatin region in melanoma cells (Fig.  1J ). Given the pivotal role of histone modification in dynamically modulating chromatin, we proceeded to investigate alterations in histone acetylation and methylation patterns at the CANT2 locus. As anticipated in the ChIP assay, all three histone markers H3K4 monomethylation (Fig.  S6A ), H3K27 acetylation (Fig.  S6B ) and H3K4 trimethylation (Fig.  1K , orange; Fig.  1L , lanes 2–3) associated with open chromatin exhibited elevated levels at the CANT2 locus in melanoma cells compared with normal PIG1 cells. Remarkably, H3K4me3 level displayed a significant increase at the CANT2 locus (Fig.  1K , orange). Consequently, we delved into the functional factors involved in the H3K4me3 modification. However, the enrichment of common histone methyltransferases, MLL1 (Fig.  S6C , panel 1, lanes 2–3; Fig.  S6D ) and SET1A (Fig.  S6C , panel 2, lanes 2–3; Fig.  S6E ), showed no discernible difference between melanoma cells and normal cells. It was surprising to observe the specific binding of MLL2 to the CANT2 locus in melanoma (Fig.  1K , green ; Fig.  1M , lanes 2–3). Moreover, co-immunoprecipitation (co-IP) assays showed MLL2 could be pulled out by baiting PHB2 in melanoma (Fig.  1N , panels 2–3, lane 3). Taken together, these data showed that PHB2 may serve as a transcriptional regulator for recruiting MLL2 to the accessible promoter of a lncRNA CANT2 , and triggering the expression of CANT2 by increased H3K4 trimethylation in melanoma.

The deletion of CANT2 diminishes tumor cell proliferation and migration in vitro

To address the role of CANT2 lncRNA in tumorigenesis, we deleted the 200-bp core promoter region in melanoma cells using the CRISPR/Cas9 method (Fig.  1A , fragment IV). As expected, CANT2 expression level was successfully knocked down to ∼ 20% of its original expression level in A375 and A875 cells (Figs. S7A, B ;  2A , lanes 1, 2, and 4). Next, we examined cell proliferation and colony formation ability in vitro. Compared with the empty vector group (NC), tumor cell proliferation was significantly reduced after the suppression of CANT2 (KO1 and KO2) (Fig.  2B, C ). Moreover, the CANT2 -KO groups presented fewer and smaller colonies (Fig.  S7C , lanes 2–3). By calculating the number of colonies, we found that tumor cell colony formation was significantly decreased after CANT2 deletion (Fig.  S7D, E ). Furthermore, wound healing assays revealed a reduction in tumor migration upon depletion of the CANT2 core promoter (Fig.  2D, E ). In contrast to the 50–60% migration area observed in the NC groups, the CANT2 -KO groups exhibited a diminished migration area ranging from 20–40% in A375 and A875 cells (Fig. 2F, G ). Consistently, transwell assay results further corroborated this observation, indicating a decreased migration ability of tumor cells following CANT2 deletion (Fig.  2H ). To confirm the role of CANT2 lncRNA in melanoma, we conducted rescue experiments by overexpressing CANT2 ( CANT2 -OE) in CANT2 -KO melanoma cells. Transfection with CANT2 overexpression plasmids restored or exceeded the original expression level of CANT2 lncRNA (Fig.  2A , lanes 3 and 5; 2I, J ). Colony formation assays indicated that the proliferation ability of CANT2 -KO melanoma cells improved after CANT2 overexpression (Fig.  2K ). Furthermore, transwell assays demonstrated that the CANT2 -OE groups exhibited increased metastasis (Fig.  S7F ). Consistently, wound healing assays revealed an elevated migration area of approximately 40–60% in CANT2 -OE melanoma cells compared to the NC groups, which showed only a 10–30% migration area (Fig.  S7G–J ). Collectively, these results suggested that CANT2 served as a oncogenic lncRNA in tumorigenesis of melanoma.

A PCR of CANT2 expression in CANT2 -KO cells transfected with or without a rescue construct ( CANT2 -OE). GAPDH was served as a positive control. Representative images from three independent experiments.  B , C CCK8 assay to measure 4-day cell growth rate in A375 ( B ) and A875 ( C ) cells after knockout of CANT2 core promoter. Triplicate experiments were performed. Data are presented as the mean ± SD using a two-tailed Pearson’s r test; ** P  < 0.01. D , E Representative images of the wound healing assay at 0 hours and 72 hours post-scratch in melanoma cells with or without CANT2 knockout. The dashed lines indicate the wound edge. Representative images from three independent experiments. F , G Quantification of the migration area. The migration area was calculated as the percentage of the wound area covered by cells at 72 hours compared to the initial wound area at 0 hours. Data are presented as mean ± SD from three independent experiments using an unpaired two-tailed t test; ** P  < 0.01; **** P  < 0.0001. H Transwell migration assay to assess the migration ability of melanoma cells after knockout of CANT2 core promoter. Representative images from three independent experiments. I , J qPCR of CANT2 expression at RNA level in melanoma cells with CANT2 knockout ( CANT2 -KO) and in CANT2 -KO cells transfected with a rescue construct ( CANT2 -OE). Relative expression levels were normalized to GAPDH . Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed t -test. * P  < 0.05; ** P  < 0.01; **** P  < 0.0001. K Colony formation assay to determine the proliferative ability of melanoma cells after CANT2 overexpression. Representative images from three independent experiments.

CANT2 enhances tumor growth and metastasis in vivo

To investigate the contribution of the CANT2 lncRNA in tumor characteristics in vivo, NC groups and CANT2 -KO groups of both A375 and A875 cells were injected subcutaneously into nude mice. We also measured the size of the resultant tumors every 3 days in subcutaneous xenografts. After 12–15 days, the mice were euthanatized, and the tumors were collected for further analysis (Fig.  3A–B ). In the CANT2 -KO groups, we found that both tumor weight ( n  = 5–6; **** P  < 0.0001; Figs.  3C ;  S8A ) and volume ( n  = 5–6; *** P  < 0.001; **** P  < 0.0001; Figs.  3D ;  S8B ) were significantly reduced compared with the NC groups. Most importantly, after suppression of CANT2 lncRNA, the survival rate of mice was significantly extended ( n  = 6; Fig.  3E, F ). To test tumor growth and migration capability after CANT2 suppression in vivo, both A375 and A875 cells were labeled with firefly luciferase to establish metastatic models in nude mice. After tail vein injection in nude mice, tumor systemic metastasis was evaluated by bioluminescence and fluorescence imaging. In the metastatic tumor model, there were fewer metastatic loci of nude mice at 30 days in the CANT2 -KO group compared with the NC groups, and luciferase signaling was significantly reduced (Fig.  3G, H ). After approximately 30 days, the mice were euthanatized, and the organs were collected for further analysis. Remarkably, histological examination showed that compared with the NC groups, the absence of CANT2 dramatically suppressed metastatic colonization and reduced the number and size of macroscopic nodules observed in the lungs (Figs.  3I ;  S8C ) and adrenal gland (Fig.  3J ). These data suggested that the CANT2 lncRNA played an oncogenic regulatory role in tumor growth and metastasis of melanoma in vivo.

A Photograph of orthotopic xenograft at 15 days after the subcutaneous injection of A375 cells with or without CANT2 core promoter deletion ( n  = 5 mice in each group). B Photograph of orthotopic xenograft at 15 days after the subcutaneous injection of A875 cells with or without CANT2 core promoter deletion ( n  = 6 mice in each group). C Bar graph showed tumor weight (mg) formed by the A375 cells with or without CANT2 core promoter deletion in a subcutaneous xenograft model. Tumor weight (mg) was measured and presented as the mean ± SD ( n  = 5 mice) using an unpaired two-tailed t test; **** P  < 0.0001. D A xenograft in vivo assay model presented tumor volume (mm 3 ) formed by the A375 cells with or without CANT2 core promoter deletion. Tumor sizes (mm 3 ) were calculated as the (length×width×width)/2 and presented as the mean ± SD ( n  = 5 mice) using a Pearson’s r test. ****, P  < 0.0001. E Survival analysis of mice following intravenous injection with A375 cells with or without CANT2 core promoter deletion ( n  = 6 mice) were analyzed using a Log-rank (Mantel-Cox) test. F Survival analysis of mice following intravenous injection with A875 cells with or without CANT2 core promoter deletion ( n  = 6 mice) were analyzed using a Log-rank (Mantel-Cox) test. G Animal imaging system demonstrated the tumor migration ability of A375 cells with or without CANT2 core promoter deletion on tumor bioluminescent signals. Representative images from five independent samples. H Animal imaging system showed the tumor migration ability of A875 cells with or without CANT2 core promoter deletion on tumor bioluminescent signals. Representative images from five independent samples. I , J Representative images of the histological analysis of lung ( I ) and kidney ( J ) seeding in mice injected intravenously with CANT2 -NC or CANT2 -KO1 A375 cells (original magnification, 1×, scale bar: 1 mm). Representative images from three independent samples.

CCBE1 serves as a regulatory target of PHB2 in melanoma

To further elucidate how CANT2 lncRNA impacted melanoma development, we concentrated on identifying and analyzing its downstream targets. We then conducted an RNA-seq (GEO accession number: GSE231936) analysis to scrutinize alterations in gene expression ensuing CANT2 suppression. Using bioinformatics analyses, we identified 249 significantly altered genes, including 174 upregulated and 75 downregulated genes in the NC group (|Fold Change | > 2, P  < 0.05, Fig.  S6A ). The KEGG (Fig.  S6B–C ) and GO (Fig.  S6D–E ) analyses revealed significant alterations in pathways related to cancer, inflammation, and the immune system upon CANT2 suppression in tumor cells. Remarkably, within this set of differentially expressed genes, CCBE1 in chr18q21.32 locus exhibited a noteworthy and significant increase in expression following CANT2 inhibition (Fig.  4A ). This observation led us to speculate that the CCBE1 is potential target gene of CANT2 . As expected, we showed that the expression of CCBE1 was significantly increased in two CANT2 -KO melanoma cells in RNA level (Fig.  4B, C ), and the results were confirmed in protein level (Fig.  4D ). We also investigated CCBE1 expression and observed the absence of CCBE1 in melanoma cells compared with normal PIG1 cells both at RNA (Fig.  4E ) and protein (Fig.  4F ) levels. Furthermore, the expression levels of CCBE1 in skin melanoma samples were notably reduced compared to those in corresponding normal tissues (Fig.  4G–H ) as indicated by data obtained from GEPIA . Next, we investigated whether PHB2 interacts with the CCBE1 promoter (Fig.  4I ). As anticipated, PHB2 was found to bind to the CCBE1 promoter (Fig.  4J ). Furthermore, we examined the binding of PHB2 in CANT2 -KO melanoma cells with or without CANT2 overexpression. ChIP experiments revealed that rescuing the expression of CANT2 lncRNA led to increased binding of PHB2 at the CCBE1 promoter in melanoma cells (Fig.  4K, L ). These discoveries suggested that CANT2 lncRNA and its downstream target gene  CCBE1 both served as regulatory candidates of PHB2 in melanoma.

A Volcano plot of differentially expressed genes in CANT2 -NC compared with CANT2 -KO A375 cells. Red, up-regulated genes; green, down-regulated genes; gray, unchanged genes. B ,  C  Real-time PCR and WB of CCBE1 expression in A375 ( B ) and A875 ( C ) cells with or without CANT2 core promoter knockout. Data are presented as mean ± SD from three independent experiments using an unpaired two-tailed t test; *** P  < 0.001 and **** P  < 0.0001. D Western blot of CCBE1 expression in melanoma cells with or without CANT2 suppression. Representative blots from three independent experiments. E ,  F CCBE1 expression at RNA ( E ) and protein level ( F ) in melanoma cells and normal PIG1 cells. GAPDH was used as negative control. Data are presented as mean ± SD from three independent experiments using an unpaired two-tailed t test; **** P  < 0.0001. G ,  H Gene expression profile ( G ) and boxplot ( H ) of CCBE1 in public SKCM dataset from GEPIA . T tumor samples, N normal samples. The |Log 2 FC| cutoff was set at 1 and the q-value cutoff at 0.01. * P  < 0.05. Jitter size was 0.4, and Log 2 (TPM + 1) was used for the log-scale. The box plot showed the minima, maxima, centre, bounds of box and whiskers. I Schematic diagram of the primer set d in ChIP assay. J ChIP analysis of PHB2 at the  CCBE1 promoter in CANT2 -NC or CANT2 -KO A375 and PIG1 cells. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. Data are presented as mean ± SD from three independent experiments using a Dunnett’s multiple comparisons test; ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001. K , L ChIP analysis of PHB2 at the  CCBE1 promoter in CANT2 -NC-NC, CANT2 -KO-NC, and CANT2 -KO-OE A375 and A875 cells. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. Data are presented as mean ± SD from three independent experiments using a Dunnett’s multiple comparisons test; * P  < 0.05; **** P  < 0.0001.

PHB2 recruits CANT2 and HDAC1 to repress CCBE1 transcription

Next, to elucidate the specific mechanism governing regulation of CCBE1 expression, we conducted chromatin isolation by RNA purification (ChIRP) using biotin-labeled oligonucleotides (Fig.  S10A ). We selected the CCBE1 promoter as the target site for detection. ChIRP-PCR analysis revealed the enrichment of CANT2 within the CCBE1 promoter region in melanoma cells (Figs.  5A , lane 1;  5B ). Nevertheless, upon CANT2 suppression, we observed the reduced enrichment of CANT2 at the CCBE1 promoter, suggesting a direct binding capability of CANT2 to the CCBE1 promoter (Figs.  5A , lane 4;  5B ). To examine whether CANT2 could interact with PHB2, we then performed ChIRP-MS, a methodology in which proteins purified through ChIRP were identified via mass spectrometry, aiming to uncover proteins that interact with CANT2 (Fig.  5C ). Upon screening and analyzing peptide signals, we identified five proteins (PHB2, TKT, eIF5A, U2A’, and Nup205) enriched in the ChIRP lysate (Fig.  S11A , and Table  S2 ). As expected, PHB2 was one of the proteins identified in ChIRP-MS, confirming our above results and further prompting us to select it for a more comprehensive investigation. Following analysis via western blot assay, we successfully confirmed the interaction between PHB2 and CANT2 (Fig.  5D , lane 1 and 3). We further conducted an RNA-ChIP experiment to examine the interaction between CANT2 and PHB2, Nup205, eIF5A, U2’A and TKT. Among these proteins, CANT2 only interacted with PHB2 in A375 and A875 cells, with no significant enrichment observed for the non-specific control U2 (Fig.  5E, F ). We then chose PHB2 for next investigation in A375 and A875 cells. Furthermore, given the significant increase of CCBE1 expression in CANT2 -KO cells, we proceeded to investigate a representative histone modification, H3K27 trimethylation, across CCBE1 locus. Subsequent ChIP-qPCR analysis revealed that H3K27 acetylation of the CCBE1 promoter was increased in CANT2 -KO melanoma cells, with markedly higher levels observed in normal PIG1 cells lacking CANT2 (Fig.  5G ). Given that Class I deacetylases, such as HDAC1 and HDAC2, are pivotal in histone deacetylation, our directed our attention to their impact on CCBE1 regulation. Previous studies have illuminated both the shared characteristics and distinct regulatory roles of HDAC1 and HDAC2, illustrating their potential for collaborative or independent regulation of cellular processes 45 . Initially, our investigation centered on the influence of HDAC1 in the PHB2-mediated CCBE1 transcription. As anticipated, HDAC1 was found to bind to the CCBE1 promoter in the presence of CANT2 lncRNA in melanoma cells (Fig.  5H ). These findings led us to hypothesize that PHB2 may facilitate the binding of HDAC1 to the CCBE1 promoter in the presence of CANT2 lncRNA. To confirm this, we initially investigated whether CANT2 directly interacted with HDAC1 through an RNA-ChIP experiment. Results showed that HDAC1 did not directly bind to CANT2 (Fig.  5I ). We then examined whether PHB2 acted as the intermediary linking CANT2 and HDAC1. Co-IP assays demonstrated that the PHB2 protein could be pulled out by baiting the HDAC1 protein (Fig.  5J , top, lane 2), and reciprocally, HDAC1 could also be pulled out by baiting the PHB2 protein (Fig.  5J , bottom, lane 3), while the IgG negative control groups exhibited weak binding. Taken together, our finding shows that CANT2 interacts PHB2 and substantially PHB2 recruits HDAC1 to the CCBE1 promoter, ultimately repressing transcription of CCBE1 through H3K27 deacetylation.

A , B ChIRP assay at the CCBE1 promoter in CANT2 -NC or CANT2 -KO A375 cells. CANT2 oligo indicated the biotinylated antisense oligonucleotides against the CANT2 lncRNA. Negative oligo indicated the scrambled oligonucleotides. Data are presented as mean ± SD from three independent experiments using a Tukey’s multiple comparisons test; **** P  < 0.0001. C Selection of ChIRP-MS for CANT2 binding proteins. D Western blot was used to verify the ChIRP-MS results. CANT2 oligo indicated the biotinylated antisense oligonucleotides against the CANT2 lncRNA. Negative oligo indicated the scrambled oligonucleotides. Representative blots from three independent experiments. E , F Real-time PCR analysis of the binding of CANT2 to PHB2, Nup205, eIF5A, U2A’, PABP and TKT using samples from the RNA-ChIP assay in A375 ( E ) and A875 ( F ) cells. IgG antibody and U2 RNA were used as negative controls. Data are presented as mean ± SD from three independent experiments using a Šídák’s multiple comparisons test; ** P  < 0.01 and **** P  < 0.0001. G , H  ChIP analysis of H3K27ac ( G ) and HDAC1 ( H ) at the CCBE1 promoter in CANT2 -NC or CANT2 -KO A375 and PIG1 cells. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. Data are presented as mean ± SD from three independent experiments using a Tukey’s multiple comparisons test; ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001. I Real-time P CR analysis of the binding of CANT2 to HDAC1 using samples from the RNA-ChIP assay in A375 cells. IgG antibody and U2 RNA were used as negative controls. Data are presented as mean ± SD from three independent experiments using a Šídák’s multiple comparisons test; **** P  < 0.0001. J Co-IP assay was performed to show the interaction between PHB2 and HDAC1 in CANT2 -NC or CANT2 -KO A375 cells. IgG was used as a negative control. Representative blots from three independent experiments.

CCBE1 is a tumor suppressor in tumorigenesis of melanoma

To validate the role of CCBE1 in tumorigenesis, we subsequently induced its overexpression in melanoma cells through the construction of plasmid vectors containing the coding sequence (CDS) of CCBE1 (Fig.  S12A–B , and  6A ). We then assessed cell proliferation and colony formation ability in vitro. Remarkably, the overexpression of CCBE1 ( CCBE1 -OE) led to a significant reduction in tumor cell proliferation compared with the NC groups (Fig.  S12C–D ). Additionally, the CCBE1 -OE groups exhibited a reduced number of colonies (Fig.  6B , lane 2). Moreover, transwell assay (Fig.  S12E , lane 2) and wound healing assay (Fig.  6C–E ) revealed that CCBE1 overexpression resulted in a notable reduction in tumor migration compared to the NC groups. Furthermore, we conducted rescue experiments to confirm the role of CCBE1 in melanoma by knocking down CCBE1 in A375 and A875 cells using shRNAs (shCCBE1) (Fig.  6F–G ). After knocking down CCBE1 in CCBE1 -OE melanoma cells, we observed an increase in tumor cell proliferation (Fig.  6H ). Additionally, CCBE1 -OE melanoma cells with shCCBE1 ( CCBE1 -OE-shCCBE1 group) exhibited enhanced metastasis compared to the NC groups, as demonstrated by transwell assay (Fig.  S12F ) and wound healing assay (Fig.  S12G–J ). These findings demonstrate that CCBE1 functions as a tumor suppressor in melanoma cells.

A Western blot of CCBE1 expression at protein level in melanoma cells after CCBE1 overexpression. Representative blots from three independent experiments. B Colony formation assay to determine the proliferative ability of melanoma cells after CCBE1 overexpression. Representative images from three independent experiments. C Representative images of the wound healing assay at 0 hours and 72 hours post-scratch in NC and CCBE1 -OE melanoma cells. The dashed lines indicate the wound edge. Representative images from three independent experiments. D , E Quantification of the migration area. The migration area was calculated as the percentage of the wound area covered by cells at 72 hours compared to the initial wound area at 0 hours. Data are presented as mean ± SD from three independent experiments using an unpaired two-tailed t test; ** P  < 0.01; **** P  < 0.0001. F , G Western blot of CCBE1 expression in melanoma cells with or without CCBE1 overexpression. Representative blots from three independent experiments. H Colony formation assay to determine the proliferative ability of melanoma cells with or without CCBE1 overexpression. Representative images from three independent experiments. I A xenograft in vivo assay model presented tumor volume (mm 3 ) formed by the A375 cells with or without CCBE1 overexpression. Tumor sizes (mm 3 ) were calculated as the (length×width×width)/2 and presented as the mean ± SD ( n  = 5 mice) using a Šídák’s multiple comparisons test. *** P  < 0.001. Representative images from five independent samples. J Bar graph showed tumor weight (mg) formed by the A375 cells with or without CCBE1 overexpression in a subcutaneous xenograft model. Tumor weight (mg) was measured and presented as the mean ± SD ( n  = 5 mice) using an unpaired two-tailed t test; * P  < 0.05. K Survival analysis of mice following intravenous injection with A375 cells with or without CCBE1 overexpression ( n  = 5 mice) using a Log-rank (Mantel-Cox) test. Triplicate experiments were performed. L Animal imaging system demonstrated the tumor migration ability of A375 cells with or without CCBE1 overexpression on tumor bioluminescent signals. Representative images from five independent samples. M ,  N  Representative images of the histological analysis of thigh ( M ) and lung ( N ) seeding in mice injected intravenously with CCBE1 -NC or CCBE1 -OE A375 cells (original magnification, 1×, scale bar: 1 mm). Representative images from three independent samples.

To further explore the impact of CCBE1 on tumor development in vivo, we subcutaneously injected CCBE1 -OE and control melanoma cells into separate groups of nude mice. We also measured the size of the resultant tumors every 3 days for 12 days in subcutaneous xenografts. Tumor size was monitored every 3 days for 12 days in subcutaneous xenografts (Figs.  6I ;  S13A–C ). Upon completion of this period, the mice were euthanized, and the tumors were collected for subsequent analysis. In the CCBE1 -OE groups, we observed a significant reduction in both tumor volume ( n  = 5–6; ** P  < 0.01; *** P  < 0.001; Figs.  6I ;  S13B ) and weight ( n  = 5–6; * P  < 0.05; **** P  < 0.0001; Figs.  6J ;  S13C ) compared with the NC groups. Significantly, the overexpression of CCBE1 led to a noteworthy extension in the survival rate of mice ( n  = 5–6; Figs.  6K ;  S13D ). To assess tumor growth and migration capability following CCBE1 overexpression in vivo, A375 and A875 cells were labeled with firefly luciferase for the establishment of metastatic models in nude mice. Subsequently, tumor systemic metastasis was assessed through bioluminescence and fluorescence imaging after tail vein injection in these mice. After approximately 30 days, the CCBE1 -OE groups exhibited fewer metastatic loci in nude mice compared to the NC groups, with significantly reduced luciferase signaling (Figs.  6L , bottom;  S13E , bottom). Remarkably, histological examination showed that compared with the NC groups, the overexpression of CCBE1 significantly suppressed metastatic colonization and reduced the number and size of macroscopic nodules observed in the thigh bone (Figs.  6M , bottom;  S13F , bottom), lung (Fig.  6N , bottom), and gastric area (Fig.  S13G , bottom). These data suggested that CCBE1 functioned as a tumor-suppressor in melanoma.

PHB2 determines the transcription of CANT2 and CCBE1 during tumorigenesis

Based on the results obtained above, we identified a series of phenomena and mechanisms where PHB2 served as a shuttle factor, simultaneously regulating the transcription of the coding gene CCBE1 and the lncRNA CANT2 from distinct genomic loci. To further validate its indispensable role in these regulatory processes, we first established melanoma cells with PHB2 knockdown, which were named as shPHB2-1 and shPHB2-2 (Fig.  7A–B ). Following the knockdown of PHB2, there was a significant reduction in the transcriptional level of CANT2 lncRNA (Fig.  7C ). Conversely, the expression of CCBE1 exhibited a substantial upregulation at both the RNA (Fig.  7D ) and protein levels (Fig.  7E ). In connection with the previously elucidated mechanism governing the regulation of CANT2 and CCBE1 , we subsequently investigated the regulatory landscape under the knockdown of PHB2. In the accessible promoter region of CANT2 (Fig.  7F ), shPHB2 tumor cells displayed reduced binding of MLL2 (Fig.  7G ) and a decreased level of H3K4 trimethylation (Fig.  7H ) compared with the empty vector group. At the CCBE1 promoter (Fig.  7I ), the suppression of PHB2 also led to a decrease in the binding of HDAC1 (Fig.  7J ), accompanied by an increase in H3K27 acetylation level (Fig.  7K ). Following analysis via western blot assay for ChIRP, we confirmed that the interaction between PHB2 and CANT2 decreased upon the suppression of PHB2 (Fig.  7L , lanes 1 and 3). Co-IP assays further demonstrated that HDAC1 could not be pulled down by baiting the PHB2 protein when PHB2 was knocked down (Fig.  7M , panel 2, lane 2). ChIRP-PCR analysis revealed that the enrichment of CANT2 at the CCBE1 promoter region was diminished in the absence of PHB2 (Fig.  7N ). Furthermore, to confirm whether PHB2 and CANT2 could directly bind to the promoter of CCBE1 and subsequently regulate its expression, we applied the method enChIP 46 , 47 , 48 . Through constructing a dCas9/pCMV expression vector of small guide RNAs (sgRNAs) with two Flag tags, which can be pulled down by the Flag antibody to isolate specific genomic regions, we modified enChIP assay to efficiently target the CCBE1 promoter region (sg CCBE1 -p groups), which allow us to directly capture the binding proteins and RNAs simultaneously (Fig.  S14A ). We first confirmed the efficiency of capturing the CCBE1 promoter (Fig.  7O–P ). By collecting RNAs and performing reverse transcription PCR, we found that in melanoma cells, CANT2 lncRNA directly bound to the CCBE1 promoter (Fig.  7Q–R ). Additionally, western blot analysis showed that PHB2 directly interacted with the promoter of CCBE1 (Fig.  7S , lane 3). These results convincingly demonstrated that PHB2 played a critical role in regulating the transcription of both the lncRNA CANT2 and the coding gene CCBE1 .

A Real-time PCR of the expression of PHB2 in A375 cells with or without PHB2 suppression. Data are presented as mean ± SD from three independent experiments using a Dunnett’s multiple comparisons test; **** P  < 0.0001. B Western blot of the expression of PHB2 at protein level in melanoma cells after PHB2 knockdown. Representative blots from three independent experiments. C , D Real-time PCR of the expression of CANT2 ( C ) and CCBE1 ( D ) in A375 cells with or without PHB2 suppression. Data are presented as mean ± SD from three independent experiments using an unpaired two-tailed t test; * P  < 0.05, ** P  < 0.01. E Western blot of the expression of CCBE1 at protein level in melanoma cells after PHB2 knockdown. Representative blots from three independent experiments. F – H ChIP analysis of PHB2, MLL2 and H3K4me3 at CANT2 core promoter in A375 cells with or without PHB2 knockdown. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. Data are presented as mean ± SD from three independent experiments using a Šídák’s multiple comparisons test; ** P  < 0.01, **** P  < 0.0001. I – K ChIP analysis of PHB2, HDAC1 and H3K27ac at the  CCBE1 promoter in A375 cells with or without PHB2 knockdown. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. Data are presented as mean ± SD from three independent experiments using a Šídák’s multiple comparisons test; **** P  < 0.0001. L Western blot was used to detect the interaction of PHB2 and CANT2 lncRNA after the suppression of PHB2. CANT2 oligo indicated the biotinylated antisense oligonucleotides against the CANT2 lncRNA. Negative oligo indicated the scrambled oligonucleotides. Representative blots from three independent experiments. M Co-IP assay was performed to show the interaction between PHB2 and HDAC1 after the suppression of PHB2. IgG was used as a negative control. Representative blots from three independent experiments. N ChIRP-PCR assay to dectect the binding of CANT2 lncRNA at the  CCBE1 promoter after the suppression of PHB2. CANT2 oligo indicated the biotinylated antisense oligonucleotides against the CANT2 lncRNA. Negative oligo indicated the scrambled oligonucleotides. Data are presented as mean ± SD from three independent experiments using a Tukey’s multiple comparisons test; **** P  < 0.0001. O – P Quality control of the enChIP assay in capturing the CCBE1 promoter via PCR. Data are presented as mean ± SD from three independent experiments using a Šídák’s multiple comparisons test; **** P  < 0.0001. Q – R RT-PCR analysis of CANT2 lncRNA binding to the CCBE1 promoter using the enChIP method. Data are presented as mean ± SD from three independent experiments using an unpaired two-tailed t test; * P  < 0.05; **** P  < 0.0001. S Western blot analysis of PHB2 binding to the CCBE1 promoter following the enChIP assay. Representative blots from three independent experiments.

The coexistence of aberrant lncRNAs and abnormal coding genes plays a critical role in malignancy development. Understanding how these coding genes and lncRNAs from various loci collectively contribute to malignancy progression poses an intriguing question in cancer research. In this study, we introduced a “one stone two birds” model to explain this co-presence phenomenon in tumorigenesis. We identified PHB2 as a shuttle factor that coordinately orchestrates the transcription of the oncogenic lncRNA CANT2 and the coding tumor-suppressor gene CCBE1 , resulting in the accelerated tumorigenesis of melanoma (Fig.  S14E ).

PHB2 is a pivotal protein in cellular biology, involved in regulating various cellular functions and has potential in various diseases, including cancers 35 , 36 , 49 , 50 . It is primarily known for its role as a crucial component of the inner mitochondrial membrane and has implications for mitochondrial structure and function 30 . Moreover, PHB2 serves as a mitophagy receptor, mediating mitophagy and affecting cancer cell growth 31 , 51 . In addition, PHB2 has also been found to function alone or interact with other transcription factors to influence gene expression in the nucleus 33 , 52 , with emerging research suggests its significance in transcriptional regulation. In our study, however, we showed that PHB2 served as a shuttle factor (the stone) coordinately orchestrated transcription of CANT2 lncRNA (the first bird) and coding tumor-suppressor gene CCBE1 (the second bird) in the tumorigenesis of melanoma. Additionally, other proteins identified in the ChIRP-MS table, such as BRD4, HNRNPM, CTCF, YY1, and cohesin, merit further investigation to explore their interactions with lncRNAs and other functional factors. Since the spatial co-presence of lncRNAs and coding genes in various tumors and other diseases, our concept of “one stone two birds” model may provide an alternative explanation to orchestrate transcriptional regulation of lncRNA and coding gene, and proposing an interesting direction for exploring more unknown functional shuttle factors.

As a canonical disease susceptibility locus, chr6p22 hosts histone gene clusters and variant lncRNA isoforms which function in different diseases. Various mechanisms govern the distinct transcription and function of the diverse coding genes and lncRNAs within the chr6p22.3 region. The deletion on chr6p22.3–p23 harboring ATXN1 , DTNBP1 , JARID2 , and NHLRC1 may be responsible for intellectual disability and autism spectrum disorders 53 . DCDC2 and KIAA0319 on chr6p22.2 act as candidate susceptibility genes of a language learning disorder developmental dyslexia 54 . In metastatic melanoma cells such as YDFR.SB3, WP and RKTJ-CB1, an onco-lncRNA CASC15 was transcribed in this locus, and it also promotes tumorigenesis in acute myeloid leukemia (AML), lung and gastric cancers 55 , 56 , 57 , 58 . Nevertheless, a pair of sense/antisense lncRNAs encoded by CASC15 and NBAT1 are tumor suppressors in neuroblastoma 59 . Surprisingly, lncRNA CASC15-S , the isoform of lncRNA CASC15 , plays the similar role to inhibit neuroblastoma development 60 . Furthermore, lncRNA CANT1 is another variant transcribed from this locus, playing a role of tumor suppressor in eye malignancies such as uveal melanoma and retinoblastoma 61 , 62 . In our study, however, we discovered an oncogenic lncRNA CANT2 that promotes tumorigenesis of melanoma. Furthermore, in breast cancer, the major histone gene cluster at chr6p22 was subdivided into three sub-clusters of histone genes that were organized into hierarchical TADs and located at TAD boundaries, forming an active chromatin hub 63 . This has raised the question of whether the higher-order chromatin structure plays a role in the chr6p22 locus and initiates melanoma tumorigenesis. Unfortunately, our analysis of visualized Hi-C maps revealed that the TAD boundary remains unaltered in melanoma cells when compared to normal skin cells. In our study, however, we uncovered that the change of chromatin accessibility governing the transcription of a lncRNA variant CANT2 at chrp22.3 locus.

It should be noted that H3K4me3 is a crucial epigenetic modification that plays a central role in gene regulation. This modification is typically associated with transcriptional start sites and open chromatin structure 64 . H3K4me3 is primarily catalyzed by MLL proteins and SET1/COMPASS methyltransferase complexes 65 . H3K4me3 helps recruit transcriptional machinery, such as RNA polymerase II, and other chromatin-modifying complexes to initiate gene expression 66 . The sudden depletion of H3K4me3 may significantly reduce overall transcriptional output, increase RNA polymerase II pausing and slowdown elongation rather than sufficiently affecting transcriptional initiation 66 . In our study, we observed that PHB2 recruited MLL2 for activating transcription of CANT2 by increasing H3K4me3. It is of great interesting to explore whether this PHB2-guided H3K4me3 pattern could applied in the transcriptional regulation of other gene or lncRNA.

CCBE1 is essential to lymphangiogenesis and presents as a promising therapeutic tool for a variety of diseases involving the lymphatic system 67 , 68 . Notably, CCBE1 has emerged as a pivotal regulator of vascular endothelial growth factor-C (VEGFC) signaling 69 . Researchers have discovered that CCBE1 enhances VEGFC proteolysis, thereby fostering tumor lymphangiogenesis and facilitating lymphatic metastasis in colorectal cancer 70 . Contrastingly, CCBE1 has been observed to impede the progression of hepatocellular carcinoma by promoting mitochondrial fusion 71 . CCBE1 exhibits predominant expression in the ovary but is downregulated in ovarian cancer cells and primary carcinomas 72 . The loss of CCBE1 expression has the potential to promote ovarian carcinogenesis by augmenting cell migration and survival 72 . In our study, we identified CCBE1 as a tumor suppressor in melanoma. Interestingly, we demonstrated that PHB2 recruits histone deacetylase HDAC1, leading to decreased H3K27 acetylation at the CCBE1 promoter and subsequent repression of CCBE1 transcription. HDAC1, a member of the class I deacetylases, functions to remove lysine-acetyl marks from histone proteins. Previous studies have shown both similarities and differences in the regulatory roles of HDAC1 and HDAC2, highlighting how they may either collaborate or operate independently to regulate cellular processes 45 . Our findings specifically emphasize the crucial role of HDAC1 in this regulatory pathway. While we cannot entirely exclude the involvement of other factors in PHB2-mediated transcriptional regulation of CCBE1 , our study presents a mechanism for the regulation of CCBE1 transcription. Future research should focus on identifying additional factors that may influence the regulation of CCBE1 .

Our research complies with all relevant ethical regulations according to Tongji University. PIG1, HEK-293T cell lines, the malignant melanoma cell lines A375 and A875 cells were purchased and authenticated from American Type Culture Collection (ATCC) and China National Collection of Authenticated Cell Cultures (NCACC), and cultured in DMEM medium (GIBCO) or 1640 medium (GIBCO). All media were supplemented with 10% FBS (GIBCO), 1% penicillin/streptomycin (GIBCO). All cells were incubated at 37°C and 5% CO 2 . All cells were confirmed to be free of mycoplasma contamination.

Animal study

All animal studies were conducted in accordance with the guidelines provided by the Institutional Animal Care and Treatment Committee of Tongji University. The animals were treated following relevant institutional and national guidelines and regulations. The maximum allowable tumor size/burden (with a diameter of less than 1 cm) was not exceeded. Mice were housed at an ambient temperature of 24 ± 2 °C, with circulating air, constant humidity of 50 ± 10%, and a 12-hour light/dark cycle. Female BALB/c nude mice (GemPharmatech) were used, as patient gender did not have a significant impact on the observations and conclusions of this study. Mice were euthanized by cervical dislocation at the end of the experiment, after which tumors and major organs were collected using surgical scissors.

Tumor xenograft nude mice model

3 × 10 6 cells ( CANT2 -NC, CANT2 -KO1 and CANT2 -KO2) in 100 μl volume of DPBS were subcutaneously injected into the right anterior subcutaneous part of 5-week-old female nude mice. Tumor volume was monitored using a vernier caliper every 3 days and calculated with the formula: length (mm)×width (mm) 2 /2. Five mice from each group were euthanatized, and the tumors were weighed. We confirm that the maximal tumor size/burden did not exceed the limits permitted (tumor volume: 1 cm 3 ) by our ethics committee or institutional review board.

Tumor metastasis nude mice model

Cells were transfected with a lentivirus encoded by Luc-vectors with neo r screening markers. Cells were selected by incubation with 0.5 mg/ml G418 solution (InvivoGen) for 2 weeks. 1.5 × 10 6 cells ( CANT2 -NC, CANT2 -KO1) in 100 μl volume of DPBS were intravenously injected through the caudal vein of 6-week-old female nude mice. 30 days later, D-Luciferin Sodium Salt (YEASEN) was injected into the right flanks of mice. Bioluminescence was detected by in vivo small animal imaging systems after anesthetization. Five mice from each group were euthanatized, and the pathological section were performed.

RNA extraction and qPCR

Total RNA was extracted from cells using TRIzol reagent (Sigma) and cDNA was synthesized using 1st Strand cDNA Synthesis SuperMix (YEASEN, 11123ES60). Real time qPCR was performed using qPCR SYBR Green Master Mix (YEASEN, 11198ES08) on Roche LightCycler 96 system. The exhibited data represents the fold change (FC) of experimental group versus control group. In brief, ΔCt was calculated as ΔCt = Ct (test gene) - Ct (Ref. gene). ΔΔCt was calculated as ΔΔCt = ΔCt (experimental group) -ΔCt (control group). The FC of a test gene in experimental group versus control group was calculated as FC = 2^(-ΔΔCt). Each gene tested in triplicates in every independent experiment, and all experiments were triplicated. Primers used are listed in Supplementary Table  1 .

The RACE assay was performed by SMARTer RACE 5’/3’ Kit (Takara Bio) following the manufacturer’s protocol. First-strand cDNA was synthesized using a modified oligo (dT) primer and SMARTScribe Reverse Transcriptase (RT) which added several non-templated residues. The SMARTer II A Oligonucleotide annealed to the tail of the cDNA and served as an extended template for SMARTScribe RT. After having 3’- and 5’-RACE-Ready cDNA samples, RACE PCR reactions and sequencing were performed to get full-length cDNA.

FAIRE assay

The FAIRE assay was conducted following a previously described protocol 73 . A total of 1 × 10 7 cells were crosslinked with 37% formaldehyde to a final concentration of 1% in PBS. The crosslinking reaction was quenched by the addition of 2.5 M glycine. The fixed cells were collected by centrifugation at 1000 g for 5 min at 4 °C and washed three times with 10 ml of 1× PBS. The pellets were resuspended in 1 ml of cold lysis buffer and sonicated to achieve an average DNA fragment size of ∼ 200–500 bp. The supernatant was transferred to a fresh 1.5-ml tube and incubated with 1 μl of DNase-free RNase A for 30 min at 37°C. Subsequently, 300 μl of phenol/chloroform/isoamyl alcohol was added, and the sample was vortexed for 10 s followed by centrifugation at 12,000 g for 5 min. The aqueous layer was transferred, and the extraction process was repeated three times, pooling all the aqueous solutions. Afterwards, 1/10 volume of 3 M sodium acetate, 2 volumes of 95% ethanol, and 1 μl of 20 mg/ml glycogen were added, and the solution was incubated at −80 °C for at least 30 min. The DNA was then purified using a DNA Clean-up Kit (AxyPrep). The PCR primers used are listed in Supplementary Table 1 .

Luciferase assay

Cells were collected and gDNA was achieved by Genomic DNA extraction Kit (TIANGEN, DP304) following the manufacturer’s protocol. Then the gDNA was used for PCR with Q5 High-Fidelity DNA Polymerases (NEB). Primers used are listed in Supplementary Table  1 . The promoter region upstream of CANT2 TSS was dissected into 5 fragments of 200 bp and were respectively cloned into the pGL3-basic vector (Promega). HEK-293T cells in a 24-well plate reached 50% confluence within 24 hours before transfection. 900 ng pGL3-basic containing different CANT2 promoter fragments or pGL3-basic and 90 ng pRL-TK (Promega) were co-transfected into HEK-293T cells using lipofectamine 3000 (Invitrogen). After 48 hours, luciferase activity was measured by Dual Luciferase Reporter Gene Assay Kit (YEASEN, 11402ES60) following the manufacturer’s protocol. Each group was repeated with 3 technical replicates. Transfections were repeated in three independent experiments. Firefly luciferase activity was normalized to Renilla luciferase activity. P values were calculated using t test.

CRISPR/Cas9-mediated CANT2 lncRNA knockout

4 sgRNAs targeting the left and right side of the core promoter of CANT2 to be deleted were identified using Optimized CRISPR Design ( http://crispor.gi.ucsc.edu ) and synthesized. SgRNA oligos were cloned respectively into lentiCRISPRv2 with BsmBI (NEB). SgRNA oligos used are listed in Supplementary Table  1 .

CCBE1 overexpression plasmid construction

The coding sequence of CCBE1 were generated by PCR and then cloned into the PGMLV-CMV-MCS-3xFlag-EF1-mScarlet-T2A-Puro vector with XhoI and BamHI (NEB). Primers used are listed in Supplementary Table  1 .

Short hairpin RNA (shRNA) targeting PHB2 construction

3 gRNAs targeting PHB2 were synthesized. SgRNA oligos were cloned respectively into PGMLV-hU6-MCS-CMV-ZsGreen1-PGK-Puro-WPRE. SgRNA oligos used are listed in Supplementary Table  1 .

Lentivirus packaging and generation of stable cell lines

The lipofectamine 3000 (Invitrogen) was incubated with Opti-MEM I Reduced Serum Medium (GIBCO) and used to transfect HEK-239T cells with 3 mg of the target plasmids, 3 mg of pMD2.D plasmids and 6 mg of PsPax plasmids. Eight hours after transfection, the medium was replaced with 10 ml of fresh medium. The supernatant containing the viruses was collected at 48 and 72 hours respectively. The virus-containing solution was filtered and concentrated. 24 hours prior to transfection, melanoma cells were seeded at 2.0 × 10 5 cells per well in a six-well plate. The medium was replaced with virus-containing supernatant supplemented with 10 ng/ml polybrene (Sigma). After 48 hours, the medium was replaced with fresh medium. Cells were selected by incubation with 4 mg/ml puromycin (Invitrogen) for 2 weeks. Colonies were derived from single cells tested for the loss of the targeted region, and later expanded for further analyses.

CCK8 cell viability assay

Cells were seeded into a flat-bottomed 96-well culture plate at 1000 cells per well with 100 μl culture medium, and incubated at 37°C with 5% CO 2 . 10 μl of CCK8 kit (YEASEN, 40203ES80) was added to each well and incubated for 2 h. Then the absorbance was measured at 450 nm with SpectraMax iD3 (Molecular Devices) for 4 consecutive days.

Colony formation assay

2000 cells were suspended in 2 ml DMEM medium and cultured in a six-well plate for 2 weeks. For quantification, the colonies grown in plates were stained with 1% crystal violet and then photographed. The number and size of the colonies were determined using ImageJ.

Wound healing assay

The migratory ability of the cells was evaluated by seeding 1 million cells into a six-well plate and cultured with DMEM medium for 24 h. Then a wound was made by manually scraping the cell monolayer with a 10 μl pipet tip and cells were incubated with FBS-free medium. Images were taken at the indicated times.

Transwell assay

The migratory ability of the cells was evaluated using a 24-well transwell plate with 8.0 µm Pore Polyester Membrane (Corning). The upper compartment contained 30,000 cells suspended in 150 μl DMEM medium containing Basement Membrane Matrix (Corning), and the lower compartment contained 600 μl DMEM medium supplemented with 10% FBS. The transwell system was stained with 0.25% crystal violet after 2 days of incubation at 37°C. The cells on the inner side of the transwell were removed by scrubbing, and the cells on the outer side were photographed.

A total of 1 × 10 8 cells were crosslinked with 3% formaldehyde at room temperature for 30 min. The reaction was quenched by adding 125 mM glycine for 5 min and then centrifuged at 13,000 g for 5 min at 4 °C. The supernatant was removed. The pellet was weighed and resuspended in 1 ml lysis buffer per 100 mg. DNA was sheared to a size of 300 bp for ChIRP-seq or 1000 bp for ChIRP-MS, respectively (Covaris M220). 1.5 μl RNA probes (100 pmol/L) targeting CANT2 exons were added to every 1 ml sample and incubated at 37 °C overnight with shaking. 50 μl MyOne Streptavidin C1 beads (Life Technologies) was pre-washed with lysis buffer for three times, and then added to the reaction and incubated at 37 °C for 2 h with shaking. Subsequently, the beads were washed 3 times with 1 ml of wash buffer for 5 min per wash. At the last wash step, 1/10 of the beads were reserved for qPCR analysis. The supernatant of the rest of beads was removed. For MS, 200 μl protein elution buffer was added to the beads and incubated at room temperature for 20 min and then at 65 °C for 10 min with shaking. The supernatant was collected and the elution was repeated for twice. Protein was purified with TCA Protein Precipitation Kit (Sangon Biotech) following the manufacturer’s instruction, and then MS was performed. For DNA sequencing, the rest of beads were suspended in 150 μl DNA elution buffer and incubated at 37 °C for 30 min with shaking. The supernatant was collected and the elution was repeated for twice. PK was added to the reaction and incubated at 65 °C for 2 h. DNA was purified and suspended in 20 μl elution buffer, and then sequencing was performed. ChIRP-MS was performed once in this study following the previous protocols 74 , 75 .

Western blot

Cells were lysed in RIPA lysis buffer (YEASEN, 20101ES60) containing 1 mM PMSF (YEASEN, 20104ES03) for 30 min, and then centrifuged at 13,000 g for 10 min at 4 °C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% (w/v) polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% BSA (YEASEN, 36101ES25) for 1 h at room temperature, the membrane was incubated with different antibodies in 5% BSA overnight at 4 °C. The membrane was then incubated with Peroxidase-Conjugated Goat Anti-Rabbit IgG or Peroxidase AffiniPure Goat Anti-Mouse IgG (YEASEN). The band signals were visualized and quantified using the Fully Automatic Chemiluminescence/Fluorescence Image Analysis System (Tanon). The following antibodies were used in this study: anti-PHB2 (1:1000, CST, 14085), anti-PHB2 (1:1000, Santa Cruz, sc-133094), anti-Histone H3 (1 μg/ml, Abcam, ab176842), anti-HDAC1 (1:1000, CST, 34589), anti-CCBE1 (1:1000, ImmunoWay, YN1730), anti-Nup205 (1:1000, Santa Cruz, sc-377047), anti-eIF5A (1:500, Santa Cruz, sc-390202), anti-U2A’ (1:1000, Santa Cruz, sc-393804), and anti-TKT (1:1000, Santa Cruz, sc-390179).

RNA-chromatin immunoprecipitation (RNA-ChIP)

RNA-ChIP was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer’s instructions. In brief, 10 7 cells were lysed with RIP lysis buffer with one freeze-thaw cycle. Cell extracts were coimmunoprecipitated with anti-PHB2, anti-Nup205, anti-eIF5A, anti-U2A’, and anti-TKT (Santa Cruz, 1:1000), and the retrieved RNA was subjected to real-time qPCR analysis. Normal mouse IgG was used as a negative control.

RNA extraction library preparation

RNA was extracted from CANT2 -NC and CANT2 -KO cells using the TRIzol reagent (Invitrogen). RNA purity and concentration were confirmed on a 2100 Bioanalyzer (Agilent Technologies) and a Qubit 2.0 fluorometer with the Qubit RNA Assay Kit (Life Technologies). 2 μg total RNA per sample was used as input material for the RNA sample preparation. Library preparation was completed using the VAHTS Total RNA-seq Library Prep Kit for Illumina (Vazyme) following the manufacturer’s recommendations. The libraries were sequenced by the Illumina PE150 paired-end sequencing platform.

ChIP was conducted using an EZ-Magna ChIP A/G kit (Millipore) according to the manufacturer’s protocol. In brief, 10 7 cells were crosslinked and then lysed. DNA was sheared to a size of 200–500 bp (Covaris M220). DNA collected was coimmunoprecipitated with anti-PHB2 (CST, 1:100), anti-HDAC1(CST, 1:100), anti-MLL2 (1:100, Proteintech, 27266-1-AP), anti-H3K4me3 (1:500, Abclonal, A22146) and anti-H3K27ac (1:500, Abclonal, A22264). Anti-normal mouse IgG was used as a negative control. After immunoprecipitation on A/G beads, DNA was purified and sequenced or amplificated with qPCR. Primers for ChIP-qPCR are listed in Supplementary Table  1 .

10 7 cells were carefully washed with pre-chilled PBS for two times, and then collected with 0.5 ml of cold RIPA lysis buffer in a clean 1.5 ml Ep tube. The tube was centrifuged at 14,000 g at 4 °C for 15 min. The supernatant was transferred to a new tube immediately. The protein was diluted to 1 μg/μl. 150 μl of A/G beads (Thermo Scientific) were washed with PBS for two times. 50 μl of washed A/G beads were added to the sample and shaking on a rotator for 10 min at 4 °C. The mixed sample was centrifuged at 14,000 g at 4 °C for 15 min again. The supernatant was transferred to a new 1.5 ml Ep tube, and mixed with 100 μl washed A/G beads. Anti-PHB2 (1:50, CST) and anti-HDAC1 (1:100, CST) were added to the sample respectively, and incubated at 4 °C overnight. The beads were washed with PBS for three times and resuspended in 40 μl RIPAP buffer. Then the sample was proceeded to western blot.

Cytoplasmic and nuclear RNA or protein isolation

Cytoplasmic and nuclear RNA or protein were extracted using PARIS Kit (Thermo Fisher, AM1921) according to the manufacturer’s instructions. The localization of the CANT2 was assayed through the amplification of CANT2 by RT-qPCR. Optimal annealing U6 was used as a nuclear localization reference, and GAPDH was used as a cytoplasmic reference. The localization of the PHB2 was assayed through the western blot. Actin-β (ACTB) was used as a cytoplasmic reference, and H3 was used as a nuclear localization reference.

We took the previous enChIP method 46 , 47 , 48 as references and made some modifications. First, we constructed a dCas9/pCMV expression vector of sgRNAs with two Flag tags, which can be pulled down by the Flag antibody to isolate specific genomic regions, thereby allowing us to directly capture the binding proteins and RNAs simultaneously. After transfecting these vectors into cells, 3/6 × 10 6 of cells were fixed with 1% formaldehyde at 37 °C for 10 min. The cells were lysed in Cell Lysis Buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% IGEPAL-CA630, cOmplete EDTA-free Protease Inhibitor Cocktail, 100 mM PMSF) for 15 min, and then in Nuclei Lysis Buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, cOmplete EDTA-free Protease Inhibitor Cocktail, 100 mM PMSF). The chromatin fraction was fragmented by sonication (500 bp–1 kb). The sonicated chromatin was diluted in 1/10 Dilution Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS, cOmplete EDTA-free Protease Inhibitor Cocktail, 100 mM PMSF), pre-cleared with 15 μg of normal mouse IgG conjugated to 150 μl of A/G beads (Thermo Scientific), and incubated with 15 μg of anti-Flag (Proteintech, 20543-1-AP) conjugated to 150 μl of A/G beads at 4°C overnight.

The beads were washed with 1 ml of Low Salt Wash Buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.1% SDS), High Salt Wash Buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.1% SDS), LiCl Wash Buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL-CA630, 0.5% sodium deoxycholate), and TE Buffer (10 mM Tris, pH 8.0, 1 mM EDTA). After washing, the beads were divided into three groups. In two groups, the beads were suspended in 285 μl of TE, 12 μl of 5 M NaCl, and Proteinase K, and then incubated at 65°C overnight or 8 h for reverse crosslinking, followed by DNA or RNA extraction using Phenol/chloroform or Trizol. The third group was suspended in RIPA buffer (including PMSF), incubated at 100°C for 10 min, and then subjected to western blot analysis.

Statistics and reproducibility

The analyses were performed on three biological replicates (n). All statistical analyses were performed using the GraphPad 9.0 software and Microsoft Excel. The significance was set at P  < 0.05. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, ns = non-significant ( P  > 0.05). All the values are presented as mean ± standard deviation (SD). Source data are provided as a Source Data file.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data generated in this study are available at the Gene Expression Omnbus GSE231936 , and the Sequence Read Archive (SRA) BioProject PRJNA970260 . The ChIRP-MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD055094 . The RNA-seq and ATAC-seq dataset from melanoma cells and SKCM dataset from GEPIA publicly available data used in this study are available in the GEO database under accession code GSE223887 37 , GSE223888 37 , GSE232375 38 , GSE188398 42 , GSE134432 43 , and GSE241445 44 .  Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 82372705 and 31870748) to H.Z., the Shanghai Oriental Elite Project (Grant No. 2000152009) to H.Z., the National Key Research and Development Program of China (Grant No. 2017YFE0196300) to H.Z., the Shanghai Natural Science Foundation (Grant No. 22ZR1466100) to H.Z., the Fundamental Research Funds for the Central Universities (Grant No. 22120230292) to H.Z., the Key Laboratory of Organ Development and Epigenetics of Jiangxi Province (Grant No. 2024SSY07141) to H.Z., the Shuguang Project of Shanghai Municipal Education Commission and Shanghai Education Development Foundation (Grant No. 17SG19) to H.Z., the Outstanding Young Medical Scholar of Shanghai Municipal Commission of Health and Family Planning (Grant No. 2017YQ067) to H.Z., the Outstanding Yong Scholar Grant of Tongji University (Grant No. PA2019000239) to H.Z., and the Startup Funding of Frontier Science Research Center for Stem Cells & Shanghai East Hospital of Tongji University (Grant No. DFRC2019003) to H.Z.

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These authors contributed equally: Tianyi Ding, Haowen Xu, Xiaoyu Zhang.

Authors and Affiliations

State Key Laboratory of Cardiology and Medical Innovation Center, Institute for Regenerative Medicine, Shanghai East Hospital, Frontier Science Research Center for Stem Cells, School of Life Science and Technology, Tongji University, Shanghai, 200092, China

Tianyi Ding, Haowen Xu, Xiaoyu Zhang, Fan Yang, Jixing Zhang, Yibing Shi, Yiran Bai, Jiaqi Yang, Chaoqun Chen, Chengbo Zhu & He Zhang

Jiangxi Province Key Laboratory of Organ Development and Epigenetics, Clinical Medical Research Center, Affiliated Hospital of Jinggangshan University, Medical Department of Jinggangshan University, Ji’an, 343009, China

School of Life Science, Jinggangshan University, Ji’an, 343009, China

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Contributions

Tianyi Ding: Conceptualization, Investigation, Methodology, Visualization, Writing—original draft, Writing—review & editing. Haowen Xu: Methodology, Visualization, Writing—original draft, Writing—review & editing. Xiaoyu Zhang: Investigation, Methodology, Data curation. Fan Yang: Resources. Jixing Zhang: Methodology. Yibing Shi: Investigation. Yiran Bai: Investigation. Jiaqi Yang: Resources. Chaoqun Chen: Resources. Chengbo Zhu: Resources. He Zhang: Project administration, Funding acquisition, Conceptualization, Supervision, Writing—original draft, Writing—review & editing. All authors have read and approved the final manuscript.

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Ding, T., Xu, H., Zhang, X. et al. Prohibitin 2 orchestrates long noncoding RNA and gene transcription to accelerate tumorigenesis. Nat Commun 15 , 8385 (2024). https://doi.org/10.1038/s41467-024-52425-z

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DOI : https://doi.org/10.1038/s41467-024-52425-z

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