Arginine Methylation-dependent TRIM47 Stability Mediated by CARM1 Promotes the Metastasis of Hepatocellular Carcinoma

Arginine Methylation-dependent TRIM47 Stability Mediated by CARM1 Promotes the Metastasis of Hepatocellular Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Arginine Methylation-dependent TRIM47 Stability Mediated by CARM1 Promotes the Metastasis of Hepatocellular Carcinoma Jia Hu, Yuzhe Tang, Xiang Meng, Xia Luo, Wen Tao Yao, Li Tian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4220751/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2024 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Abstract The tripartite motif (TRIM) protein family has been shown to play important roles in the occurrence and development of various tumors. However, the biological functions of TRIM47 and its regulatory mechanism in hepatocellular carcinoma (HCC) remain unexplored. Here, we showed that TRIM47 was upregulated in HCC tissues compared with adjacent normal tissues, especially at advanced stages, and associated with poor prognosis in HCC patients. Functional studies demonstrated that TRIM47 enhanced the migration and invasion ability of HCC cells in vitro and in vivo . Mechanistically, TRIM47 promotes HCC metastasis through interacting with SNAI1 and inhibiting its degradation by proteasome. Moreover, TRIM47 was di-methylated by CARM1 at its arginine 210 (R210) and arginine 582 (R582), which protected TRIM47 from the ubiquitination and degradation mediated by E3 ubiquitin ligase complex CRL4 CRBN . Collectively, our study reveals a pro-metastasis role of TRIM47 in HCC, unveils a unique mechanism controlling TRIM47 stability by CARM1 mediated arginine methylation, and highlights the role of the CARM1-CRL4 CRBN -TRIM47-SNAI1 axis in HCC metastasis. This work may provide potential therapeutic targets for metastatic HCC treatment. Biological sciences/Cancer/Metastasis Biological sciences/Biochemistry/Proteins/Oncogene proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Tripartite motif-containing (TRIM) family proteins have a conserved domain architecture characterized by a RING domain in N-terminal, a B-box domain, a coiled-coil domain, and a variable C-terminal domain 1 . Dysregulation of TRIM proteins is closely associated with tumorigenesis and tumor progression 2 . TRIM47, also known as GOA (Gene overexpressed in astrocytoma protein), was upregulated and possessed oncogenic function in multiple types of tumors, such as non-small cell lung carcinoma (NSCLC) 3 , colorectal cancer (CRC) 4 and prostate cancer (PC) 5 . Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of cancer-related mortality worldwide 6 . A bioinformatics study reported that TRIM47 expression was associated with poor prognosis of HCC. A TRIM family gene-based signature including TRIM47 and another 5 TRIM genes performed well in Overall survival (OS) prediction for HCC 7 . These studies indicated that TRIM47 might play an important role in HCC progression. However, the detailed roles of TRIM47 and its regulatory mechanism in HCC remain elusive. Protein arginine methylation is a prevalent post-translational modification, which is catalyzed by a group of enzymes termed protein arginine methyltransferases (PRMTs) 8 . Coactivator-associated arginine methyltransferase 1 (CARM1), a type I PRMTs, is deregulated in numerous cancers and plays critical roles in cancer progression by catalyzing asymmetric di-methylation of histone or nonhistone substrate proteins 9 . For example, CARM1 promoted breast cancer metastasis by methylating chromatin remodeling factor BAF155 10 . Arginine methylation of MDH1 by CARM1 inhibits glutamine metabolism and suppresses pancreatic ductal adenocarcinoma (PDAC) progression 11 . However, independent studies reported opposite functional roles of CARM1 in HCC. CARM1 was previously reported to suppress the glycolysis in liver cancer cells by mediating arginine 234 (R234) methylation of GAPDH, thus inhibiting the proliferation of liver cancer cells 12 . This conflicts with another study demonstrating that CARM1 indicates poor prognosis and promotes HCC progression by activating AKT/mTOR signaling 13 . These conflicting results lead to a confused understanding of CARM1’s functions in HCC, thus needing to explore its underlying molecular mechanism. Here, we found that TRIM47 was upregulated in tumor tissues and associated with poor clinical outcomes of HCC patients. In vitro and in vivo studies revealed that TRIM47 facilitated the migration and invasion of HCC cells. Mechanistically, TRIM47 promoted epithelial-mesenchymal transition (EMT) by interacting with SNAI1 and maintained its stability. Moreover, we identified that TRIM47 was a novel substrate of CARM1, while the methylation by CARM1 protected TRIM47 from proteasomal degradation mediated by E3 ubiquitin ligase complex CRL4 CRBN . This work may provide a basis for finding novel targets and designing therapeutic strategies against metastatic HCC. 2. Materials and Methods 2.1 Antibodies and Reagents Antibodies used in this study were as follows: anti-TRIM47 (Proteintech, 26885-1-AP), CARM1 (Proteintech, 55246-1-AP), anti-GAPDH (Proteintech, 10494-1-AP), anti-Ubiquitin (Proteintech, 10201-2-AP), anti-Myc (Abclonal, AE010), anti-FLAG (Abclonal, AE005), Asymmetric di-methyl arginine antibody (Cell Signaling Technology, 13522), anti-E-cadherin (Proteintech,20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-RHOA (Proteintech, 10749-1-AP), anti-Rac1 (Proteintech, 24072-1-AP), anti-AXIN2 (Proteintech, 20540-1-AP), anti-β-Catenin (Proteintech, 51067-2-AP), anti-GST (Proteintech,10000-0-AP), anti-CRBN (Proteintech, 28494-1-AP), anti-SNAI1 (Proteintech, 13099-1-AP), anti-SNAI2 (Cell Signaling Technology, 9585), anti-Vimentin (Cell Signaling Technology, 5741), anti-ZEB1 (Cell Signaling Technology, 3396), anti-ZO-1(Cell Signaling Technology, 5406). Other reagents used in this study were: Cycloheximide (MedchemExpress, HY-12320), CARM1-IN-1 (MedchemExpress, HY-12759), MG132 (MedchemExpress, HY-13259), Chloroquine (Sigma-Aldrich, C6628-25G). 2.2 Cell Culture and Transfection HEK293T and human HCC cell lines HepG2, Huh7 were obtained from American Type Culture Collection (ATCC). SMMC7721 cell line was purchased from Warner Bio with STR. All cells used in this study were cultured in RPMI 1640 medium (Gibco, 31800022) and DMEM medium (Gibco, 12800017) containing 10% fetal bovine serum (VISTECH, SE100-011). Plasmids were transiently transfected into cells by using Neofect TM DNA reagent (Neofect, TF20121201) according to the manufacturer’s instruction. 2.3 Plasmids Construction Human TRIM47 cDNA was cloned into the p3×FLAG-CMV-10 Vector (sigma aldrich) to generate p3×FLAG-CMV-TRIM47. Human CARM1, CRBN and SNAI1 cDNA was cloned into pcDNA.3.1 Vector (Invitrogen). The following mutational TRIM47 plasmids were constructed based on p3×FLAG-CMV-TRIM47 plasmid: p3×FLAG-CMV-TRIM47 R210K , p3×FLAG-CMV-TRIM47 R582K , p3×FLAG-CMV-TRIM47 R210/582K . TRIM47 and TRIM47 R210/582K stably overexpression cell lines were constructed using the pLenti-3×Flag-CMV plasmid. Oligonucleotides specific shRNA against TRIM47 or CARM1 were synthesized and cloned into pLKO.1 Vector (Addgene Plasmid, #10878). All plasmids used in our study were confirmed by DNA sequencing. The shRNA sequences were listed in Table S1 . 2.4 Lentivirus Production and Transduction Indicated lentiviral plasmids were transfected into HEK293T cells. After 48h post transfection, the supernatant containing lentiviral particles was harvested. HCC cells were infected with the lentivirus in the presence of polybrene (8 µg/mL) for 24h and then selected by puromycin (0.5 µg/mL) for one week. The overexpression or knockdown efficiency was detected by western blot. 2.5 Cell migration, invasion and wound healing assay The cell migration and invasion assay were performed by using 8-µm Boyden chambers (Corning Inc, 3422). For migration assay, 1×10 5 SMMC7721 cells or Huh7 cells were seeded in the upper chamber with serum-free RPMI 1640 medium. The lower chamber was added with RPMI 1640 medium containing 10% FBS. After 12 h for SMMC7721 or 6 h for Huh7, the migrated cells were fixed with 4% paraformaldehyde, stained with crystal violet and counted. For invasion assay, the upper chamber was pre-coated with matrigel matrix (BD Science, 356234). 2×10 5 SMMC7721 cells were seeded in the upper chamber with serum-free RPMI 1640 medium. The lower chamber was added with RPMI 1640 medium containing 10% FBS. After 72 h, the invaded cells were fixed with 4% paraformaldehyde, stained with crystal violet and counted. For wound healing assay, indicated cells were seeded in 6-well plate and grown to 90% confluence. A linear scratch was made by a sterile 200 µl tip. Cells were cultured in RPMI 1640 medium containing 1% FBS to close the wound for 48 h. The scratch area was analyzed by Image J software. 2.6 Animal experiments 1×10 6 indicated SMMC7721 cells (shNC, shTRIM47#1 and shTRIM47#2) were injected into Balb/c nude mice (male, 6 weeks) through tail vein. After 8 weeks, the mice were sacrificed and pulmonary metastatic nodules were counted. The animal studies were approved by the Animal Ethics Committee of Wuhan University of Science and Technology. The mice used in our study were housed under specific pathogen-free (SPF) conditions. 2.7 RNA Extraction and Semi-Quantitative RT-PCR RNA was extracted by RNA extraction kit (Abclonal, RK30120) and cDNA was synthesized using a Transcription Reagent Kit (Abclonal, RK20429) according to the manufacturer’s instruction. PCR analysis of mRNA expression was conducted using a 2 × Taq Plus Master Mix (Vazyme, P211-01). The PCR products were separated by agarose gel electrophoresis. 2.8 Immunofluorescence and F-actin staining Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). For immunofluorescence, cells were then blocked with 10% FBS, incubated with the indicated primary antibodies and secondary antibodies. For F-actin staining, cells were incubated with Rhodamine-conjugated Phalloidins for 1 h. nuclei was stain with DAPI. 2.9 Western Blot Cells were harvested and lysed with RIPA buffer (Beyotime Biotechnology, P0013C) containing protease inhibitors. The protein concentration was detected with BCA kit (Thermo Scientific, A55864). The cell lysate was separated by SDS-PAGE and then transferred onto Immobilon-P (PVDF) membranes (Merck Millipore, ISEQ00010). After blocking with 5% skimmed milk, membranes were incubated with indicated antibodies. The protein bands were visualized by chemiluminescence system. 2.10 Co-immunoprecipitation assay (Co-IP) Cells were harvest and lysed with RIPA buffer (Beyotime Biotechnology, P0013D) containing protease inhibitors. The cell lysates were incubated with indicated antibodies overnight at 4°C. Then protein A/G magnetic beads (Biolinkedin) were added and incubated for another 2 hours. The protein-bound beads were washed with washing buffer for 3 times, boiled with protein loading buffer and analyzed by western blot. 2.11 GST-PAK1 PBD Pull Down Assay and GTP-RhoA activation assay GST-PAK1 PBD protein was expressed in E.coli strain BL21 and purified. Equal amounts of GST-PAK1 PBD protein were incubated with glutathione Sepharose 4B beads (GE Healthcare) and indicated cell lysates. Then the beads were washed with washing buffer for 5 times, boiled with protein loading buffer and analyzed by western blot. The GTP-RhoA activation assay were performed with the Rho Activation Assay Biochem Kit (Cytoskeleton) according to the manufacture’s instruction. 2.12 Statistical Analysis Kaplan-Meier method was performed to analyze survival rate and the survival differences was calculated with log-rank test. Unpaired Student’s t-tests or one-way ANOVA were used to determined other comparisons according to the number of groups. Date was presented as the mean ± standard deviation (SD). Statistical significance was set at P < 0.05. All analyses were performed with GraphPad Prism software. 3. Results 3.1 TRIM47 is a pro-metastatic factor in HCC To evaluate the clinical significance of TRIM47 in human HCC, we analyzed the publicly available HCC expression profiles in The Cancer Genome Atlas (TCGA) and GSE76427 database. TRIM47 was upregulated in HCC tumor tissues compared with normal tissues, especially at advanced stages (stage IV vs stage I/II/III in TCGA) (Fig. 1 A-B, Fig. S1 A). Kaplan-Meier survival analysis revealed that HCC patients with high TRIM47 levels usually had poorer overall survival (OS) (Fig. 1 C, Fig. S1 B). These data suggest that TRIM47 may possess oncogenic activity in HCC. Next, we explored the function of TRIM47 in HCC progression. We detected TRIM47 expression in four HCC cell lines (HepG2, Huh7, SMMC7721 and Bel7402) and found that SMMC7721 cells expressed TRIM47 at a relatively high level while Huh7 and Bel7402 cells at a low level (Fig. S1 C). SMMC7721 (high expression of TRIM47) and Huh7 (low expression of TRIM47) cell lines were thus chosen for further studies. Knockdown of TRIM47 in SMMC7721 cells significantly inhibited cell migration and invasion in vitro (Fig. 1 D-F, Fig. S1 D-E). Conversely, TRIM47 overexpression promoted cell migration ability in Huh7 and SMMC7721 cells (Fig. 1 G-I). Moreover, TRIM47 knockdown decreased the foci number of lung metastasis in vivo (Fig. 1 J-K). These findings indicate that TRIM47 is a pro-metastatic factor in HCC. 3.2 CARM1 interacts with TRIM47 and maintains its stabilization To dissect the upstream regulatory factors and pro-metastatic mechanism of TRIM47 in HCC, we performed an immunoprecipitation-coupled mass spectrometry screen. CARM1, a type I PRMTs playing critical roles in cancer progression by methylating histone or nonhistone substrates, was identified as a putative TRIM47-interacting protein (Fig. 2 A). Noticeably, TP53, another protein reported to interact with TRIM47 14 , was also hitted in our screening, indicating the reliability of our screening. The binding between TRIM47 and CARM1 were confirmed by reciprocal co-IP assay (Fig. 2 B). Next, we determined whether the expression of CARM1 or TRIM47 was affected by their interactions. Knockdown of TRIM47 had no effect on CARM1 protein levels (Fig. S2 A). However, TRIM47 protein levels was significantly decreased upon CARM1 depletion, while its mRNA expression was unchanged (Fig. 2 C, Fig. S2 B). Treatment with specific CARM1 methyltransferase inhibitor HY-12759 also reduced TRIM47 protein levels in a dose-dependent manner (Fig. 2 D). The cycloheximide chase assay showed that the protein half-life of TRIM47 was remarkably shortened after CARM1 knockdown (Fig. 2 E). These results suggest that CARM1 acts upstream of TRIM47 to control TRIM47’s protein stability through post-translational modifications. Of note, decreased TRIM47 protein levels by CARM1 knockdown were rescued when treated HCC cells with proteasome inhibitor MG132, but not with lysosome inhibitor CQ (Fig. 2 F, Fig. S2 C), suggesting that CARM1 protects TRIM47 protein from proteasome-mediated degradation. Consistently, the ubiquitination of TRIM47 was enhanced by CARM1 knockdown or HY-12759 treatment (Fig. 2 G-H). Taken together, these results indicate that CARM1 stabilizes TRIM47 protein by inhibiting its ubiquitination. 3.3 TRIM47 is an arginine methylation substrate of CARM1 As a type I PRMTs, CARM1 catalyzes the formation of asymmetric di-methylarginine in its substrate proteins 9 . We found that TRIM47 was indeed arginine-methylated by using an anti-asymmetric di-methylarginine (α-ADME) antibody (Fig. 3 A). CARM1 knockdown or HY-12759 treatment markedly decreased the methylation of TRIM47 (Fig. 3 B-C). To define which arginine residues were methylated by CARM1, we performed mass spectrometric analysis of immunopurified TRIM47 protein from HCC cells and identified arginine 210 (R210) as a putative asymmetric di-methylation site (Fig. S3A). A previous proteomic study reported that arginine 582 (R582) might be another methylated arginine residue of TRIM47 15 . To determine whether R210 and R582 are the major methylated site in TRIM47, we mutated these two sites into lysine (TRIM47 R210K or TRIM47 R582K ) that conferred resistance to arginine methylation. Compared with wild-type (WT) TRIM47, both TRIM47 R210K and TRIM47 R582K mutants showed a substantial, but incomplete reduction of asymmetric di-methylation levels (Fig. S3B). The methylation signal was also markedly inhibited for the R210 and R582 double mutant (TRIM47 R210/582K ) (Fig. 3 D). These results indicate that both R210 and R582 are the major, if not the sole, methylation site of TRIM47. Interestingly, R582 is located in a conserved glycine-arginine-methionine (PGM) rich substrate motif reported for the recognition of CARM1, while R210 is not (Fig. 3 E). We further tested whether the methylation of R210 and R582 contributed to TRIM47 stability and ubiquitination status. TRIM47 R210/582K mutant exhibited higher ubiquitination levels than that of WT TRIM47 (Fig S3C). The ubiquitination level of WT TRIM47 was sharply enhanced by CARM1 knockdown, whereas TRIM47 R210/582K mutant showed little or no change (Fig. 3 F). Consistently, HY-12759 significantly decreased WT TRIM47 protein levels, but not TRIM47 mutants (TRIM47 R210K , TRIM47 R582K and TRIM47 R210/582K ) (Fig. 3 G, Fig. S3D). We also assessed the effect of TRIM47 arginine mutants on the metastasis of HCC. TRIM47 R210K and TRIM47 R582K mutant-expressing cells were less metastatic compared to the WT TRIM47-expressing cells (Fig. S3E-G). Noticeably, TRIM47 R210/582K mutant absolutely abolished the pro-metastatic ability of TRIM47 in HCC (Fig. 3 H-J, Fig. S3E-G). Together, these data indicated that the methylated modification by CARM1 stabilizes TRIM47 protein levels and promotes the metastasis of HCC. 3.4. CARM1 suppresses HCC metastasis in a TRIM47-dependent manner CARM1 functions as an oncogene or a tumor suppressor depending on cancer types 16 , 17 . However, the roles of CARM1 in dynamic HCC progression are controversial 12 , 13 . We analyzed the HCC cohort in TCGA and found that HCC patients at the advanced stages expressed higher CARM1 than early staged patients (stage III + IV vs stage I + II) (Fig. 4 A). Kaplan-Meier survival analysis revealed that high levels of CARM1 was associated with poor survival in HCC patients (Fig. 4 B, Fig. S4A). Silencing of CARM1 or CARM1 inhibitor treatment suppressed the migration ability of SMMC7721 cells while CARM1 overexpression promoted cell migration, which were similar to the phenotypes observed for TRIM47 (Fig. 4 C-H, Fig. S4B-D). Moreover, TRIM47 overexpression completely rescued the migration inhibition induced by CARM1 knockdown (Fig. 4 I-K). Thus, we concluded that CARM1 promotes HCC metastasis in a TRIM47-dependent manner. 3.5 TRIM47 methylation inhibits the ubiquitylation of TRIM47 by CUL4 CRBN Previous results suggested that CARM1 stabilized TRIM47 protein levels by inhibiting its ubiquitylation and degradation (Fig. 2 F-H). We intended to identify the E3 ubiquitin ligase that mediated the degradation of TRIM47. The above immunoprecipitation-coupled mass spectrometry screen conducted by us found that CRBN, a substrate adaptor for the CRL4 E3 ubiquitin ligase, was a potential TRIM47 interacting protein (Fig. 5 A). Co-IP experiments showed that TRIM47 indeed interacted with CRBN (Fig. 5 B). TRIM47 protein level was decreased by CRBN overexpression, which could be blocked by MG132 (Fig. 5 C-D). Consistently, CRBN overexpression promoted the ubiquitination of TRIM47 (Fig. 5 E). Since CRL4 E3 ubiquitin ligases employed CRBN as an adaptor to recognize substrate protein 18 , we detected one of the CUL4 paralogues, CUL4A for the ubiquitination and protein stability of TRIM47. CUL4A overexpression also significantly enhanced the ubiquitination and degradation of TRIM47 (Fig. S5A-B). Using the ubiquitin mutants with the same lysine at position 48 or 63 and the remaining lysine replaced by arginine (K48 or K63), we found that CRBN enhanced K48-linked ubiquitination of TRIM47 (Fig. 5 F, Fig. S5C). Moreover, HY15729 strengthened the interaction between CRBN and TRIM47 in SMMC7721 cells, leading the enhancement of TRIM47 ubiquitination. We further investigated whether CARM1-mediated methylation affected TRIM47 ubiquitylation regulated by CUL4 CRBN . CRBN overexpression only decreased the protein level of WT TRIM47, whereas TRIM47 R210/582A remained unchanged (Fig. 5 H). This novel observation reveals a crosstalk between methylation and ubiquitylation in the orchestration of TRIM47 protein stability. 3.6. TRIM47 promotes HCC metastasis by protecting SNAI1 from proteasome-mediated degradation We next explored the potential mechanisms by which CARM1-TRIM47 axis suppressed HCC metastasis. TRIM47 knockdown significantly decreased the number of F-actin stress fibers, which is the hallmark of the cell migration (Fig. 6 A-B). We examined several pathways and molecular that influence cell metastasis, including canonical WNT/β-catenin signaling 19 , RhoA/ROCK1 signaling 20 , RAC1 pathway 21 and EMT inducing transcription factors 22 . Interestingly, only SNAI1 protein levels were significantly decreased in TRIM47 silenced cells (Fig. 6 C, Fig. S6A-C). Knockdown of CARM1 also inhibited SNAI1 protein expression (Fig. 6 D). Interestingly, Co-IP experiments showed that SNAI1 is a binding partner of TRIM47 (Fig. 6 E). Of note, Treatment with MG132, not with CQ, could rescue the reduced SNAI1 protein levels by TRIM47 knockdown (Fig. 6 G, Fig. S6D), suggesting that TRIM47 knockdown promotes SNAI1 degradation via the ubiquitin-proteasome pathway. To validate this, we performed protein ubiquitination assays. The ubiquitination of SNAI1 was enhanced by TRIM47 knockdown (Fig. 6 F). SNAI1 overexpression extensively reversed the suppressive effect of TRIM47 depletion on migration of SMMC7721 cells (Fig. 6 H-G). Collectively, these results indicate that TRIM47 promotes HCC cells migration by protecting SNAI1 from proteasome-mediated degradation. 4. Discussion and Conclusion TRIM47 is over-expressed and acts as an oncogene in multiple types of tumors. TRIM47 facilitates breast cancer proliferation and endocrine therapy resistance by activating NF-κB signaling 23 . Up-regulated TRIM47 is associated with poor outcomes and promotes the proliferation and metastasis of CRC 4 . However, the biological functions of TRIM47 and the possible underlying mechanisms in HCC have not been elucidated. In this study, we showed that the TRIM47 was up-regulated in advanced HCC and associated with poor survival of HCC patients. TRIM47 was able to promote HCC metastasis in vitro and in vivo . Therefore, TRIM47 acted as a pro-metastatic factor in HCC, which was consistent with previous study demonstrated that a TRIM family gene-based signature including TRIM47 and another 5 TRIM genes could be used to predict poor prognosis of HCC with high accuracy 7 . Although TRIM47's role in a variety of tumors has been extensively reported, little is known about its regulatory mechanisms in cancer. A study reported that LMO7 promoted the lysine 48 (K48)-linked ubiquitination and degradation of TRIM47 to alleviate hepatic steatosis, inflammation, and fibrosis 24 . Our study identified TRIM47 as a previously uncharacterised substrate of CARM1 and CARM1 methylated TRIM47 at its arginine 210 and arginine 582. The canonical CARM1 methylation motif is PMG rich motifs 15 . The TRIM47 R582 methylation residue fit the consensus motif while TRIM47 R210 do not. The crosstalk between methylation and other modifications regulates protein stability, activity, and intracellular location. PRMT5 methylated Mxi1 and facilitated its ubiquitin-mediated degradation in lung cancer 25 . The methylation of FOXO1 at its residues Arg 248 and Arg 250 by PRMT1 blocks AKT dependent phosphorylation of FOXO1, thereby increasing the stability and transcriptional activity of FOXO1 26 . Our study found that the methylation of TRIM47 R210/582 could be used to mark it for degradation by the CUL4 CRBN E3 ubiquitin ligase complex. Interestingly, besides stabilizing the protein levels of TRIM47, TRIM47 R210/582 methylation directly affect its ability to promote HCC cells migration. CARM1 methylates histone and non-histone proteins to function as an oncogene or tumor suppressor in different types of malignant tumors, suggesting that the function of CARM1 in cancer is context-dependent 11 , 27 , 28 . Compared with defined roles in other cancers, the function of CARM1 in HCC is controversial, as both pro-tumorigenic and onco-suppressor functions were reported 12 , 13 , 29 . Here, we found that CARM1 had a similar pro-metastasis phenotype with TRIM47 in HCC. High expression of CARM1 was correlated with poor prognosis in HCC patients and CARM1 was able to promote HCC metastasis in vitro and in vivo . Moreover, the promotion of HCC metastasis mediated by CARM1 was largely dependent on TRIM47. Our data strongly supports CARM1 as an oncogene in HCC. However, large-scale cohort studies and detailed immunohistochemical analysis of TRIM47 and CARM1 in HCC clinical samples are still needed for future investigation, which may facilitate understanding their roles and clinical correlation in HCC progression. As an E3 ubiquitin ligase, TRIM47 usually promoted the ubiquitination and degradation of its binding proteins, such as SMAD4 and FOXO1 4, 30 . However, we observed that TRIM47 interacted with SNAI1 and protected it from ubiquitin-mediated degradation. SNAI1 is an important transcription factor of EMT, which suppresses the transcription of E-cadherin by combining with the E-box sequence of E-cadherin’s promoter 31 . EMT is characterized by reduced cell-cell adhesion, lossing of cell polarity and the acquisition of mesenchymal features, which is considered to be a prerequisite for tumor metastasis 32 , 33 . A study reported that TRIM47 knockdown inhibited EMT through the inactivation of Wnt/β-catenin pathway in glioma 34 . To our surprise, TRIM47 knockdown did not affect Wnt/β-catenin pathway in HCC cells. The ubiquitination of SNAI1 was regulated by several E3 ubiquitin ligase, including FBXO11 35 , GSK3β 36 , and FBXL14 37 . The interaction between TRIM47 and SNAI1 may prevent the ubiquitylation and degradation of SNAI1 by other E3 ubiquitin ligases. In conclusion, we display for the first time that CARM1-CUL4 CRBN -TRIM47-SNAI1 cascade is involved in HCC metastasis. Our data are consistent with a model in which TRIM47 methylation by CARM1 inhibits CUL4 CRBN -mediated ubiquitylation of TRIM47. Accumulated TRIM47 thus interacts with SANI1 and protecting it from proteasomal degradation (Fig. 7 ). Our findings underscore the roles of TRIM47 and CARM1 in HCC metastasis, unveils a novel mechanism that crosstalk between arginine methylation and ubiquitylation orchestrates TRIM47-mediated HCC metastasis. The newly defined CARM1-CUL4 CRBN -TRIM47-SNAI1 regulatory axis may open new avenues for metastatic HCC therapy. Declarations Acknowledgements We thank Yuan Cao and Piao Zou at Analytical & Testing Center, Wuhan University of Science and Technology for their assistance in the experiments. Conflict of interest The authors declare that they have no competing interests. Author contributions Hu Jia, Zhu Haichuan and Xiao Juan designed research; Tang Yuzhe, Meng Xiang, Luo Xia, Yao Wentao, Tian Li, Zhang Zijian, Zhao Yuan performed research; Tang Yuzhe, Meng Xiang and Luo Xia analyzed data; Hu Jia and Zhu Haichuan wrote the paper; Hu Jia revised the manuscript. Ethics A pproval All animal interventions were approved by approved by the Animal Ethics Committee of Wuhan University of Science and Technology\ Funding This work was supported by the Natural Science Foundation Program of China Programs (82373038) and Innovation and Development Joint Funds of the National Natural Science Foundation of Hubei (No. 2022CFD109). References Di Rienzo M, Romagnoli A, Antonioli M, Piacentini M, Fimia GM. TRIM proteins in autophagy: selective sensors in cell damage and innate immune responses. Cell Death Differ 2020, 27 (3) : 887-902. Huang N, Sun X, Li P, Liu X, Zhang X, Chen Q , et al. TRIM family contribute to tumorigenesis, cancer development, and drug resistance. Exp Hematol Oncol 2022, 11 (1) : 75. Han Y, Tian H, Chen P, Lin Q. TRIM47 overexpression is a poor prognostic factor and contributes to carcinogenesis in non-small cell lung carcinoma. Oncotarget 2017, 8 (14) : 22730-22740. Liang Q, Tang C, Tang M, Zhang Q, Gao Y, Ge Z. 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WNT/beta-catenin signaling in hepatocellular carcinoma: The aberrant activation, pathogenic roles, and therapeutic opportunities. Genes Dis 2024, 11 (2) : 727-746. Zeng Y, Cao Y, Liu L, Zhao J, Zhang T, Xiao L , et al. SEPT9_i1 regulates human breast cancer cell motility through cytoskeletal and RhoA/FAK signaling pathway regulation. Cell Death Dis 2019, 10 (10) : 720. Liu S, Yu M, He Y, Xiao L, Wang F, Song C , et al. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology 2008, 47 (6) : 1964-1973. Giannelli G, Koudelkova P, Dituri F, Mikulits W. Role of epithelial to mesenchymal transition in hepatocellular carcinoma. J Hepatol 2016, 65 (4) : 798-808. Azuma K, Ikeda K, Suzuki T, Aogi K, Horie-Inoue K, Inoue S. TRIM47 activates NF-kappaB signaling via PKC-epsilon/PKD3 stabilization and contributes to endocrine therapy resistance in breast cancer. Proc Natl Acad Sci U S A 2021, 118 (35). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4220751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":296813654,"identity":"440f5fb5-b87f-44a0-b8b4-7db2584ec8c4","order_by":0,"name":"Jia Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3OsWoCQRCA4TkWzmaJ7Yr6DgMLK4Lcs0w4sNoilVgG0krqhLzEgi8wsoWND3CQIqa5KsUFmxSCbiCF1bplwP1gmmX+ZQCy7B9CAGLAWdX/eyge05KHeT34XeXEJOh84Tg1mSj65B8UQjdWHzqYjR2Ldh9Lpi9EmxWWd6axRjHMteNygtHDGiKWKIV5tyYc5u8dy1JdSzZHVMX6zeqO4ZSWeIlYuKHFcBgnJLs9+RFSrT7ahdphrV99aeLJ1tbfX8dT1V/5dbdcVuPn7VMbTQAkXf4QRsT3gx5fXcmyLLtxZ5EpULqXqdunAAAAAElFTkSuQmCC","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jia","middleName":"","lastName":"Hu","suffix":""},{"id":296813655,"identity":"b59dc876-2186-4081-ab3a-bebb079418a5","order_by":1,"name":"Yuzhe Tang","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuzhe","middleName":"","lastName":"Tang","suffix":""},{"id":296813656,"identity":"09261364-af42-4e3d-8c92-17e6597d4b73","order_by":2,"name":"Xiang Meng","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Meng","suffix":""},{"id":296813657,"identity":"3b9e5511-7484-4242-8f74-c53f72a942f9","order_by":3,"name":"Xia Luo","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Luo","suffix":""},{"id":296813658,"identity":"6cd8ca14-86d1-4a97-bc0c-277a424ea785","order_by":4,"name":"Wen Tao Yao","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"Tao","lastName":"Yao","suffix":""},{"id":296813659,"identity":"32f764ce-81a1-445f-bf87-980411fef9e0","order_by":5,"name":"Li Tian","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Tian","suffix":""},{"id":296813660,"identity":"3e591c5f-1b47-4069-8850-fd86da8639f3","order_by":6,"name":"Zijian Zhang","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zijian","middleName":"","lastName":"Zhang","suffix":""},{"id":296813661,"identity":"b2ae2934-c929-4a8c-8b84-32fa78ddf763","order_by":7,"name":"Yuan Zhao","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhao","suffix":""},{"id":296813662,"identity":"85ab6bab-2b56-4669-86bd-fea2834a51ea","order_by":8,"name":"Haichuan Zhu","email":"","orcid":"https://orcid.org/0000-0001-7545-7919","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Haichuan","middleName":"","lastName":"Zhu","suffix":""},{"id":296813663,"identity":"319912c5-98a2-43e4-a537-05aff40a7015","order_by":9,"name":"Juan Xiao","email":"","orcid":"","institution":"Affiliated Hospital of Hubei University of Arts and Science","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-04-05 05:05:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4220751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4220751/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-024-02244-4","type":"published","date":"2024-11-20T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57722735,"identity":"08b13da4-91ee-43e3-8634-54b8ea8da9ce","added_by":"auto","created_at":"2024-06-04 19:10:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9588088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM47 promotes HCC cells migration and invasion\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e \u003cstrong\u003eA\u003c/strong\u003e Analysis of TRIM47 mRNA expression in HCC primary tumor tissues and normal liver tissues from TCGA database. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eB\u003c/strong\u003e Analysis of TRIM47 mRNA expression in different tumor stages of HCC patients from TCGA database. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eC\u003c/strong\u003e Kaplan-Meier survival analysis of overall survival (OS) stratified by TRIM47 expression in HCC tumor tissues from TCGA database. \u003cem\u003eP \u003c/em\u003e= 0.0375. \u003cstrong\u003eD\u003c/strong\u003e Western blot analysis of TRIM47 protein levels in TRIM47 stably knockdown (shTRIM47#1 and shTRIM47#2) or control (shNC) SMMC7721 cells. \u003cstrong\u003eE-F\u003c/strong\u003e The migration assay (upper panel) and invasion assay (bottom panel) of SMMC7721 cells with TRIM47 stably knockdown (\u003cstrong\u003eE\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eF\u003c/strong\u003e). Scale bars, 50μm. Data are shown as mean ± SD. n = 3 samples per group, four fields per sample. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eG\u003c/strong\u003e Western blot analysis of TRIM47 protein levels in SMMC7721 cells transiently transfected with Vector or FLAG-TRIM47. \u003cstrong\u003eH-I \u003c/strong\u003eThe migration assay of SMMC7721 cells transiently transfected with Vector or FLAG-TRIM47 (\u003cstrong\u003eH\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eI\u003c/strong\u003e). Scale bars, 50μm. Data are shown as mean ± SD. n = 3 samples per group, four fields per sample. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eJ-K\u003c/strong\u003e 2×10\u003csup\u003e6\u003c/sup\u003e TRIM47 stably knockdown (shTRIM47#1 and shTRIM47#2) or control (shNC) SMMC7721 cells were injected into nude mice (n=5 per group) via tail vain. The representative haematoxylin and eosin (H\u0026amp;E) images (\u003cstrong\u003eJ\u003c/strong\u003e) and quantification of metastatic foci in lungs (\u003cstrong\u003eK\u003c/strong\u003e) were shown. Black arrows indicated the metastatic foci. Scale bars, 100μm. Data are shown as mean ± SD. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/fc4963a6b2b25e9bd47093f5.png"},{"id":57722327,"identity":"e7560e30-196d-495a-8f6a-22e7d4c11944","added_by":"auto","created_at":"2024-06-04 19:02:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2085656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCARM1 is a binding partner of TRIM47.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eHEK293T cells were transfected with FLAG-TRIM47 for 48 h. Cell extracts were subjected to immunoprecipitated with anti-FLAG antibody or a control IgG. CARM1 was identified via mass spectrometry. \u003cstrong\u003eB\u003c/strong\u003e HEK293T cells were co-transfected with FLAG-TRIM47 and MYC-CARM1 for 48 h. Total cell extracts were immunoprecipitated with anti-FLAG or anti-MYC antibodies. FLAG-TRIM47 and MYC-CARM1 were detected by western blot. \u003cstrong\u003eC \u003c/strong\u003eWestern blot analysis of TRIM47 protein levels in CARM1 stably knockdown SMMC7721 cells.\u003cstrong\u003eD \u003c/strong\u003eWestern blot analysis of TRIM47 expression in SMMC7721 cells treated with CARM1 inhibitor HY12759 for the indicated times. \u003cstrong\u003eE \u003c/strong\u003eCycloheximide chase analysis of TRIM47 degradation in CARM1 stably knockdown SMMC7721 cells. Therelative bandintensity of TRIM47 was quantified and plotted. \u003cstrong\u003eF \u003c/strong\u003eWestern blot analysis of TRIM47 protein levels in CARM1 stably knockdown (shCARM1#1 and shCARM1#2) or control (shNC) SMMC7721 cells treated with DMSO or 10 μM MG132 for 4 h. \u003cstrong\u003eG \u003c/strong\u003eCARM1 stably knockdown (shCARM1#1) or control (shNC) SMMC7721 cells were co-transfected with FLAG-TRIM47 and HA-ubiquitin plasmids for 48h. Total cell extracts were immunoprecipitated with anti-FLAG antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e were detected with anti-ubiquitin and anti-FLAG antibodies. \u003cstrong\u003eH \u003c/strong\u003eSMMC7721 cells were transfected with FLAG-TRIM47 plasmid for 48 h and treated with HY12759 for another 8 h. Total cell extracts were immunoprecipitated with anti-FLAG antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e were detected with anti-ubiquitin and anti-FLAG antibodies.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/f613175dbd822f886c90a5a7.png"},{"id":57722329,"identity":"1b34d4cd-6157-4d60-a59e-68c0b4a9ffe3","added_by":"auto","created_at":"2024-06-04 19:02:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4402456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCARM1 di-methylates TRIM47 at its arginine 210 and arginine 582. A\u003c/strong\u003e SMMC7721 cells were transfected with FLAG-TRIM47 for 48 h. Cell extracts were subjected to immunoprecipitated with anti-FLAG antibody or control IgG. Arginine methylation of immunopurified TRIM47 was detected by western blot. \u003cstrong\u003eB\u003c/strong\u003e CARM1 stably knockdown (shCARM1#1) or control (shNC) SMMC7721 cells were transfected with Vector or FLAG-TRIM47 for 48 h. Cell extracts were subjected to immunoprecipitated with anti-FLAG antibody. Arginine methylation of immunopurified TRIM47 was detected by western blot. \u0026nbsp;\u003cstrong\u003eC \u003c/strong\u003eSMMC7721 cells were transiently transfected with Vector or FLAG-TRIM47 for 48 h, then treated with 10μM HY12759 for another 8h. Arginine methylation of immunopurified TRIM47 was detected by western blot. \u003cstrong\u003eD\u003c/strong\u003e SMMC7721 cells were transfected with FLAG-TRIM47 or FLAG-TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e for 48 h. Cell extracts were subjected to immunoprecipitated with anti-FLAG antibody. Arginine methylation of immunopurified TRIM47 was detected by western blot. \u003cstrong\u003eE \u003c/strong\u003eThe schematic representation of TRIM47 constructs (Upper panel). The conserved glycine-arginine-methionine (PGM) rich substrate motif reported for the recognition of CARM1 (bottom panel). \u003cstrong\u003eF \u003c/strong\u003eCARM1 stably knockdown (shCARM1#1) or control (shNC) SMMC7721 cells were co-transfected with FLAG-TRIM47 or FLAG-TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e and HA-ubiquitin plasmids for 48h. Cell extracts were immunoprecipitated with anti-FLAG antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e\u0026nbsp;were detected with anti-ubiquitin and anti-FLAG antibodies.\u0026nbsp; \u003cstrong\u003eG \u003c/strong\u003eSMMC7721 cells were transfected with FLAG-TRIM47 or TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e for 48h and treated with HY12759 for another 8 h. Western blot analysis of TRIM47 protein levels.\u003cstrong\u003e H \u003c/strong\u003eSMMC7721 cells were transfected with FLAG-TRIM47 plasmid for 48h and treated with HY12759 for another 8h. Cell extracts were immunoprecipitated with anti-FLAG antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e\u0026nbsp;were detected with anti-ubiquitin and anti-FLAG antibodies.\u003cstrong\u003e H\u003c/strong\u003e Western blot analysis of TRIM47 protein levels in SMMC7721 and Huh7 cells transiently transfected with Vector, FLAG-TRIM47 or FLAG-TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e. \u003cstrong\u003eI-J \u003c/strong\u003eThe migration assay of SMMC7721 and Huh7 cells transiently transfected with Vector, FLAG-TRIM47 or FLAG-TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e. (\u003cstrong\u003eI\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eJ\u003c/strong\u003e). Scale bars, 50μm. Data shown as mean ± SD. n = 3 samples per group, four fields per sample. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ns, \u003cem\u003eno significance\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/3f9f60f663be4c074985878d.png"},{"id":57722324,"identity":"1d28cfbb-f609-44ad-9b11-5ce5f6c8ad8f","added_by":"auto","created_at":"2024-06-04 19:02:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6618423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCARM1 promotes HCC cells migration depend on TRIM47\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e \u003cstrong\u003eA\u003c/strong\u003e Analysis of CARM1 mRNA levels in different tumor stages (stag III+IV vs stag I+II) of HCC patients from TCGA database. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eB\u003c/strong\u003e Kaplan-Meier survival analysis of overall survival (OS) stratified by CARM1expression in the HCC tissues from TCGA database. \u003cem\u003eP \u003c/em\u003e= 0.046.\u003cstrong\u003e C\u003c/strong\u003eWestern blot analysis of CARM1 protein levels in CARM1 stably knockdown (shCARM1#1 and shCARM1#2) or control (shNC) SMMC7721 cells. \u003cstrong\u003eD-E \u003c/strong\u003eThe migration assay of SMMC7721 cells with CARM1 stably knockdown (\u003cstrong\u003eD\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eE\u003c/strong\u003e). Scale bars, 50μm. Data shown as mean ± SD. n = 3 samples per group, four fields per sample. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eF \u003c/strong\u003eWestern blot analysis of TRIM47 protein levels in SMMC7721 cells treated with HY12795 (10μM) for 8 h.\u003cstrong\u003e G-H \u003c/strong\u003eThe migration assay of SMMC7721 cells treated with HY12795 (10μM) for 8 h (\u003cstrong\u003eG\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eH\u003c/strong\u003e). Scale bars, 50μm. Data shown as mean ± SD. n = 3 samples per group, four fields per sample. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003eI-K \u003c/strong\u003eCARM1 stable knockdown SMMC7721 cells were transfected with Vector or FLAG-TRIM47. Cells were harvested and subjected to western blot (\u003cstrong\u003eI\u003c/strong\u003e) and migration assay (\u003cstrong\u003eJ\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eK\u003c/strong\u003e). Scale bars, 50μm. Data shown as mean ± SD. n = 3 samples per group, four fields per sample. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001;ns, \u003cem\u003eno significance\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/fa20c79373f35eb3b29ceb46.png"},{"id":57722326,"identity":"206561a5-081f-41e5-ae5b-4ee225923ab8","added_by":"auto","created_at":"2024-06-04 19:02:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1883074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRBN promotes the ubiquitination of TRIM47.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e HEK293T cells were transfected with FLAG-TRIM47 for 48 h. Cell extracts were subjected to immunoprecipitated with anti-FLAG antibody or a control IgG. CRBN was identified via mass spectrometry. \u003cstrong\u003eB\u003c/strong\u003e HEK293T cells were co-transfected with FLAG-TRIM47 and MYC-CRBN for 48 h. Cell extracts were immunoprecipitated with anti-FLAG or anti-MYC antibodies. FLAG-TRIM47 and MYC-CRBN were detected by western blot. \u003cstrong\u003eC \u003c/strong\u003eWestern blot analysis of TRIM47 protein levels in SMMC7721 cells transfected with MYC-CRBN for 48 h.\u003cstrong\u003e D \u003c/strong\u003eSMMC7721 cells were transfected with MYC-CRBN plasmid for 48h and treated with MG132 (10 μM) for another 4 h. Western blot analysis of TRIM47 protein levels. \u003cstrong\u003eE \u003c/strong\u003eSMMC7721 cells were co-transfected with MYC-CRBN and HA-ubiquitin plasmids for 48h. Cell extracts were immunoprecipitated with anti-TRIM47 antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e\u0026nbsp;were detected with anti-ubiquitin and anti-TRIM47 antibodies. \u003cstrong\u003eF\u003c/strong\u003e Immunoprecipitation analysis of TRIM47 ubiquitination in SMMC7721 cells co-transfected with MYC-CRBN and HA-ubiquitin (K48) or HA-ubiquitin (K63).\u003cstrong\u003e \u003c/strong\u003eCell extracts were immunoprecipitated with anti-TRIM47 antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e\u0026nbsp;were detected with anti-HA and anti-TRIM47 antibodies. \u003cstrong\u003eG \u003c/strong\u003eSMMC7721 cells were treated with HY12759 for 8 h. Total cell extracts were immunoprecipitated with anti- TRIM47 antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e\u0026nbsp;were detected with anti-ubiquitin, anti-TRIM47 and anti-CRBN antibodies. \u003cstrong\u003eH \u003c/strong\u003eWestern blot analysis of TRIM47 protein levels in TRIM47 stably knockdown SMMC7721 cells co-transfected with FLAG-TRIM47 or FLAG-TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e and MYC-CRBN plasmids for 48h.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/6397cad3cab8b9611f9fd8e6.png"},{"id":57722736,"identity":"1df11758-e299-436b-8bcf-36b621548589","added_by":"auto","created_at":"2024-06-04 19:10:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5875276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM47 promotes HCC metastasis via SNAI1.\u003c/strong\u003e \u003cstrong\u003eA-B\u003c/strong\u003e Representative images of F-actin (red) staining in control (shNC) and TRIM47 stably knockdown (shTRIM47#1 and shTRIM47#2) SMMC7721 cells (\u003cstrong\u003eA\u003c/strong\u003e). Quantification of stress fibers per cell (\u003cstrong\u003eB\u003c/strong\u003e). Scale bars, 200 μm. Data are shown as mean ± SD. n = 10 cells per condition, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eC\u003c/strong\u003e Western blot analysis of EMT related proteins, including CDH1, CDH2, SNAI1, SNAI2, VIMENTIN, ZEB-1 and ZO-1 in TRIM47 stably knockdown SMMC7721 cells. \u003cstrong\u003eD\u003c/strong\u003e Western blot analysis of SNAI1 protein levels in CARM1 stably knockdown SMMC7721 cells.\u003cstrong\u003e E \u003c/strong\u003eHEK293T cells were co-transfected with FLAG-TRIM47 and MYC-SNAI1 for 48 h. Cell extracts were immunoprecipitated with anti-FLAG or anti-MYC antibodies. FLAG-TRIM47 and MYC-SNAI1 were detected by western blot. \u003cstrong\u003eF \u003c/strong\u003eCell extracts from TRIM47 stably knockdown (shTRIM47#1) or control (shNC) SMMC7721 cells were immunoprecipitated with anti-SNAI1 antibody. The immuno\u003cem\u003eprecipitates\u003c/em\u003e were detected by with anti-ubiquitin and anti-SNAI1antibodies. \u003cstrong\u003eG \u003c/strong\u003eTRIM47 stably knockdown (shTRIM47#1 and shTRIM47#2) or control (shNC) SMMC7721 cells were treated with DMSO or 10 μM MG132 for 6 h. Western blot analysis of SNAI1 protein levels. \u003cstrong\u003eH-J \u003c/strong\u003eTRIM47 stably knockdown SMMC7721 cells were transfected with Vector or MYC-SNAI1. Cells were harvested and subjected to Western blot (\u003cstrong\u003eH\u003c/strong\u003e) and migration assay (\u003cstrong\u003eI\u003c/strong\u003e). The average number of cells per field were calculated (\u003cstrong\u003eJ\u003c/strong\u003e). Scale bars, 50μm. Data shown as mean ± SD. n = 3 samples per group, four fields per sample. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/011c838c49cd0e71a4ba1e14.png"},{"id":57722330,"identity":"9f661bda-cc9d-44bf-af55-4dab67fb6d91","added_by":"auto","created_at":"2024-06-04 19:02:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":420459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model showing the regulation of TRIM47 by CARM1.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/95cfa50141076bdd3a2553c9.png"},{"id":69516389,"identity":"2a5e43fe-1e3d-4289-8e5d-484cce6da9ab","added_by":"auto","created_at":"2024-11-21 08:09:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":41854125,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/66e0b555-d799-4140-8230-4a2b35279b67.pdf"},{"id":57722331,"identity":"9b86927d-4e01-413d-8c91-67f2d7f26798","added_by":"auto","created_at":"2024-06-04 19:02:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18187157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryMaterialsandOriginalWBs.docx","url":"https://assets-eu.researchsquare.com/files/rs-4220751/v1/c0d0b6d06fc8c810cec7f15d.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Arginine Methylation-dependent TRIM47 Stability Mediated by CARM1 Promotes the Metastasis of Hepatocellular Carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTripartite motif-containing (TRIM) family proteins have a conserved domain architecture characterized by a RING domain in N-terminal, a B-box domain, a coiled-coil domain, and a variable C-terminal domain\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Dysregulation of TRIM proteins is closely associated with tumorigenesis and tumor progression\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. TRIM47, also known as GOA (Gene overexpressed in astrocytoma protein), was upregulated and possessed oncogenic function in multiple types of tumors, such as non-small cell lung carcinoma (NSCLC)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, colorectal cancer (CRC)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and prostate cancer (PC)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of cancer-related mortality worldwide\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. A bioinformatics study reported that TRIM47 expression was associated with poor prognosis of HCC. A TRIM family gene-based signature including TRIM47 and another 5 TRIM genes performed well in Overall survival (OS) prediction for HCC\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These studies indicated that TRIM47 might play an important role in HCC progression. However, the detailed roles of TRIM47 and its regulatory mechanism in HCC remain elusive.\u003c/p\u003e \u003cp\u003eProtein arginine methylation is a prevalent post-translational modification, which is catalyzed by a group of enzymes termed protein arginine methyltransferases (PRMTs)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Coactivator-associated arginine methyltransferase 1 (CARM1), a type I PRMTs, is deregulated in numerous cancers and plays critical roles in cancer progression by catalyzing asymmetric di-methylation of histone or nonhistone substrate proteins\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. For example, CARM1 promoted breast cancer metastasis by methylating chromatin remodeling factor BAF155\u003csup\u003e10\u003c/sup\u003e. Arginine methylation of MDH1 by CARM1 inhibits glutamine metabolism and suppresses pancreatic ductal adenocarcinoma (PDAC) progression\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, independent studies reported opposite functional roles of CARM1 in HCC. CARM1 was previously reported to suppress the glycolysis in liver cancer cells by mediating arginine 234 (R234) methylation of GAPDH, thus inhibiting the proliferation of liver cancer cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This conflicts with another study demonstrating that CARM1 indicates poor prognosis and promotes HCC progression by activating AKT/mTOR signaling\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These conflicting results lead to a confused understanding of CARM1\u0026rsquo;s functions in HCC, thus needing to explore its underlying molecular mechanism.\u003c/p\u003e \u003cp\u003eHere, we found that TRIM47 was upregulated in tumor tissues and associated with poor clinical outcomes of HCC patients. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies revealed that TRIM47 facilitated the migration and invasion of HCC cells. Mechanistically, TRIM47 promoted epithelial-mesenchymal transition (EMT) by interacting with SNAI1 and maintained its stability. Moreover, we identified that TRIM47 was a novel substrate of CARM1, while the methylation by CARM1 protected TRIM47 from proteasomal degradation mediated by E3 ubiquitin ligase complex CRL4\u003csup\u003eCRBN\u003c/sup\u003e. This work may provide a basis for finding novel targets and designing therapeutic strategies against metastatic HCC.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Antibodies and Reagents\u003c/h2\u003e \u003cp\u003eAntibodies used in this study were as follows: anti-TRIM47 (Proteintech, 26885-1-AP), CARM1 (Proteintech, 55246-1-AP), anti-GAPDH (Proteintech, 10494-1-AP), anti-Ubiquitin (Proteintech, 10201-2-AP), anti-Myc (Abclonal, AE010), anti-FLAG (Abclonal, AE005), Asymmetric di-methyl arginine antibody (Cell Signaling Technology, 13522), anti-E-cadherin (Proteintech,20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-RHOA (Proteintech, 10749-1-AP), anti-Rac1 (Proteintech, 24072-1-AP), anti-AXIN2 (Proteintech, 20540-1-AP), anti-β-Catenin (Proteintech, 51067-2-AP), anti-GST (Proteintech,10000-0-AP), anti-CRBN (Proteintech, 28494-1-AP), anti-SNAI1 (Proteintech, 13099-1-AP), anti-SNAI2 (Cell Signaling Technology, 9585), anti-Vimentin (Cell Signaling Technology, 5741), anti-ZEB1 (Cell Signaling Technology, 3396), anti-ZO-1(Cell Signaling Technology, 5406). Other reagents used in this study were: Cycloheximide (MedchemExpress, HY-12320), CARM1-IN-1 (MedchemExpress, HY-12759), MG132 (MedchemExpress, HY-13259), Chloroquine (Sigma-Aldrich, C6628-25G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell Culture and Transfection\u003c/h2\u003e \u003cp\u003eHEK293T and human HCC cell lines HepG2, Huh7 were obtained from American Type Culture Collection (ATCC). SMMC7721 cell line was purchased from Warner Bio with STR. All cells used in this study were cultured in RPMI 1640 medium (Gibco, 31800022) and DMEM medium (Gibco, 12800017) containing 10% fetal bovine serum (VISTECH, SE100-011). Plasmids were transiently transfected into cells by using Neofect\u003csup\u003eTM\u003c/sup\u003eDNA reagent (Neofect, TF20121201) according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Plasmids Construction\u003c/h2\u003e \u003cp\u003eHuman TRIM47 cDNA was cloned into the p3\u0026times;FLAG-CMV-10 Vector (sigma aldrich) to generate p3\u0026times;FLAG-CMV-TRIM47. Human CARM1, CRBN and SNAI1 cDNA was cloned into pcDNA.3.1 Vector (Invitrogen). The following mutational TRIM47 plasmids were constructed based on p3\u0026times;FLAG-CMV-TRIM47 plasmid: p3\u0026times;FLAG-CMV-TRIM47\u003csup\u003eR210K\u003c/sup\u003e, p3\u0026times;FLAG-CMV-TRIM47\u003csup\u003eR582K\u003c/sup\u003e, p3\u0026times;FLAG-CMV-TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e. TRIM47 and TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e stably overexpression cell lines were constructed using the pLenti-3\u0026times;Flag-CMV plasmid. Oligonucleotides specific shRNA against TRIM47 or CARM1 were synthesized and cloned into pLKO.1 Vector (Addgene Plasmid, #10878). All plasmids used in our study were confirmed by DNA sequencing. The shRNA sequences were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Lentivirus Production and Transduction\u003c/h2\u003e \u003cp\u003eIndicated lentiviral plasmids were transfected into HEK293T cells. After 48h post transfection, the supernatant containing lentiviral particles was harvested. HCC cells were infected with the lentivirus in the presence of polybrene (8 \u0026micro;g/mL) for 24h and then selected by puromycin (0.5 \u0026micro;g/mL) for one week. The overexpression or knockdown efficiency was detected by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell migration, invasion and wound healing assay\u003c/h2\u003e \u003cp\u003eThe cell migration and invasion assay were performed by using 8-\u0026micro;m Boyden chambers (Corning Inc, 3422). For migration assay, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e SMMC7721 cells or Huh7 cells were seeded in the upper chamber with serum-free RPMI 1640 medium. The lower chamber was added with RPMI 1640 medium containing 10% FBS. After 12 h for SMMC7721 or 6 h for Huh7, the migrated cells were fixed with 4% paraformaldehyde, stained with crystal violet and counted.\u003c/p\u003e \u003cp\u003eFor invasion assay, the upper chamber was pre-coated with matrigel matrix (BD Science, 356234). 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e SMMC7721 cells were seeded in the upper chamber with serum-free RPMI 1640 medium. The lower chamber was added with RPMI 1640 medium containing 10% FBS. After 72 h, the invaded cells were fixed with 4% paraformaldehyde, stained with crystal violet and counted.\u003c/p\u003e \u003cp\u003eFor wound healing assay, indicated cells were seeded in 6-well plate and grown to 90% confluence. A linear scratch was made by a sterile 200 \u0026micro;l tip. Cells were cultured in RPMI 1640 medium containing 1% FBS to close the wound for 48 h. The scratch area was analyzed by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Animal experiments\u003c/h2\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e indicated SMMC7721 cells (shNC, shTRIM47#1 and shTRIM47#2) were injected into Balb/c nude mice (male, 6 weeks) through tail vein. After 8 weeks, the mice were sacrificed and pulmonary metastatic nodules were counted. The animal studies were approved by the Animal Ethics Committee of Wuhan University of Science and Technology. The mice used in our study were housed under specific pathogen-free (SPF) conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA Extraction and Semi-Quantitative RT-PCR\u003c/h2\u003e \u003cp\u003eRNA was extracted by RNA extraction kit (Abclonal, RK30120) and cDNA was synthesized using a Transcription Reagent Kit (Abclonal, RK20429) according to the manufacturer\u0026rsquo;s instruction. PCR analysis of mRNA expression was conducted using a 2 \u0026times; Taq Plus Master Mix (Vazyme, P211-01). The PCR products were separated by agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.8 Immunofluorescence and F-actin staining\u003c/h3\u003e\n\u003cp\u003eCells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). For immunofluorescence, cells were then blocked with 10% FBS, incubated with the indicated primary antibodies and secondary antibodies. For F-actin staining, cells were incubated with Rhodamine-conjugated Phalloidins for 1 h. nuclei was stain with DAPI.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Western Blot\u003c/h2\u003e \u003cp\u003eCells were harvested and lysed with RIPA buffer (Beyotime Biotechnology, P0013C) containing protease inhibitors. The protein concentration was detected with BCA kit (Thermo Scientific, A55864). The cell lysate was separated by SDS-PAGE and then transferred onto Immobilon-P (PVDF) membranes (Merck Millipore, ISEQ00010). After blocking with 5% skimmed milk, membranes were incubated with indicated antibodies. The protein bands were visualized by chemiluminescence system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Co-immunoprecipitation assay (Co-IP)\u003c/h2\u003e \u003cp\u003eCells were harvest and lysed with RIPA buffer (Beyotime Biotechnology, P0013D) containing protease inhibitors. The cell lysates were incubated with indicated antibodies overnight at 4\u0026deg;C. Then protein A/G magnetic beads (Biolinkedin) were added and incubated for another 2 hours. The protein-bound beads were washed with washing buffer for 3 times, boiled with protein loading buffer and analyzed by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 GST-PAK1\u003csup\u003ePBD\u003c/sup\u003e Pull Down Assay and GTP-RhoA activation assay\u003c/h2\u003e \u003cp\u003eGST-PAK1\u003csup\u003ePBD\u003c/sup\u003e protein was expressed in \u003cem\u003eE.coli\u003c/em\u003e strain BL21 and purified. Equal amounts of GST-PAK1\u003csup\u003ePBD\u003c/sup\u003e protein were incubated with glutathione Sepharose 4B beads (GE Healthcare) and indicated cell lysates. Then the beads were washed with washing buffer for 5 times, boiled with protein loading buffer and analyzed by western blot.\u003c/p\u003e \u003cp\u003eThe GTP-RhoA activation assay were performed with the Rho Activation Assay Biochem Kit (Cytoskeleton) according to the manufacture\u0026rsquo;s instruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical Analysis\u003c/h2\u003e \u003cp\u003eKaplan-Meier method was performed to analyze survival rate and the survival differences was calculated with log-rank test. Unpaired Student\u0026rsquo;s t-tests or one-way ANOVA were used to determined other comparisons according to the number of groups. Date was presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses were performed with GraphPad Prism software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 TRIM47 is a pro-metastatic factor in HCC\u003c/h2\u003e \u003cp\u003eTo evaluate the clinical significance of TRIM47 in human HCC, we analyzed the publicly available HCC expression profiles in The Cancer Genome Atlas (TCGA) and GSE76427 database. TRIM47 was upregulated in HCC tumor tissues compared with normal tissues, especially at advanced stages (stage IV vs stage I/II/III in TCGA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Kaplan-Meier survival analysis revealed that HCC patients with high TRIM47 levels usually had poorer overall survival (OS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). These data suggest that TRIM47 may possess oncogenic activity in HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we explored the function of TRIM47 in HCC progression. We detected TRIM47 expression in four HCC cell lines (HepG2, Huh7, SMMC7721 and Bel7402) and found that SMMC7721 cells expressed TRIM47 at a relatively high level while Huh7 and Bel7402 cells at a low level (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). SMMC7721 (high expression of TRIM47) and Huh7 (low expression of TRIM47) cell lines were thus chosen for further studies. Knockdown of TRIM47 in SMMC7721 cells significantly inhibited cell migration and invasion \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-E). Conversely, TRIM47 overexpression promoted cell migration ability in Huh7 and SMMC7721 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). Moreover, TRIM47 knockdown decreased the foci number of lung metastasis \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-K). These findings indicate that TRIM47 is a pro-metastatic factor in HCC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 CARM1 interacts with TRIM47 and maintains its stabilization\u003c/h2\u003e \u003cp\u003eTo dissect the upstream regulatory factors and pro-metastatic mechanism of TRIM47 in HCC, we performed an immunoprecipitation-coupled mass spectrometry screen. CARM1, a type I PRMTs playing critical roles in cancer progression by methylating histone or nonhistone substrates, was identified as a putative TRIM47-interacting protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Noticeably, TP53, another protein reported to interact with TRIM47\u003csup\u003e14\u003c/sup\u003e, was also hitted in our screening, indicating the reliability of our screening. The binding between TRIM47 and CARM1 were confirmed by reciprocal co-IP assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we determined whether the expression of CARM1 or TRIM47 was affected by their interactions. Knockdown of TRIM47 had no effect on CARM1 protein levels (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). However, TRIM47 protein levels was significantly decreased upon CARM1 depletion, while its mRNA expression was unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Treatment with specific CARM1 methyltransferase inhibitor HY-12759 also reduced TRIM47 protein levels in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The cycloheximide chase assay showed that the protein half-life of TRIM47 was remarkably shortened after CARM1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results suggest that CARM1 acts upstream of TRIM47 to control TRIM47’s protein stability through post-translational modifications. Of note, decreased TRIM47 protein levels by CARM1 knockdown were rescued when treated HCC cells with proteasome inhibitor MG132, but not with lysosome inhibitor CQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC), suggesting that CARM1 protects TRIM47 protein from proteasome-mediated degradation. Consistently, the ubiquitination of TRIM47 was enhanced by CARM1 knockdown or HY-12759 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). Taken together, these results indicate that CARM1 stabilizes TRIM47 protein by inhibiting its ubiquitination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 TRIM47 is an arginine methylation substrate of CARM1\u003c/h2\u003e \u003cp\u003eAs a type I PRMTs, CARM1 catalyzes the formation of asymmetric di-methylarginine in its substrate proteins\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We found that TRIM47 was indeed arginine-methylated by using an anti-asymmetric di-methylarginine (α-ADME) antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). CARM1 knockdown or HY-12759 treatment markedly decreased the methylation of TRIM47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). To define which arginine residues were methylated by CARM1, we performed mass spectrometric analysis of immunopurified TRIM47 protein from HCC cells and identified arginine 210 (R210) as a putative asymmetric di-methylation site (Fig. S3A). A previous proteomic study reported that arginine 582 (R582) might be another methylated arginine residue of TRIM47\u003csup\u003e15\u003c/sup\u003e. To determine whether R210 and R582 are the major methylated site in TRIM47, we mutated these two sites into lysine (TRIM47\u003csup\u003eR210K\u003c/sup\u003e or TRIM47\u003csup\u003eR582K\u003c/sup\u003e) that conferred resistance to arginine methylation. Compared with wild-type (WT) TRIM47, both TRIM47\u003csup\u003eR210K\u003c/sup\u003e and TRIM47\u003csup\u003eR582K\u003c/sup\u003e mutants showed a substantial, but incomplete reduction of asymmetric di-methylation levels (Fig. S3B). The methylation signal was also markedly inhibited for the R210 and R582 double mutant (TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results indicate that both R210 and R582 are the major, if not the sole, methylation site of TRIM47. Interestingly, R582 is located in a conserved glycine-arginine-methionine (PGM) rich substrate motif reported for the recognition of CARM1, while R210 is not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We further tested whether the methylation of R210 and R582 contributed to TRIM47 stability and ubiquitination status. TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e mutant exhibited higher ubiquitination levels than that of WT TRIM47 (Fig S3C). The ubiquitination level of WT TRIM47 was sharply enhanced by CARM1 knockdown, whereas TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e mutant showed little or no change (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Consistently, HY-12759 significantly decreased WT TRIM47 protein levels, but not TRIM47 mutants (TRIM47\u003csup\u003eR210K\u003c/sup\u003e, TRIM47\u003csup\u003eR582K\u003c/sup\u003e and TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, Fig. S3D). We also assessed the effect of TRIM47 arginine mutants on the metastasis of HCC. TRIM47\u003csup\u003eR210K\u003c/sup\u003e and TRIM47\u003csup\u003eR582K\u003c/sup\u003e mutant-expressing cells were less metastatic compared to the WT TRIM47-expressing cells (Fig. S3E-G). Noticeably, TRIM47\u003csup\u003eR210/582K\u003c/sup\u003e mutant absolutely abolished the pro-metastatic ability of TRIM47 in HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-J, Fig. S3E-G). Together, these data indicated that the methylated modification by CARM1 stabilizes TRIM47 protein levels and promotes the metastasis of HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. CARM1 suppresses HCC metastasis in a TRIM47-dependent manner\u003c/h2\u003e \u003cp\u003eCARM1 functions as an oncogene or a tumor suppressor depending on cancer types\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the roles of CARM1 in dynamic HCC progression are controversial\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We analyzed the HCC cohort in TCGA and found that HCC patients at the advanced stages expressed higher CARM1 than early staged patients (stage III + IV vs stage I + II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Kaplan-Meier survival analysis revealed that high levels of CARM1 was associated with poor survival in HCC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Fig. S4A). Silencing of CARM1 or CARM1 inhibitor treatment suppressed the migration ability of SMMC7721 cells while CARM1 overexpression promoted cell migration, which were similar to the phenotypes observed for TRIM47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-H, Fig. S4B-D). Moreover, TRIM47 overexpression completely rescued the migration inhibition induced by CARM1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-K). Thus, we concluded that CARM1 promotes HCC metastasis in a TRIM47-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 TRIM47 methylation inhibits the ubiquitylation of TRIM47 by CUL4\u003csup\u003eCRBN\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003ePrevious results suggested that CARM1 stabilized TRIM47 protein levels by inhibiting its ubiquitylation and degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). We intended to identify the E3 ubiquitin ligase that mediated the degradation of TRIM47. The above immunoprecipitation-coupled mass spectrometry screen conducted by us found that CRBN, a substrate adaptor for the CRL4 E3 ubiquitin ligase, was a potential TRIM47 interacting protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Co-IP experiments showed that TRIM47 indeed interacted with CRBN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). TRIM47 protein level was decreased by CRBN overexpression, which could be blocked by MG132 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Consistently, CRBN overexpression promoted the ubiquitination of TRIM47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Since CRL4 E3 ubiquitin ligases employed CRBN as an adaptor to recognize substrate protein\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, we detected one of the CUL4 paralogues, CUL4A for the ubiquitination and protein stability of TRIM47. CUL4A overexpression also significantly enhanced the ubiquitination and degradation of TRIM47 (Fig. S5A-B). Using the ubiquitin mutants with the same lysine at position 48 or 63 and the remaining lysine replaced by arginine (K48 or K63), we found that CRBN enhanced K48-linked ubiquitination of TRIM47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, Fig. S5C). Moreover, HY15729 strengthened the interaction between CRBN and TRIM47 in SMMC7721 cells, leading the enhancement of TRIM47 ubiquitination. We further investigated whether CARM1-mediated methylation affected TRIM47 ubiquitylation regulated by CUL4\u003csup\u003eCRBN\u003c/sup\u003e. CRBN overexpression only decreased the protein level of WT TRIM47, whereas TRIM47\u003csup\u003eR210/582A\u003c/sup\u003e remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). This novel observation reveals a crosstalk between methylation and ubiquitylation in the orchestration of TRIM47 protein stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6. TRIM47 promotes HCC metastasis by protecting SNAI1 from proteasome-mediated degradation\u003c/h2\u003e \u003cp\u003eWe next explored the potential mechanisms by which CARM1-TRIM47 axis suppressed HCC metastasis. TRIM47 knockdown significantly decreased the number of F-actin stress fibers, which is the hallmark of the cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). We examined several pathways and molecular that influence cell metastasis, including canonical WNT/β-catenin signaling\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, RhoA/ROCK1 signaling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, RAC1 pathway\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and EMT inducing transcription factors\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Interestingly, only SNAI1 protein levels were significantly decreased in TRIM47 silenced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Fig. S6A-C). Knockdown of CARM1 also inhibited SNAI1 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Interestingly, Co-IP experiments showed that SNAI1 is a binding partner of TRIM47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Of note, Treatment with MG132, not with CQ, could rescue the reduced SNAI1 protein levels by TRIM47 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, Fig. S6D), suggesting that TRIM47 knockdown promotes SNAI1 degradation via the ubiquitin-proteasome pathway. To validate this, we performed protein ubiquitination assays. The ubiquitination of SNAI1 was enhanced by TRIM47 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). SNAI1 overexpression extensively reversed the suppressive effect of TRIM47 depletion on migration of SMMC7721 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-G). Collectively, these results indicate that TRIM47 promotes HCC cells migration by protecting SNAI1 from proteasome-mediated degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion and Conclusion","content":"\u003cp\u003eTRIM47 is over-expressed and acts as an oncogene in multiple types of tumors. TRIM47 facilitates breast cancer proliferation and endocrine therapy resistance by activating NF-κB signaling\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Up-regulated TRIM47 is associated with poor outcomes and promotes the proliferation and metastasis of CRC\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the biological functions of TRIM47 and the possible underlying mechanisms in HCC have not been elucidated. In this study, we showed that the TRIM47 was up-regulated in advanced HCC and associated with poor survival of HCC patients. TRIM47 was able to promote HCC metastasis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Therefore, TRIM47 acted as a pro-metastatic factor in HCC, which was consistent with previous study demonstrated that a TRIM family gene-based signature including TRIM47 and another 5 TRIM genes could be used to predict poor prognosis of HCC with high accuracy\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough TRIM47's role in a variety of tumors has been extensively reported, little is known about its regulatory mechanisms in cancer. A study reported that LMO7 promoted the lysine 48 (K48)-linked ubiquitination and degradation of TRIM47 to alleviate hepatic steatosis, inflammation, and fibrosis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Our study identified TRIM47 as a previously uncharacterised substrate of CARM1 and CARM1 methylated TRIM47 at its arginine 210 and arginine 582. The canonical CARM1 methylation motif is PMG rich motifs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The TRIM47\u003csup\u003eR582\u003c/sup\u003e methylation residue fit the consensus motif while TRIM47\u003csup\u003eR210\u003c/sup\u003e do not. The crosstalk between methylation and other modifications regulates protein stability, activity, and intracellular location. PRMT5 methylated Mxi1 and facilitated its ubiquitin-mediated degradation in lung cancer\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The methylation of FOXO1 at its residues Arg 248 and Arg 250 by PRMT1 blocks AKT dependent phosphorylation of FOXO1, thereby increasing the stability and transcriptional activity of FOXO1\u003csup\u003e26\u003c/sup\u003e. Our study found that the methylation of TRIM47\u003csup\u003eR210/582\u003c/sup\u003e could be used to mark it for degradation by the CUL4\u003csup\u003eCRBN\u003c/sup\u003e E3 ubiquitin ligase complex. Interestingly, besides stabilizing the protein levels of TRIM47, TRIM47 \u003csup\u003eR210/582\u003c/sup\u003e methylation directly affect its ability to promote HCC cells migration.\u003c/p\u003e\u003cp\u003eCARM1 methylates histone and non-histone proteins to function as an oncogene or tumor suppressor in different types of malignant tumors, suggesting that the function of CARM1 in cancer is context-dependent\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Compared with defined roles in other cancers, the function of CARM1 in HCC is controversial, as both pro-tumorigenic and onco-suppressor functions were reported\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Here, we found that CARM1 had a similar pro-metastasis phenotype with TRIM47 in HCC. High expression of CARM1 was correlated with poor prognosis in HCC patients and CARM1 was able to promote HCC metastasis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Moreover, the promotion of HCC metastasis mediated by CARM1 was largely dependent on TRIM47. Our data strongly supports CARM1 as an oncogene in HCC. However, large-scale cohort studies and detailed immunohistochemical analysis of TRIM47 and CARM1 in HCC clinical samples are still needed for future investigation, which may facilitate understanding their roles and clinical correlation in HCC progression.\u003c/p\u003e\u003cp\u003eAs an E3 ubiquitin ligase, TRIM47 usually promoted the ubiquitination and degradation of its binding proteins, such as SMAD4 and FOXO1\u003csup\u003e4, 30\u003c/sup\u003e. However, we observed that TRIM47 interacted with SNAI1 and protected it from ubiquitin-mediated degradation. SNAI1 is an important transcription factor of EMT, which suppresses the transcription of E-cadherin by combining with the E-box sequence of E-cadherin’s promoter\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. EMT is characterized by reduced cell-cell adhesion, lossing of cell polarity and the acquisition of mesenchymal features, which is considered to be a prerequisite for tumor metastasis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. A study reported that TRIM47 knockdown inhibited EMT through the inactivation of Wnt/β-catenin pathway in glioma\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To our surprise, TRIM47 knockdown did not affect Wnt/β-catenin pathway in HCC cells. The ubiquitination of SNAI1 was regulated by several E3 ubiquitin ligase, including FBXO11\u003csup\u003e35\u003c/sup\u003e, GSK3β\u003csup\u003e36\u003c/sup\u003e, and FBXL14\u003csup\u003e37\u003c/sup\u003e. The interaction between TRIM47 and SNAI1 may prevent the ubiquitylation and degradation of SNAI1 by other E3 ubiquitin ligases.\u003c/p\u003e\u003cp\u003eIn conclusion, we display for the first time that CARM1-CUL4\u003csup\u003eCRBN\u003c/sup\u003e-TRIM47-SNAI1 cascade is involved in HCC metastasis. Our data are consistent with a model in which TRIM47 methylation by CARM1 inhibits CUL4\u003csup\u003eCRBN\u003c/sup\u003e-mediated ubiquitylation of TRIM47. Accumulated TRIM47 thus interacts with SANI1 and protecting it from proteasomal degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our findings underscore the roles of TRIM47 and CARM1 in HCC metastasis, unveils a novel mechanism that crosstalk between arginine methylation and ubiquitylation orchestrates TRIM47-mediated HCC metastasis. The newly defined CARM1-CUL4\u003csup\u003eCRBN\u003c/sup\u003e-TRIM47-SNAI1 regulatory axis may open new avenues for metastatic HCC therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe\u0026nbsp;thank\u0026nbsp;Yuan Cao and Piao Zou at Analytical \u0026amp; Testing Center, Wuhan University of Science and Technology for their assistance in the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu Jia, Zhu Haichuan\u0026nbsp;and Xiao Juan\u003csup\u003e\u0026nbsp;\u003c/sup\u003edesigned research;\u0026nbsp;Tang Yuzhe, Meng Xiang, Luo Xia, Yao Wentao, Tian Li, Zhang Zijian, Zhao Yuan\u0026nbsp;performed research;\u0026nbsp;Tang Yuzhe, Meng Xiang\u0026nbsp;and\u0026nbsp;Luo Xia analyzed data;\u0026nbsp;Hu Jia and Zhu Haichuan\u0026nbsp;wrote the paper; Hu Jia\u0026nbsp;revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics A\u003c/strong\u003e\u003cstrong\u003epproval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal interventions were approved by\u0026nbsp;approved by the Animal Ethics Committee of Wuhan University of Science and Technology\\\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation Program of China Programs (82373038) and Innovation and Development Joint Funds of the National Natural Science Foundation of Hubei (No. 2022CFD109).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDi Rienzo M, Romagnoli A, Antonioli M, Piacentini M, Fimia GM. 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However, the biological functions of TRIM47 and its regulatory mechanism in hepatocellular carcinoma (HCC) remain unexplored. Here, we showed that TRIM47 was upregulated in HCC tissues compared with adjacent normal tissues, especially at advanced stages, and associated with poor prognosis in HCC patients. Functional studies demonstrated that TRIM47 enhanced the migration and invasion ability of HCC cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, TRIM47 promotes HCC metastasis through interacting with SNAI1 and inhibiting its degradation by proteasome. Moreover, TRIM47 was di-methylated by CARM1 at its arginine 210 (R210) and arginine 582 (R582), which protected TRIM47 from the ubiquitination and degradation mediated by E3 ubiquitin ligase complex CRL4\u003csup\u003eCRBN\u003c/sup\u003e. Collectively, our study reveals a pro-metastasis role of TRIM47 in HCC, unveils a unique mechanism controlling TRIM47 stability by CARM1 mediated arginine methylation, and highlights the role of the CARM1-CRL4\u003csup\u003eCRBN\u003c/sup\u003e-TRIM47-SNAI1 axis in HCC metastasis. This work may provide potential therapeutic targets for metastatic HCC treatment.\u003c/p\u003e","manuscriptTitle":"Arginine Methylation-dependent TRIM47 Stability Mediated by CARM1 Promotes the Metastasis of Hepatocellular Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 19:02:23","doi":"10.21203/rs.3.rs-4220751/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c3982d6-da64-4fd4-8870-ba9941959b53","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31306754,"name":"Biological sciences/Cancer/Metastasis"},{"id":31306755,"name":"Biological sciences/Biochemistry/Proteins/Oncogene proteins"}],"tags":[],"updatedAt":"2024-11-21T08:08:48+00:00","versionOfRecord":{"articleIdentity":"rs-4220751","link":"https://doi.org/10.1038/s41420-024-02244-4","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2024-11-20 05:00:00","publishedOnDateReadable":"November 20th, 2024"},"versionCreatedAt":"2024-06-04 19:02:23","video":"","vorDoi":"10.1038/s41420-024-02244-4","vorDoiUrl":"https://doi.org/10.1038/s41420-024-02244-4","workflowStages":[]},"version":"v1","identity":"rs-4220751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4220751","identity":"rs-4220751","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}