The Role Of TRIM31 In The Metastasis And Tumor Progression Process Of Hepatocellular Carcinoma

Part oneThe Role of TRIM31 in Metastatic Hepatocellular CarcinomaAimsIt is well known that when epithelial cells are deprived of anchorage, they undergo detachment-induced apoptosis, also known as anoikis. However, many cancer cells acquire anoikis resistance and maintain survival without adhering strongly to substratum. Anoikis-resistance is a hallmark of cancer and the prerequisite for metastasis.TRIM31, an E3 ubiquitin ligase, is a member of the tripartite motif-containing protein (TRIM) family composed of a RING-finger, a B-box and a coiled-coil motif. TRIM family proteins and their alterations are involved in a broad range of biological and pathological processes such as cell growth, genetic diseases, apoptosis, transcriptional regulation and cancer development. However, the role of TRIM31 in hepatocellular carcinoma (HCC) progression remains to be clarified.Methods and Materials1. Establishment of anchorage-dependent and independent hepatoma modelHuman BEL7402, SMMC7721. HepG2 and Huh7 hepatoma cells were maintained in RPMI-1640 medium supplemented with 10% FBS.6-well plate was coated with 1.5ml of poly-HEMA solution for construction of anchoraged deprived cell model. Coated plates were allowed to air dry under sterile conditions in a laminar flow hood. Before use, poly-HEMA-coated dishes were washed with PBS for 3 times. Hepatocarcinoma cells grown on normal plates and poly-HEMA-coated plates were established as anchorage-dependent and independent models, respectively.2. Effect of TRIM31 on anchorage dependent and independent hepatocarcinoma cells (1) Anchorage-dependent and independent cells were transfected with plasmids and small interference RNAs and cultured for 24h, followed by morphology investigation. (2) Cell proliferation, colony formation, and invasion assaySMMC7721 cells and BEL7402 cells were plated in 96 well plates at the density of 104 cells/well, and the proliferation status of HCC cells were detected by CCK-8 Kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacture’s instruction at the indicated time points. For colony formation assay, HCC cells were plated into 6-well plates with the density of 1000 cells/well and incubated for 7-10 days; formed colonies were visualized and analyzed after staining with 0.005% crystal violet (dissolved in methanol). For invasion assay, transwell migration plates (Corning, NY, USA) with 8μm pore size were coated with matrigel.10000 Cells in 100μl RPMI medium without FBS were placed into the upper chamber, and 600μl medium containing 10% FBS were put to the lower chamber. After 12 hours of incubation, the inserts were removed and all the residual cells on the upper side were scraped with a cotton swab. Migrated cells on the lower side of the insert were fixed with 4% formalin for 15 minutes, washed twice with PBS, stained with 0.1% crystal violet for 5 minutes, and analyzed under the microscope.3. Western blot and Immunoprecipitation assay were used for detection of the signaling pathway and molecular target of TRIM31 in anchorage-deprived HCC cells. Results1. Both of the mRNA and protein levels of TRIM31 were significantly up-regulated in HCC cells after anchorage deprival, which indicated that TRIM31 may play a role in anoikis-resistance of HCC cells.2. Knockdown of TRIM31 induced cell death of HCC cells after anchorage deprival, further analysis showed that apoptosis was induced in these anchorage deprived cells, which indicated that TRIM31 mediated anoikis-resistance of HCC cells. Furthermore, the anoikis-resistance related malignant behaviors were also mediated by TRIM31.3. Ectopic overexpression of TRIM31 promoted activation of AMPK pathway and induced increased activation of its downstream molecules.4. The AMPK inhibitor compound C treatment abrogated the anoikis-resistance effect of TRIM31, which indicated that TRIM31 induced anoikis-resistance of HCC cells through activation of AMPK pathway.5. TRIM31 directly bound to p53, an upstream inhibitor of AMPK pathway, promoted its ubiquitination and degradation, thus further overactivated AMPK signaling pathway and induced anoikis-resistance of HCC cells.6. Exogenous transfection of p53 efficiently rescued TRIM31-induced anoikis-resistance and other malignant behaviors of HCC cells.ConclusionOur study showed for the first time that p53 was a direct ubiquitous degraded target of TRIM31. TRIM31 could mediate anoikis resistance of HCC cells and further induce other accompanied malignant behaviors during this process by directly target p53 for degradation.Innovations and significances1. This is the first time to reveal that TRIM31 could medicate anoikis-resistance of HCC cells through overactivation of AMPK pathway and further promote tumor progression.2. This is the first time to show that TRIM31 could directly target P53 for ubiquitous degradation and further overactivate AMPK pathway, thus contributes to anoikis-resistance of HCC cells.Part twoThe Role of TRIM31 in Progression of Hepatocellular CarcinomaAimsLiver cancer is the third leading cause of cancer-related deaths worldwide and hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver. The mortality rate of HCC is high due to unclear pathogenesis, late diagnosis and limited treatment option. The rapidly growing tumor displays heterogeneity of histopathologic characteristics while the molecular pathogenesis undergoing is still not fully understood. Therefore, it is critical to clarify its molecular mechanism and identify the therapeutic target for effective manipulation of this disease. Tripartite motif (TRIM) 31 is a member of the tripartite motif-containing protein family, and its role in hepatocellular carcinoma (HCC) progression remains to been clarified.Methods and Materials1. Patient samplesPaired HCC tissue samples and their corresponding distal non-cancerous liver tissues from 212 HCC patients were collected for analysis of TRIM31 expression in clinical HCC patients. Among these samples,104 paired HCC tissues and their corresponding non-cancerous liver tissues were used to analyze the expression and location of TRIM31 by immunohistochemistry. To validate the IHC data, another cohort of 108 pairs of HCC tissues and corresponding distal non-cancerous liver tissues were used for analysis of TRIM31 protein expression by Western blot.2. Cell culture, SiRNA, plasmids constructs and transfectionHepatocellular carcinoma SMMC7721 and BEL7402 cell lines were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China) and were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. Two small interference RNAs targeting TRIM31 (Si-TRIM31) were transfected to the HCC cells to block TRIM31 expression. Nonspecific random sequences were used as non-specific negative control (Si-NC).3. Cell proliferation, colony formation, and invasion assaySMMC7721 cells and BEL7402 cells were plated in 96 well plates and the proliferation status of the HCC cells were detected by CCK-8 Kit. For colony formation assay, HCC cells were plated into 6-well plates with the density of 1000 cells/well and incubated for 7-10 days; formed colonies were visualized and analyzed. For invasion assay, transwell migration plateswith 8μm pore size were coated with matrigel.10000 Cells in 100μl RPMI medium without FBS were placed into the upper chamber, and 600μl medium containing 10% FBS were put to the lower chamber. Migrated cells on the lower side of the insert were fixed, stained and analyzed under the microscope.4. Immunohistochemistry (IHC), western blot, and immunoprecipitationImmunohistochemistry (IHC) staining of the target proteins and evaluations of the intensities of staining were performed as described before. Western blot analysis of the target proteins in HCC cells and liver tissues were performed as previously described. For immunoprecipitation (IP) assay, cells were allowed to grow in 10cm dishes and transfected with the appropriate plasmids. Whole-cell extracts were collected and lysed in IP buffer as described previousl. All the other antibodies for IP assay were the same as those used in western blot assay.5. RNA purification and Real-time PCR assayTotal RNA was extracted from liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 2 μg of RNA using a Reverse Transcription Kit (Toyobo Co., Osaka, Japan). The PCR cycling conditions were as follows:pre-denaturing at 95℃ for 10 min, followed by 40 cycles of denaturing at 95℃ for 10 s, annealing at 58℃ for 20 s, and extension at 72℃ for 10s.6. Construction of xenografted tumor modelSMMC7721 cell suspensions (1×10’ cells) in a total volume of 100μl were injected subcutaneously into the left flanks of 5-week-old male BALB/C nude mice (Huafukang Biotechnology, Beijing, China). When visible tumor appeared, the mice were randomly divided into Si-TRIM31 transfection group and Si-NC control group. This injection was performed every other day before the mice were sacrificed and the tumors were isolated.7. Statistical analysisData were assessed using software packages SPSS version 16.0(SPSS Inc., IL, USA). Statistical analyses were prepared using GraphPad Prism version 5.04 (GraphPad Software, La Jolla, CA, USA). P value<0.05 was considered statistically significant.Results1. Both of the mRNA and protein levels of TRIM31 were significantly up-regulated in human tumor samples compared with their corresponding adjacent non-cancerous liver tissues, and advanced HCC tissues were prone to have higher levels of TRIM31 expression.2. Knockdown of TRIM31 suppressed malignant behaviors of HCC cells, including cell proliferation, clone formation, and cell invasion; whereas overexpression of TRIM31 promotes malignant behaviors of HCC cells both in vivo and in vitro.3. Ectopic overexpression of TRIM31 promoted activation of mTORC1 pathway and induced increased activation of its downstream molecules, including S6K1, S6 and 4EBP1, which further induced the up-regulation of its downstream target molecule HIF-la.4. TRIM31 directly bound to the TSC1/2 complex, an upstream inhibitor of mTORC1 pathway, promoted k48 linked ubiquitous degradation of TSC1-TSC2 complex via its ring domain; thus further overactivated mTORC1-HIF1αsignaling pathway and facilitate HCC progression.5. Exogenous transfection of TSC1 and TSC2 efficiently rescued TRIM31-induced malignant behaviors of HCC cells.6. Negative regulation of TSC1-TSC2 complex by TRIM31 was further verified in animal model and clinical HCC specimen.Conclusion:Our study showed that TRIM31 was overexpressed in HCC cells and its overexpression contributed to HCC progression through promoting the overactivation of mTORC1-HIF1α pathway by directly targeting TSC1/2 complex for degradation. This study indicated a novel therapeutic strategy against HCC by targeting TRIM31.