Association And Mechanism Studies Of TRIM16-E121D Polymorphism With The Risk And Prognosis Of Hepatocellular Carcinoma

Background and objectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide.Its morbidity ranks the sixth in malignant tumor diseases.There are about 840,000 new cases and about 782,000 people died from the disease each year.The main risk factors for the development of HCC are chronic hepatitis B and C virus infection,excessive drinking,obesity,smoking,and type 2 diabetes.Since the completion of the Human Genome Project,SNP(single nucleotide polymorphism)has been used as the third type of genetic markers.There have been continuous studies to find SNPs related to risk of different diseases through GWAS(genome-wide association studies).For example,Professor Jiang found genetic vatiants in STAT4 and HLA-DQ genes(rs7574865 in STAT4 and rs9275319 in HLA-DO)associated with the risk of hepatitis B virus-related HCC through GWAS.The tripartite-motif(TRIM)protein family belongs to the E3 ubiquitin ligase which includes at least 70 members.Many TRIM family proteins play important roles in a broad of biological processes,including cell proliferation,cellular signalling,DNA repair,tumour development and progression.TRIM16 is a member of this family,which has been found to inhibit occurrence and development of malignant tumors.The purpose of this study was to investigate the role and mechanism of TRIM 16 and its coding sequencing(CDS)region SNPs in the pathogenesis and development of HCC.Methods1.Screening susceptible SNPs within the CDS region of TRIM 16.SNPs of TRIM 16 were obtained from dbSNP and 1000 Genomes databases.SNPs in the CDS region of TRIM16 with minimum allele frequencies(MAF)? 0.05 were further screened out.Polyphen was used to predict SNPs of TRIM16 damaging the function of the protein.Then the candidate SNPs were assessed for the associations with HCC risk in our previously published GW AS dataset containing whole-genome genotyping data of 1156 HCC cases and 1349 controls as well as with HCC prognosis in another cohort including 300 HCC patients with more than three years of follow-up.2.Investigating the role and mechanism of SNPs within TRIM16 in the occurrence and development of HCC CCK-8,Edu,cell migration and invasion,and Western blot experiments were used to verify the biological function of these SNPs in liver cancer cells.qRT-PCR and Western blot were utilized to explore the molecular biological mechanism.Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)assay were used to find and verify proteins that bind to TRIM 16.Ubiquitination and Western blot experiments were used to detect the regulatory relationship between TRIM 16 and their interacting protein.Results1.The SNP rs2074890 within the CDS region of TRIM16 is associated with risk of HCC(odds ratio=0.81,P=0.023)as well as the HCC prognosis(hazard ratio=0.44,P=0.034).The rs2074890 results in a single base substitution that leads to an animo acid substitution:Glutamicacid at position 121(corresponding to TRIM16121E)to Asparticacid(corresponding to TRIM16121D).2.Both TRIM16121E and TRIM16121D can inhibit the proliferation,migration and invasion of liver cancer cells.Meanwhile,it was also found that TRIM16121D has stronger ability to inhibit the proliferation,migration and invasion of liver cancer cells than TRIM16121E.3.TRIM16 has ability to mediate K48-linked ubiquitination to degrade?-catenin,and the inhibition ability of TRIM16121Dis stronger than TRIM16121E.4.TRIM16121E and TRIM16121D inhibit the activity of Wnt/?-catenin pathway to affect the proliferation,migration and invasion of HCC cell lines.TRIM16121D has stronger ability to inhibit the activity of Wnt/?-catenin signaling pathway than TRIM16121E.In addition,TRIM16121E and TRIM16121D mainly decreases nuclear?-catenin in HCC cells.ConclusionSNP-rs2074890(E121D)is associated with the risk of HCC(odds ratio=0.81,P=0.023)as well as the HCC prognosis(hazard ratio=0.44,P=0.034).Subjects carrying rs2074890 TT or GT genotype show lower risk to HCC and better prognosis than those carrying rs2074890 GG genotype.The rs2074890 results in a single base substitution that leads to an animo acid substitution:Glutamicacid at position 121(corresponding to TRIM16121E)to Asparticacid(corresponding to TRIM16121D).TRIM16121D has stronger ability to decrease proliferation,migration and invasion of HCC cells.TRIM16 binds to ?-catenin and regulates the protein level of ?-catenin by promoting K48-ubiquitination of ?-catenin.And the ubiquitylation of ?-catenin by K48-mediated linkage was enhanced more in TRIM16121D cells than in TRIM16121E.TRIM16121E and TRIM16121D inhibiting the Wnt/?-catenin signaling pathway to affect HCC cells'phenotype.In addition,TRIM16121D has stronger ability to inhibit the activity of Wnt/?-catenin pathway than TRIM16121E.