Identification And Functional Validation Of The Novel Mutated Gene In Human Hepatocellular Carcinoma

HCC is a kind of malignant tumor with poor prognosis. HCC is associated with HBVinfections especially in China. The mechanism of HCC pathogenesis, including the geneticmechanism, is still unclear. In order to find the genetic mechanism in HCC developmentand progression, we performed the exome sequencing on10primary HCC samples withPVTT, and then identified novel mutations of important genes in human hepatocellularcarcinoma, including ARID1A and SAMD9L mutations, we also validated their effects onprogression, migration, tumorigenesis and cell cycle in HCC cells.Genes carrying two confirmed non-silent mutations were considered as the candidatemutated genes in HCC. We first evaluated the mutation frequency of the candidate genesin additional paired HBV-associated HCC samples via PCR-based Sanger sequencing ontheir protein-coding exons. The proliferation, colony formation, migration and invasion ofHCC cells altered by knockdown of ARID1A, SAMD9L and other genes were examined.Furthermore, SAMD9L expression was investigated through reverse transcriptionpolymerase chain reaction (RT-PCR) in HCC samples and cell lines. Upon RNAinterference against SAMD9L, Cell cycle and5-bromo-2’-deoxyuridine (BrdU)incorporation were analyzed by flow cytometry and immunofluorescent assay. Fortumorigenicity assay, HCC cell sublines with stably silenced SAMD9L were establishedand inoculated into nude mice. TOP/FOP Flash system was used to envaluate the activityof Wnt pathway, and Western blot was used to test β-catenin protein level after SAMD9Lknockdown.Results showed that, among110pairs of HCC samples with HBV infection, ARID1Amutations were found in14samples, with a mutation frequency12.7%. SAMD9Lmutations were found in7of overall130pairs of HCC samples with a mutation frequency 5.38%. ARID1A knockdown by siRNA can promote cell proliferation, colony formation onsoft-agar, migration and invasion in MHCC-97L and MHCC-97H cells. SAMD9Lexpression was significantly down-regulated in approximately60%HCC specimens ascompared to adjacent non-cancerous livers. SAMD9L knockdown obviously promoted cellproliferation and colony formation of HCC cell lines. Interestingly, SAMD9L silencefacilitated G1-S transition of cell cycle progression. Furthermore, SK-hep-1andMHCC-97H cells with stable SAMD9L knockdown exhibited promoted tumorigenicity inathymic mice. SAMD9L knockdown can increase cells in S phase during cell progressionand activate Wnt/β-catenin pathway in HCC cells.