The Malignant Behavior And Molecular Mechanism Of Hepatoma Carcinoma Cell Regulated By ETV6

Background: Hepatocellular carcinoma(HCC)is a common type of hepatocellular carcinoma,and the fatality rate of HCC is at the forefront of all cancers.HCC lacks typical symptoms in the early stage,most patients diagnosed with liver cancer in the middle and late stages.Therefore,it is of great significance to study the molecular level changes and regulatory mechanisms during the occurrence and development of hepatocellular carcinoma so as to provide a target for the diagnosis of hepatocellular carcinoma.ETV6,also known as TEL,is a member of the E26 transformation-specific(ETS)family.The proteins of this family mainly contain two functional domains.One PNT(N-terminal)domain,which is mainly involved in protein-protein interaction and regulates the intranuclear transport of ETV6 protein itself.Another C-terminal ETS domain is responsible for binding DNA.ETV6 is widely expressed in embryonic development and adult stage,and it has been proved to be a strong transcription suppressor gene,which is related to the occurrence and development of leukemia、lung cancer、renal cell carcinoma.However,the function and regulatory mechanism of ETV6 in HCC are not clear.Objective: 1.To explore the clinical correlation between ETV6 expression and hepatocellular carcinoma;2.To study the effects of ETV6 knockdown on the biological behavior of HCCLM3 and Hu H7 cells;3.To study the effects of ETV6 overexpression on the biological behavior of HCCLM3 and Hu H7 cells and its mechanism.Methods: 1.WB assay was used to detect the expression of ETV6 protein in HCCLM3 and Hu H7 cells;2.Knock down ETV6 in HCCLM3 and Hu H7 cells;WB assay was used to detect the interference efficiency of si RNA on the expression of ETV6 in HCCLM3 and Hu H7 cells;MTT assay was used to detect the effect of ETV6down-regulation on the proliferation of HCCLM3 and Hu H7 cells;Plate cloning assay was used to detect the effect of ETV6 down-regulation on the cloning ability of HCCLM3 and Hu H7 cells;Transwell assay was used to detect the effect of ETV6down-regulation HCCLM3 and Hu H7 migration and invasion;Ghost pencil cyclic peptide staining assay to detect the effect of ETV6 down-regulation on the cytoskeleton of HCCLM3 and Hu H7;3.Lentivirus infection method to construct HCCLM3 and Hu H7 cell lines with over-expression of ETV6;MTT method to detect the effect of ETV6 over-expression on the proliferation of HCCLM3 and Hu H7;Transwell method to detect the effect of ETV6 over-expression on the migration and invasion of HCCLM3 and Hu H7;plate cloning assay to detect the effect of ETV6 over-expression on HCCLM3 and Hu H7 migration The effects of ETV6 overexpression on the cloning ability of HCCLM3 and Hu H7 were measured.The effect of ETV6 overexpression on the cytoskeleton of HCCLM3 and Hu H7 cells was detected by Ghost Pencil Cyclic Peptide staining.4.The expression levels of E-cadherin and Vimentin in HCCLM3 and Hu H7 cells were detected by WB method.Results: 1.23 clinical samples of hepatocellular carcinoma showed that ETV6 was highly expressed in the tissues of hepatocellular carcinoma patients;2.Obtaining HCCLM3 and Hu H7 cell lines with obvious down-expression of ETV6,the down-regulation efficiency was 62% 和 45%;The low expression of ETV6 inhibited the proliferation,cloning,migration and invasion of HCCLM3 and Hu H7 cells,and also inhibited the formation of intracellular microfilaments and extracellular pseudopods;3.The ETV6 overexpression efficiency of HCCLM3-p CDH-ETV6 and Hu H7-p CDH-ETV6 cell lines was 100% and 62%.4.Overexpression of ETV6 enhanced the cloning,migration and invasion ability of Hu H7 and HCCLM3 cells in vitro,and promoted the formation of F-actin and extracellular pseudopod,but had no significant effect on proliferation ability.At the same time,the expression level of E-cadherin in epithelial cells decreased 30% and 35%,while the expression of Vimentin in mesenchymal cells increased 87% and 153%.Conclusion: 1.ETV6 is highly expressed in hepatocellular carcinoma samples;2.ETV6 knockdown inhibits the proliferation,clone formation,migration and invasion of hepatocellular carcinoma cells,and inhibits the formation of cytoskeleton;3.Overexpression of ETV6 promotes the cloning,migration and invasion of hepatocellular carcinoma cells,promotes the formation of cytoskeleton,and has a slight effect on cell proliferation.;4.Overexpression of ETV6 inhibits the expression of E-cadherin and promotes Vimentin expression.It can promote EMT and enhance the ability of migration and invasion of HCCLM3 and Hu H7 cells.