HBx Gene Regulates S100A4 Protein To Promote Proliferation Of Hepatocellular Carcinoma Cells And Its Clinical Prognosis

Objective:Hepatitis B virus(HBV)infection is one of the main risk factors for the development of human hepatocellular carcinoma(HCC).The integration of HBV DNA into the genome was observed in 85%of HBV-related HCCs.Although most HBV genes are not expressed in HCC tissues,HBV-X(HBx)protein is detected in most HBV-related HCC tissues,suggesting that HBx protein plays an important role in the development of HBV-related HCC.HBx protein is a multifunctional regulatory factor,which is closely related to the pathogenesis of hepatocellular carcinoma.However,the molecular mechanism of carcinogenicity of HBx protein is still not fully understood.The aim of this study is to find genes related to HBx regulating the pathogenesis of hepatocellular carcinoma and to determine its clinical significance in the pathogenesis of hepatocellular carcinoma.At the same time,the mechanism of IFN-alpha2b in the treatment of hepatitis B or HCC was studied to explore whether IFN-alpha2b could treat the disease by inhibiting the expression of HBx-related genes.Methods:1.Establishment of a cell line with high expression of HBxThe lentiviruses carrying HBx gene were transferred into primary hepatocellular carcinoma cells to make them highly expressed,and the changes of cell function were verified,such as the changes of proliferation rate in vitro,tumorigenesis rate in vivo and the release of inflammatory factors.2.Validation of the relationship between HBx expression and S100A4 expression levelThe differentially expressed genes in cells of high expression group and negative control group were analyzed by transcriptome sequencing.The level of differentially expressed proteins was verified by Western blotting.shRNA knockdown was performed on the up-regulated S100A4 protein in HBx-overexpressing cell lines,and cell function changes were detected to prove the correlation between the differentially expressed genes and cell function.At the same time,the relationship between S100A4 and cell function was further validated by changing cell lines.3.Analysis of the correlation between S100A4 and clinical prognosis of hepatocellular carcinomaImmunohistochemical technique was used to detect the expression of S100A4 in 180 cases of hepatocellular carcinoma.The correlation between S100A4 and the prognosis of hepatocellular carcinoma patients was verified by the survival time of patients.Result1.HBx lentivirus transfection:Lentivirus carrying HBx gene was transferred into primary hepatocellular carcinoma cells.The proliferation rate of HBx-expressing cells was significantly faster than that of control group by cell proliferation test and nude mice tumorigenesis test,and the release of inflammatory factors such as IL-lbeta also showed an upward trend.2.Differential gene analysis:Screening differentially expressed genes according to transcriptome sequencing analysis results.The up-regulated genes of hepatocellular carcinoma cells in HBx overexpression group were S100A4,IRF-7,RPS-27,etc.3.High expression of S100A4 and its functional verification3.1 Protein imprinting technology to verify the expression of S100A4 gene:S100A4 is an important member of S100 family proteins,its function is to increase the progression and metastasis of tumors.The up-regulated expression of S100 A4 protein in hepatocellular carcinoma cells with high expression of HBx suggested that the over-expression of S100A4 protein might be related to the faster proliferation of hepatocellular carcinoma cells.3.2 The relationship between the expression intensity of S100A4 and cell function:shRNA was used to knock down the expression of S100A4 in hepatocellular carcinoma cells of HBx overexpression group,and then functional verification showed that the proliferation rate of hepatocellular carcinoma cells after knockdown of S100A4 was significantly slowed down.Subsequently,the hepatocellular carcinoma cell lines were replaced for verification,and the same results were obtained.3.3 The relationship between the expression intensity of S100A4 and the prognosis of hepatocellular carcinoma patients:S100A4 was highly expressed in the cytoplasm of hepatocellular carcinoma patients,and the expression intensity was negatively correlated with the survival time.Conclusion1.HBx overexpression can accelerate cell proliferation,and transcriptome sequencing results show that HBx overexpression can up-regulate the expression of S100A4 protein.2.There was a significant correlation between protein expression and cell proliferation rate.And down-regulate S100A4 by shRNA could slow down cell proliferation.lt is suggested that S100A4 may accelerate the proliferation of cancer cells,and that HBx gene may alter cell function by regulating the expression of S100A4 protein.3.The expression of S100A4 in cancer tissues was significantly higher than that in adjacent tissues,and was significantly correlated with the size of tumors and the expression of PD-L1.The expression of S100A4 is significantly correlated with the clinical prognosis of patients with hepatocellular carcinoma,suggesting that S100A4 may be a good target for the treatment of hepatocellular carcinoma.