The Investigation Of Invasion Mechanism Of HBx In HCC

Hepatocellular carcinoma (HCC) is one of the most common primary malignant tumor, and remains a leading cause of death. It ranks fifth in overall frequency and third in annual mortality rate worldwide. The infection of hepatitis B virus (HBV) and/or hepatitis C virus (HCV) has been identified as a major environmental risk factors to be closely associated with HCC, which account for more than 80% of HCC cases. Although, the underlying mechanism remains unclear, much of the research on HBV hepatocarcinogenesis has been focused on the HBx gene. The aim is to investigate the effect of invasion which HBx protein exert on hepatocellular carcinoma cells and initially explore the mechanism of tumor invasion regulated by NDRG1.1. Firstly, recombination adenovirus expressing HBx, NDRG1 cDNA and NDRG1 siRNA were successfully constructed. After plaque purification and PCR identification of the amplifying adenovirus, and the Western-blot analysis of SMMC7721 cells transiently infected with them, these results demonstrated the recombinant adenovirus have been successfully produced, and had the favorable infected ability. The stable transfectants cell lines AGS/pSuppressor-NDRG1-siRNA and AGS/pSuppressor have been successfully established which down-regulated NDRG1 and control.2. After infected SMMC7721 cells with recombinant adenovirus Ad-HBx,Ad-HBx + Ad-NDRG1,Ad-HBx + Ad-NDRG1/siRNA,Ad-NDRG1,Ad-NDRG1/siRNA and Ad-LacZ, the results were analyzed using different experimental methods. (1)Matrigel results showed the invasive ability of cells infected with HBx was improved, and the promoted invasive ability was overall inhibited by exogenous NDRG1, and increased by NDRG1 siRNA. (2)The results of Gelatin Zymograph demonstrated the expression of MMP9 was increased by HBx, but not much higher, and exogenous NDRG1 inhibited intracellular MMP9, as well as inhibited the induced MMP9 expression by HBx to much lower lever. (3)The results of Western-blot showed HBx and NDRG1 siRNA could also induce MT1-MMP expression, but NDRG1 siRNA could not increased the expression of MT1-MMP which induced by HBx; The induced expression of MT1-MMP by HBx was totally inhibited by NDRG1. However, the expression of MMP2 was induced by HBx, which slightly inhibited by NDRG1 and NDRG1-siRNA. (4)NDRG1 siRNA could markly induce the invasion of AGS which inhibited by exogenous NDRG1, meanwhile upregulate the expression of MT1-MMP and MMP2.3.The results of Western-blot showed the inhibited expression of NDRG1 was depended on the expression of HBx, moreover HBx induced the expression of COX-2 which was decreased by both NDRG1 and NDRG1 siRNA,and weakly inhibited the expression ofβ-catenin in SMMC7721. Additionally, in AGS cells, NDRG1 siRNA inhibited the expression of COX-2 andβ-catenin.4.After transiently transfected SMMC7721 which infected with Ad-HBx, Ad-HBx + Ad-NDRG1/siRNA,Ad-HBx + Ad-NDRG1,Ad-NDRG1/siRNA,Ad-NDRG1and Ad-LacZ, and stable transfectants AGS cell lines with TOPFlash and Renilla luciferase reporters, the results showed HBx could active the transcriptional activity of TCF/LEF, which was totally inhibited by NDRG1; moreover, Ad-HBx + Ad-NDRG1,Ad-NDRG1/siRNA and Ad-NDRG1 could also inhibit the transcriptional activity of TCF/LEF in SMMC7721; however, only Ad-NDRG1 inhibited the improved invasion by HBx. In AGS cells, NDRG1 could also inhibit the transcriptional activity of TCF/LEF. These results indicate Wnt/β-catenin pathway doesn't paly an important role in induced invasion by HBx, there is relationship between the changed expression ofβ-catenin and E-cadherin, so whether the pathway affect the tumor invasion needs more further investigation.In summary, the tumor invasion was induced by HBx, and totally inhibited by NDRG1. The improved invasion may achieve through the expression MT1-MMP and MMP9 induced by HBx,and the induced expression of MT1-MMP and MMP9 could be inhibited by NDRG1, there exists the possibility between improved invasion and changed expression ofβ-catenin. The study provides a new experimental basis to further elucidate the roles of HBx and NDRG1 in pathogenesis of HCC.