Effects Of Hepatitis B Virus X Protein On The Expressions Of MMPs And TIMPs

The invasion and metastasis of hepatocellular carcinoma (HCC) was closely correlated with matrix metalloproteinases (MMPs). Hepatitis B virus X protein (HBx) could enhance the invasion and metastasis of hepatoma carcinoma cells by inducing the expressions of MMP3, MMP9 and MMP14 (MT1-MMP). There were currently known 23 kinds of MMPs in human cells and their enzymatic activities were inhibited by tissue inhibitors of metalloproteinases (TIMPs). The aim of this study is to comprehensively evaluate the effects of HBx protein on the expressions of MMPs and TIMPs .The first part of this study was aimed to construct HBx recombinant eukaryotic expression vector and confirm the expression of HBx in HepG2 hepatocytes. So the HBx gene from genotype B hepatitis B virus , i.e., XB, was cloned into the eukaryotic expression vector pcDNA3.1/HisC in EcoR? and Xho? sites, the recombinant plasmid pcDNA3.1/HisC-XB was transiently transfected into HepG2 cells, and the western Blot using anti-Xpress antibody was used to confirm the expression of HBx.In the second part of this study, HepG2 cells were transfected with pcDNA3.1/HisC and pcDNA3.1/HisC-XB seperately, then selected by G418 at the final concentration of 800μg ml-1 to obtain G418 resistant clones. Semiquantitative RT-PCR showed that HBx could enhance the transcriptions of MMP2, MMP7, MMP13, MMP14, MMP16, MMP17, MMP19, MMP23, MMP24, TIMP1, TIMP4, but inhibit the transcriptions of MMP1, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP15, MMP20, TIMP2, TIMP3. Gelatin Zymography and Collagen Zymography revealed that HBx could promote the expression of Pro-MMP2 and the activation of MMP9, but inhibit the expression of total MMP9. Reverse Gelatin Zymography demonstrated that HBx could promote the expressions of TIMP1 and TIMP4 but inhibit those of TIMP2 and TIMP3.In conclusion, the effects of HBx on the expressions of MMPs and TIMPs was sophisticated, the exact mechanism of these effects attributing to the enhancement of the invasion and metastasis of hepatoma carcinoma cells need to be further elucidated.