Experimental Study On The Inhibition Effect Of SiRNA Expressing Vector-based RNA Interference Targeting HBV X In Human Hepatocellular Carcinoma Cell Line HepG2.2.15

Objective:To observe the hepatocarcinogenic effect of HBV x, the most potential oncogenic factor, interference on cell growth and the expression of hTERT and MMP-2 genes in human hepatocellular carcinoma HepG2.2.15 cells, which constitutively produce HBV x. Methods:(1) Identification and amplification of HBV x gene expression vector pSIHBV/X. (2) The human hepatocellular carcinoma HepG2.2.15 cell culture. (3) pSIHBV/X and the cloning vector containing enhanced green fluorescent protein gene(pEGFP-N1) was transiently co-transfected into HepG2.2.15 cells in order to determine the transfection efficiency. (4) Evaluation of silence efficiency by using RT-PCR method. (5) Determination of the levels of HBsAg and HBeAg in cell supernatant by ELISA. (6) The cell proliferation evaluated by MTT. (7) The cell apoptosis detected by Annexin V-FITC/PI double staining method. (8) The mRNA expression levels of hTERT and MMP-2 assayed by Real-time PCR. (9) The protein expression levels of hTERT and MMP-2 detected by Western blot. Results:The results of sequencing showed that pSIHBV/X was constructed successfully. The transfection efficiency was 27% judged by pEGFP-N1. The efficiency of RNA interference of HBV x gene was 53.6%. The levels of both HBsAg and HBeAg in HepG2.2.15 cells supernatant were significantly decreased after the transfection of pSIHBV/X, which differences have statistical significance compared with the control group(P<0.01). The cell proliferation was inhibited and the cell apoptosis was promoted after the transfection of pSIHBV/X, which differences have statistical significance compared with the control group (P<0.01). The expression levels of mRNA and protein of both hTERT and MMP-2 in HepG2.2.15 cells decreased at different level, which differences have statistical significance compared with the control group(P<0.05). Conclusions:Our results display that the reduction of the levels of HBsAg and HBeAg, the inhibition of cell proliferation and the promotive effect of the cell apoptosis in HepG2.2.15 cells after HBV x gene RNA interference. Meanwhile, HBV x gene RNA interference can also inhibit the expression of both cancer marker gene hTERT and metastasis of cancer marker gene MMP-2 in HepG2.2.15 cells.