PreC/C Gene-targeting RNA Interference Suppresses Hepatitis B Virus Replication And Expression In Human Hepatoma Cells

ObjectHBV infection is a serious public health issue globe-wide. Throughout the world thereare an estimated0.35billion people chronically infected with HBV, of whom about onemillion die each year from HBV-related liver cirrhosis, liver failure, and hepatocarcinoma(HCC). Approved therapies include interferon (IFN) and nucleoside analogues, which fail toachieve complete viral clearance besides being liable to provoke side effects such asdrug-resistance.To explore the antiviral efficacy of small interfering RNAs (siRNAs)/shRNAtargeting preC/C of HBV in human hepatoma cells Huh-7and HepG2.2.15cells.MethodsThree21nucleotide(nt)siRNAs for treating HBV preC/C gene were designed andsynthesized according to the HBV genome in GenBank accession numbers (U95551);simultaneously, one21-nt-long non-homologous siRNA was also designed randomly fornegative control. They were cloned into vector pU6for constructing shRNA-expressingplasmids pU6-C1, pU6-C2, pU6-C3and control pU6-C4. To assess the function of siRNAs, areporter gene system was constructed. The HBV preC/C gene was synthesized by PCR withpT-HBV1.3as the template. The preC/C gene was then inserted into the enhanced greenfluorescent protein expression vector (EGFP-N1) in order to construct the recombinantplasimid pEGFP-preC/C (E-C), which carries the EGFP reporter gene. The threeshRNA-expressing plasmids—pU6-C1, pU6-C2, or pU6-C3—was each then cotransfectedinto Huh-7cells along with either reporter gene expression vector E-C or the controls; orthese three plasmids—pU6-C1, pU6-C2, or pU6-C3—was each cotransfected intoHepG2.2.15cells along with the controls. First, upon determination of the number of cellsexhibiting EGFP expression in Huh-7cells as detected by an BH-2fluorescence microscopeand FACS-440flow cytometry at different times after cotransfection, the investigators evaluated the inhibitory efficiency of the three shRNA-expressing plasmids by an EGFPreporter system in cultured cells. Subsequently, the expression amount of HBsAg and HBeAgin HepG2.2.15cell supernatant at24h,48h,72h and96h post-cotransfection was detected byenzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect theexpression of HBsAg and HBcAg at72h post-cotransfection in HepG2.2.15cells. The copylevel of HBV mRNA transcripts cDNA in HepG2.2.15cells was further investigated throughquantitative real-time polymerase chain reaction (Real-time PCR).ResultsIn comparison with single plasmid transfection pEGFP-N1or E-C, fluorescencemicroscope examination and flow cytometry detection at48hours after cotransfectionindicated that the expression of the reporter gene EGFP in cotransfected group Huh-7cellinvolving pU6-C1, pU6-C2or pU6-C3resulted in an80%reduction in EGFP signal relativeto the controls (P <0.01). It was also found through immunofluorescence that the expressionof HBsAg and HBcAg in HepG2.2.15cells was reduced markedly (P＜0.01), that the copylevel of HBV mRNA transcripts cDNA as detected at48hours after cotransfection byquantitative real-time PCR was reduced respectively by73.9±1.2%(P=0.029；P＜0.05),48.2±1.8%and35.8±1.4%(P=0.037，0.040；P＜0.05)relative to the control, that it conformedwith that detected by fluorescence microscope/flow cytometry, ELISA, andimmunofluorescence (P＜0.01). Thereby further corroborating the antiviral efficacy of RNAi.The efficacy was obvious at48h, reaching a peak at72h.ConclusionFor the first time it has been found that RNAi induced by siRNA/shRNA targeting HBVpreC/C gene is effective and specific in inhibiting HBV replication and expression in humanhepatoma cells Huh-7and HepG2.2.15cells. Our data suggest that RNAi may provide aneffective, viable approach in gene therapy to treating major infectious diseases such asHBV/HCV/HIV infection.