Background RNA interference （RNAi） is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs （siRNAs） have sufficiently inhibited hepatit...Background RNA interference （RNAi） is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs （siRNAs） have sufficiently inhibited hepatitis B virus （HBV） replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. Methods A mutant RNAi vector （pSI-C mut） with two base pairs different from the original target gene sequence at the RNAi vector （pSI-C） was constructed according to the method described in this study, A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload, pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein （GFP） gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay （ELISA） assay was used to measure the concentration of HBV surface antigen （HBsAg） in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein （HBcAg） in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR （RT-PCR） analysis. Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection （ p. i. ）, the OD values were shown to be 5.07 ± 1.07 in infecting group and 0.62 ± 0. 59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group （ P 〈 0. 01 ）. Liver intracellular synthesis of viral core protein was sharply reduced to 0. 9% ±0. 1%, compared with 5.4% ± 1.2% of positive hepatocytes in infecting group （P 〈0. 01 ）, and the transcriptional level of HBV C mRNA was greatly reduced by 84. 7%. However, the irrelevant RNAi control plasmid （pSIGFP）, and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. Conclusion pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.展开更多
Objectives To construct a hepatoma directed gene delivery system which could transfer preS2 antisense RNA to liver cancer cells specifically, and to explore a new therapeutic strategy for hepatocellular carcinoma by ...Objectives To construct a hepatoma directed gene delivery system which could transfer preS2 antisense RNA to liver cancer cells specifically, and to explore a new therapeutic strategy for hepatocellular carcinoma by blocking hepatitis B virus (HBV) with antisense RNA targeting hepatocellular carcinoma. Methods GE7 and HA20 were synthesized and mixed with pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system named AFP-enhancing 4-element complex. Nude mice bearing hepatocelluar carcinoma cells HepG2.2.15 were injected with AFP-enhancing 4-element complex via a tail vein. Total RNA from tissues was extracted, and reversal transcription-ploymerase chain reaction (RT-PCR) was used to detect the expression of preS2. Different doses of AFP-enhancing 4-element complex was injected into nude mice at different time points, and tumor diameter was measured.Results AFP-enhancing 4-element complex was constructed successfully. RT-PCR showed preS2 antisense RNA delivered by AFP-enhancing 4-element complex only expressed in liver tumor HepG2.2.15 cells of the mice. After the treatment of AFP-enhancing 4-element complex with dose of 0.2 μg per mouse (once a week for 4 weeks), the mean tumor diameter of nude mice was significantly shorter than that of the control groups (0.995±0.35 cm vs 2.125±0.25 cm, P<0.01). Conclusions An HBV antisense RNA gene delivery system targeting hepatocellular carcinoma, AFP-enhancing 4-element complex, was constructed successfully. PreS2 antisense RNA expressed specifically in hepatocelluar carcinoma cells significantly inhibits tumor growth of mice bearing hepatocarcinoma HepG2.2.15 and may have therapeutic potential in HBV related hepatocarcinoma.展开更多
文摘Background RNA interference （RNAi） is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs （siRNAs） have sufficiently inhibited hepatitis B virus （HBV） replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. Methods A mutant RNAi vector （pSI-C mut） with two base pairs different from the original target gene sequence at the RNAi vector （pSI-C） was constructed according to the method described in this study, A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload, pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein （GFP） gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay （ELISA） assay was used to measure the concentration of HBV surface antigen （HBsAg） in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein （HBcAg） in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR （RT-PCR） analysis. Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection （ p. i. ）, the OD values were shown to be 5.07 ± 1.07 in infecting group and 0.62 ± 0. 59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group （ P 〈 0. 01 ）. Liver intracellular synthesis of viral core protein was sharply reduced to 0. 9% ±0. 1%, compared with 5.4% ± 1.2% of positive hepatocytes in infecting group （P 〈0. 01 ）, and the transcriptional level of HBV C mRNA was greatly reduced by 84. 7%. However, the irrelevant RNAi control plasmid （pSIGFP）, and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. Conclusion pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.
基金ThisstudywassupportedbyagrantfromtheNationalNaturalScienceFoundationofChina (No 39970 333)
文摘Objectives To construct a hepatoma directed gene delivery system which could transfer preS2 antisense RNA to liver cancer cells specifically, and to explore a new therapeutic strategy for hepatocellular carcinoma by blocking hepatitis B virus (HBV) with antisense RNA targeting hepatocellular carcinoma. Methods GE7 and HA20 were synthesized and mixed with pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system named AFP-enhancing 4-element complex. Nude mice bearing hepatocelluar carcinoma cells HepG2.2.15 were injected with AFP-enhancing 4-element complex via a tail vein. Total RNA from tissues was extracted, and reversal transcription-ploymerase chain reaction (RT-PCR) was used to detect the expression of preS2. Different doses of AFP-enhancing 4-element complex was injected into nude mice at different time points, and tumor diameter was measured.Results AFP-enhancing 4-element complex was constructed successfully. RT-PCR showed preS2 antisense RNA delivered by AFP-enhancing 4-element complex only expressed in liver tumor HepG2.2.15 cells of the mice. After the treatment of AFP-enhancing 4-element complex with dose of 0.2 μg per mouse (once a week for 4 weeks), the mean tumor diameter of nude mice was significantly shorter than that of the control groups (0.995±0.35 cm vs 2.125±0.25 cm, P<0.01). Conclusions An HBV antisense RNA gene delivery system targeting hepatocellular carcinoma, AFP-enhancing 4-element complex, was constructed successfully. PreS2 antisense RNA expressed specifically in hepatocelluar carcinoma cells significantly inhibits tumor growth of mice bearing hepatocarcinoma HepG2.2.15 and may have therapeutic potential in HBV related hepatocarcinoma.
