基于microRNA的RNA干扰抗乙型肝炎病毒的实验研究
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摘要
乙肝病毒感染是危害极大的全球性公共卫生问题,全世界约3.5亿感染者,每年死于乙肝及其并发症的人数超过100万人,目前仍缺乏有效的治疗药物。
RNA干扰技术的应用使人们在抗病毒治疗领域有了新的发现,现已显示出潜在的应用前景。microRNA在小分子RNA研究中扮演着重要角色,可以通过与靶mRNA完全互补产生特异性切割或与3’UTR区非特异互补而使翻译过程受到抑制,从而达到沉默靶基因的目的。
本研究利用RNA pol II启动子产生的miR155框架体系,构建针对HBV S区,X区的内源性miRNA,并经分子克隆方法产生针对S区的双靶点串联表达载体,体外转染hepG2.2.15细胞,结果表明对HBV的复制与表达均显示抑制作用。为体内研究奠定了基础。
Infection of Hepatitis B virus (HBV) is a severely world-wide public health problem. With more than 350 million people infected, Hepatitis B is widely prevalent and severely. Each year about 1 million deaths are commonly associated with cirrhosis, severe hepatitis or hepatocellular carcinoma (HCC).Although hepatitis B vaccines have been used for many years worldwide, HBV infection is still a big challenge to many countries and there is no ideal agent in the field of anti-HBV. Although recently some effective antiviral drugs have been developed, all of the treatments have their own shortages. Until now, interferon-αand nucleoside analogs, such as Iamivudine, are of the few drugs capable of inhibiting HBV replication. However, The recombinant interferons are suitable to few cases with HBV infection, and the low response efficiencies of patients with IFN in virology result in their limited usage. In addition lamivudine has been shown to have a inhibition of HBV replication, but the treatment may be failed due to the drug resistance causing by the escaping mutants with prolonged treatment. So, the need for new effective anti-HBV therapies is pressing.
After antisensenucleic acids and ribozyme technology, RNA interference (RNAi) is a new gene therapy tool which play a important role in researching of virus, cancer, and genome function. It has high specificity and efficacy, by which double-stranded directs sequence-specific post-transcriptional silencing of homologous genes and the effective molecular is siRNA or miRNA. MicroRNAs (miRNAs) are an abundant class of small single-stranded non-coding RNAs (19-30 nucleotides long) that serve widespread functions in post-transcriptional gene silencing, induce the sequence-specific degradation of homologous messenger RNA (mRNA).
The HBV genome is a partially double-stranded DNA molecule which has 3.2kb and replicates by reverse transcription of a pre-genomic RNA intermediate in a manner similar to that of the retroviruses. HBV genome overlaps obviously so that the synthesis of HBV proteins could be effectively inhibited by cutting its mRNA and pregenomic RNA on proper site.
HepG2.2.15 cell line is a derivative of the human HepG2 hepatoma cell line, contains integrated HBV ayw DNA. This cell line can produce HBV mRNAs, antigens and viral particles and serve as a satisfactory in vitro model for investigation of HBV infection.
Because polⅢis limited in application for its tissue specificity expression, we utilized pcDNA6.2-GW/EmGFP-miR vector which contain polⅡpromoter and express endogenous miRNA.
In the present study, we developed miRNAs that specifically target the S antigen and X gene of HBV and explored the inhibitory effect of miRNAs on HBV replication and expression. The aim of this study was to provide some evidences for anti-HBV therapy by miRNA.
Based on the sequence of HBV in GenBank two miRNAs were synthesized and cloned into pcDNA6.2-GW/EmGFP-miR. At the same time, unrelated miRNA was designed as control. After the construction of the recombinant expression vectors was confirmed by enzyme digestion, electrophoresis and gene sequencing, transfection into HepG 2.2.15 cells was carried out by liposome.
The expression of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells was assayed by enzyme-linked immunosorbentassay ( ELISA), the HBV DNA in the supernatant of HepG 2.2.15 cells was examined by Fluorescence Quantitative PCR(FC-PCR). The inhibitory effect of HBV gene expression and replication by the miRNAs was observed with different concentration of miRNAs and at the different time point post-transfection.
The results are as following:
1. One day after HepG 2.2.15 cells plating, HBsAg and HBeAg could be detected in cell culture media. The amount of HBsAg and HBeAg secretion increased with plating time prolonging.
2. The expression vectors were transfected into HepG2 2.2.15 cells. The expression of HBsAg and HBeAg in the supernatant was inhibited at the different time point post-transfection. The peak of inhibitory effect was at 48h.
3. The expression vectors were transfected into HepG 2.2.15 cells and inhibited the replication of HBV-DNA in the supernatant at the different time point post-transfection. The peak of inhibitory effect was at 48h.
In summary, HBV-specific miRNA and exhibited high efficiency and specificity to suppress HBV gene replication and expression in HepG2.2.15 cells. Also, our results indicated that the anti-HBV effects of miRNAs were sequence- and dose-dependent.
RNA干扰技术的应用使人们在抗病毒治疗领域有了新的发现,现已显示出潜在的应用前景。microRNA在小分子RNA研究中扮演着重要角色,可以通过与靶mRNA完全互补产生特异性切割或与3’UTR区非特异互补而使翻译过程受到抑制,从而达到沉默靶基因的目的。
本研究利用RNA pol II启动子产生的miR155框架体系,构建针对HBV S区,X区的内源性miRNA,并经分子克隆方法产生针对S区的双靶点串联表达载体,体外转染hepG2.2.15细胞,结果表明对HBV的复制与表达均显示抑制作用。为体内研究奠定了基础。
Infection of Hepatitis B virus (HBV) is a severely world-wide public health problem. With more than 350 million people infected, Hepatitis B is widely prevalent and severely. Each year about 1 million deaths are commonly associated with cirrhosis, severe hepatitis or hepatocellular carcinoma (HCC).Although hepatitis B vaccines have been used for many years worldwide, HBV infection is still a big challenge to many countries and there is no ideal agent in the field of anti-HBV. Although recently some effective antiviral drugs have been developed, all of the treatments have their own shortages. Until now, interferon-αand nucleoside analogs, such as Iamivudine, are of the few drugs capable of inhibiting HBV replication. However, The recombinant interferons are suitable to few cases with HBV infection, and the low response efficiencies of patients with IFN in virology result in their limited usage. In addition lamivudine has been shown to have a inhibition of HBV replication, but the treatment may be failed due to the drug resistance causing by the escaping mutants with prolonged treatment. So, the need for new effective anti-HBV therapies is pressing.
After antisensenucleic acids and ribozyme technology, RNA interference (RNAi) is a new gene therapy tool which play a important role in researching of virus, cancer, and genome function. It has high specificity and efficacy, by which double-stranded directs sequence-specific post-transcriptional silencing of homologous genes and the effective molecular is siRNA or miRNA. MicroRNAs (miRNAs) are an abundant class of small single-stranded non-coding RNAs (19-30 nucleotides long) that serve widespread functions in post-transcriptional gene silencing, induce the sequence-specific degradation of homologous messenger RNA (mRNA).
The HBV genome is a partially double-stranded DNA molecule which has 3.2kb and replicates by reverse transcription of a pre-genomic RNA intermediate in a manner similar to that of the retroviruses. HBV genome overlaps obviously so that the synthesis of HBV proteins could be effectively inhibited by cutting its mRNA and pregenomic RNA on proper site.
HepG2.2.15 cell line is a derivative of the human HepG2 hepatoma cell line, contains integrated HBV ayw DNA. This cell line can produce HBV mRNAs, antigens and viral particles and serve as a satisfactory in vitro model for investigation of HBV infection.
Because polⅢis limited in application for its tissue specificity expression, we utilized pcDNA6.2-GW/EmGFP-miR vector which contain polⅡpromoter and express endogenous miRNA.
In the present study, we developed miRNAs that specifically target the S antigen and X gene of HBV and explored the inhibitory effect of miRNAs on HBV replication and expression. The aim of this study was to provide some evidences for anti-HBV therapy by miRNA.
Based on the sequence of HBV in GenBank two miRNAs were synthesized and cloned into pcDNA6.2-GW/EmGFP-miR. At the same time, unrelated miRNA was designed as control. After the construction of the recombinant expression vectors was confirmed by enzyme digestion, electrophoresis and gene sequencing, transfection into HepG 2.2.15 cells was carried out by liposome.
The expression of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells was assayed by enzyme-linked immunosorbentassay ( ELISA), the HBV DNA in the supernatant of HepG 2.2.15 cells was examined by Fluorescence Quantitative PCR(FC-PCR). The inhibitory effect of HBV gene expression and replication by the miRNAs was observed with different concentration of miRNAs and at the different time point post-transfection.
The results are as following:
1. One day after HepG 2.2.15 cells plating, HBsAg and HBeAg could be detected in cell culture media. The amount of HBsAg and HBeAg secretion increased with plating time prolonging.
2. The expression vectors were transfected into HepG2 2.2.15 cells. The expression of HBsAg and HBeAg in the supernatant was inhibited at the different time point post-transfection. The peak of inhibitory effect was at 48h.
3. The expression vectors were transfected into HepG 2.2.15 cells and inhibited the replication of HBV-DNA in the supernatant at the different time point post-transfection. The peak of inhibitory effect was at 48h.
In summary, HBV-specific miRNA and exhibited high efficiency and specificity to suppress HBV gene replication and expression in HepG2.2.15 cells. Also, our results indicated that the anti-HBV effects of miRNAs were sequence- and dose-dependent.
引文
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