Xiaoman Liu1, Zhuo Yang, Tao Feng. 1. Department of Biochemistry and Molecular Biology, College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China. lxm1926@hotmail.com
Abstract
OBJECTIVE: We used a minicircle DNA vector system to express small interfering RNA (siRNA) and studied the inhibition of hepatitis B virus (HBV) replication and gene expression in vitro. METHODS: siRNA targeting HBV S gene (siHBS) was designed , synthesized and cloned into a minicircle DNA vector pMC. BESPX-MCS2. After sequencing, we transformed the recombinant pMC-H1-siHBS-U6 into E. coli ZYCY10P3S2T, and induced the degradation of its bacterial backbone by adding L-arabinose into the bacterial growth medium. As expected, a minicircle RNA interference (RNAi) vector pmc-H1-siHBS-U6 was generated only consisting of gene expression cassette. Then pmc-H1-siHBS-U6 was co-transfected into Huh-7 cells with HBV expression vector pHBV1.3. ELISA and Real-time PCR were performed to evaluate the inhibition effect of the secretion of HBsAg and HBeAg and the levels of HBV DNA and mRNA in Huh-7 cells. RESULTS: We Successfully established the minicircle-based RNAi vector pmc-H1-siHBS-U6, which can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells for two to three weeks. Real-time PCR results show that HBV DNA and mRNA levels were also down-regulated about 71% and 80%. CONCLUSION: The minicircle DNA-based RNAi vector pmc-H1-siHBS-U6 can suppress HBV replication and gene expression specifically, efficiently and steadily. Thus, this study provided us a new siRNA delivery system and a new gene therapy strategy of HBV infection.
OBJECTIVE: We used a minicircle DNA vector system to express small interfering RNA (siRNA) and studied the inhibition of hepatitis B virus (HBV) replication and gene expression in vitro. METHODS: siRNA targeting HBV S gene (siHBS) was designed , synthesized and cloned into a minicircle DNA vector pMC. BESPX-MCS2. After sequencing, we transformed the recombinant pMC-H1-siHBS-U6 into E. coli ZYCY10P3S2T, and induced the degradation of its bacterial backbone by adding L-arabinose into the bacterial growth medium. As expected, a minicircle RNA interference (RNAi) vector pmc-H1-siHBS-U6 was generated only consisting of gene expression cassette. Then pmc-H1-siHBS-U6 was co-transfected into Huh-7 cells with HBV expression vector pHBV1.3. ELISA and Real-time PCR were performed to evaluate the inhibition effect of the secretion of HBsAg and HBeAg and the levels of HBV DNA and mRNA in Huh-7 cells. RESULTS: We Successfully established the minicircle-based RNAi vector pmc-H1-siHBS-U6, which can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells for two to three weeks. Real-time PCR results show that HBV DNA and mRNA levels were also down-regulated about 71% and 80%. CONCLUSION: The minicircle DNA-based RNAi vector pmc-H1-siHBS-U6 can suppress HBV replication and gene expression specifically, efficiently and steadily. Thus, this study provided us a new siRNA delivery system and a new gene therapy strategy of HBV infection.
Authors: Sofia Stenler; Oscar Pb Wiklander; Maria Badal-Tejedor; Janne Turunen; Joel Z Nordin; David Hallengärd; Britta Wahren; Samir El Andaloussi; Mark W Rutland; C I Edvard Smith; Karin E Lundin; Pontus Blomberg Journal: Mol Ther Nucleic Acids Date: 2014-01-07 Impact factor: 10.183