Research Paper Acta Microbiologica Sinica 52 2 191-197 4 February 2012 ISSN 0001-6209 CN 11-1995 /Q http / /journals. im. ac. cn /actamicrocn sirna 1 2 1* 1 400016 2 100084 RNA small interfering RNA sirna hepatitis B virus HBV HBV S sirna pmc. BESPX-MCS2 pmc-h1-sihbs-u6 E. coli ZYCY10P3S2T L- RNA pmc-h1-sihbs-u6 RNA HBV phbv1. 3 Huh-7 1-7 ELISA Huh-7 HBsAg HBeAg Real-time RT-PCR RNA HBV DNA mrna HBV S sirna pmc-h1-sihbs-u6 Huh-7 HBsAg HBeAg 2-3 Real-time PCR HBV DNA mrna 71% 80% sirna HBV RNA HBV HBV RNA hepatitis B virus HBV RNA Huh-7 Q933 A 0001-6209 2012 02-0191-07 hepatitis B virus HBV RNA HBV RNAi HBV RNA pregenomic RNA pgrna α- sirna HBV RNA RNA mrna interference RNAi post-transcriptiona1 gene silencing RNA 1 - PTGS 2 HBV RNA HBV 81071770 * Tel + 86-23-68485627 E-mail fengtao9_9@ yahoo. com 1986 - E-mail lxm1926@ hotmail. com 2011-09-05 2011-12-14
192 Xiaoman Liu et al. /Acta Microbiologica Sinica 2012 52 2 3 RNA Escherichia coli DH5α Huh-7 RNA 1. 1. 2 Huh-7 DMEM 10% FBS 100 μg /ml 4 100 U /ml 37 5% CO 2 TRIzol Lipofectamine 2000 5-6 Invitrogen Opti-MEM GIBCO Thermo PCR T4 7-10 DNA DNA Marker TaKaRa DNA Qiagen 11 Vigorous Real-time PCR 12 Promega L- HBV Qiagen DNA Tanon1600 Bio-RAD Real-time PCR ABI 7300 PCR 13-19 RNA HBV 1. 2 HBV S sirna 1 1. 1 1. 1. 1 pmc. RNAi 1 BESPX-MCS2 pmc sirna sihbs ZYCY10P3S2T E. coli SBI H1 1. 2. 1 HBS sirna HBV1. 3 sirna HBV S BLAST HBV HBV 673-697 S Invitrogen HBV EGFP siegfp 20 - U6 psuper-u6 21 1 sirna 1 Table 1 sirna Restriction site Sequences 5 3 sihbs siegfp The Sequences of sihbs and siegfp sihbs BglⅡ Forward sequence GATCAAAAACTCAGTTTACTAGTGCCATTTG TTCTTCAAGAGAG AACAAATGGCACTAGTAAACTGAGTTTTT SalⅠ Reverse sequence TCGAAAAAACTCAGTTTACTAGTGCCATTTG TTCTCTCTTGAAGAACAAATGGCACTAGTAAACTGAGTTTTT siegfp BglⅡ Forward sequence GATCAAAAAAAGAACGGCATCAAGGTGAA CTTCAAGAGAGTTCACCTTGATGCCGTTCTTTTTTT SalⅠ Reverse sequence TCGAAAAAAAAGAACGGCATCAAGGTGAA CTCTCTTGAAGTTCACCTTGATGCCGTTCTTTTTTT 1. 2. 2 sirna psuper-u6 psuper-u6 5' BglⅡ SalI psuper-sihbs-u6 psuper-siegfp-u6 T4 DNA DH5a Spe Ⅰ
sirna. / 2012 52 2 193 NaOH 10 ml 20% L- 3 ± SD 1% 30 250 rpm /min 5 h P < 0. 05 RNA pmc-h1-sihbs-u6 pmc-h1-siegfp- U6 pmc-h1-u6 2 1. 4 RNA Huh-7 Huh-7 12 2. 1 sirna psuper-sihbs / siegfp-u6 37 5% CO 2 70% - 90% opti-mem pmc-h1-sihbs-u6 sirna 1 psuper-u6 0. 5 ng 1 ng 2 ng phbv1. 3 0. 5 ng SpeⅠ EcoRI 1 1 2 1 4 1 600 bp Huh-7 37 5% CO 2 520 bp 6 h 10% DMEM 2 37 5% CO 2 psuper-u6 EcoRI 600 bp sirna pmc-h1-520 bp sihbs siegfp-u6 pmc-h1-u6 3 siegfp DNA 1. 2. 3 psuper-u6 24-168 h 7 d pmc SpeⅠ - 80 EcoRI H1-siHBS-U6 H1-siEGFP-U6 ELISA H1-U6 psuper-u6 RNA pmc 1. 6 HBV DNA pmc-h1-sihbs-u6 pmc-h1-siegfp-u6 pmc-h1-u6 1. 3 sirna RNA 24 h 48 h 72 h HBV DNA copies /ml RNA 1. 7 HBV mrna DNA Trizol Invitrogen 72h RNA cdna cdna 100 μl 5 ml ABI7300 PCR 50 μg /ml LB 37 250 rpm / HBS β-actin. PCR 95 10 min min 6-8 h 1 1000 94 10 s 60 60 s 40 CT 100 ml 50 μg /ml TB 37 250 rpm /min 16-18 hod 600 4-5 100 ml LB 4 ml 1N 1. 5 HBsAg HBeAg HBsAg HBeAg QIAGEN HBV ABI7300 PCR 2 - CT 100% 1. 8 SPSS10. 0 1 sihbs Fig. 1 The oligonucleotides sihbs1 top and sihbs2 below annealed to generate double-stranded sihbs.
194 Xiaoman Liu et al. /Acta Microbiologica Sinica 2012 52 2 DNA HBV S sirna 2 psuper -sihbs /EGFP-U6 SpeI EcoRI Fig. 2 psuper-sihbs /siegfp /-U6 was digested by SpeI and EcoRI. lane M. TaKaRa DL10 000 DNA marker lane 1. psuper -sihbs-u6 lane 2. psuper-siegfp-u6 lane 3. psuper-u6 as the empty vector control. 2. 2 RNA H1-siHBS-U6 H1-siEGFP-U6 3 H1-U6 pmc RNA RNA pmc-h1-sihbs-u6 3-A 600 bp 2. 3 HBsAg HBeAg pmc-h1-sihbs-u6 phbv1. 3 1 1 2 1 4 1 Huh-7 ELISA RNA Fig. 3 The construction generation and identification of Minicircle RNAi vector A. Diagram of production process of a minicircle DNA vector. B. pmc-h1-sihbs-u6 was digested by Bgl Ⅱ and EcoNI restriction enzymes before electrophoresis. lane M. TaKaRa DL10 000 DNA maker. lane +. pmc-h1-sihbs-u6 with Bgl Ⅱ and DNA EcoNI restriction enzymes. lane. pmc-h1-sihbs-u6 without BglII and EcoNI restriction enzymes. 4 sirna BglⅡ DNA EcoNI 2. 4 sirna HBV-DNA 3-B pmc-h1-sihbs-u6 QIAGEN HBV HBV-DNA ABI7300 PCR 24 h 48 h HBsAg HBeAg 72 h HBV DNA HBsAg HBeAg pmc-h1-sihbs-u6 HBV- pmc-h1-sihbs-u6 4 1 DNA 24 h 48 h 72 h 72 h 64% 67% 71% HBsAg HBeAg P < 0. 05 72. 5% 89% 168 h pmc-h1-siegfp-u6 pmc- H1-U6 2 sirna HBV- P < 0. 05 DNA pmc-h1-siegfp-u6 pmc-h1-u6
sirna. / 2012 52 2 195 CT 2 - CT pmc-h1-sihbs-u6 Huh-7 HBV mrna 72 h 80% 5 P < 0. 05 pmc- H1-siEGFP-U6 pmc-h1-u6 5 sirna HBV mrna 5 Real-time RT-PCR sirna 4 ELISA HBV S mrna sirna HBsAg A HBeAg B Fig. 5 Inhibition of HBV S gene mrna by RNAi based on Minicircle DNA vector delivery in Huh-7 cell pools. The amount of HBV S gene Fig. 4 Inhibition of HBsAg A and HBeAg B by RNAi mrna was measured by Real-time RT-PCR. The data shown represent based on Minicircle DNA vector delivery in Huh-7 cell the mean values ± S. D. based on three independent experiments. pools. The amount of HBsAg and HBeAg in the culture Compared with control groups *** P < 0. 001. medium was measured in 1-7 days by ELISA. The data shown represent the mean values ± S. D. based on three 3 independent experiments. Compared with control groups * P < 0. 05 ** P < 0. 02 *** P < 0. 001. DNA minicircle DNA 1997 2 Real-time PCR sirna Huh-7 HBV DNA DNA Table 2 Inhibition of HBV DNA by RNAi based on Minicircle DNA vector delivery in Huh-7 cell pools. The amount of HBV DNA was measured by Real-time PCR. The data shown represent CpG the mean values ± S. D. based on three independent DNA experiments. 10 6 copies /ml ± SD Species Number 24h 48h 72h pmc-h1-siegfp-u6 3 5. 33 ± 0. 8 * 7. 86 ± 0. 9 10. 56 ± 1. 2 pmc-h1u6 3 5. 18 ± 0. 5 7. 57 ± 0. 3 11. 34 ± 1. 3 pmc-h1-sihbs-u6 3 1. 88 ± 1. 1 ** 2. 57 ± 0. 9 ** 3. 16 ± 0. 4 *** Chen * 10 6 copies /ml ± SD Compared with control groups ** P < 0. 02 P < 0. 001. Kay 2003 14 2005 22 2010 23 2. 5 sirna Huh-7 HBV mrna ΦC31 pmc-h1-sihbs-u6 72h I-SceI ΦC31 RNA cdna HBS I-SceI β-actin ABI7300 PCR
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sirna. / 2012 52 2 197 18 Vaysse L Gregory LG Harbottle RP Perouzel E Tolmachov O Coutelle C. Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo. Journal of Gene Medical 2006 8 6 754-763. 19 Wu J Xiao X Zhao P Xue G Zhu X Zheng L Zeng Y Huang W. Minicircle-IFNgamma induces antiproliferative and antitumoral effects in human nasopharyngeal carcinoma. Clinical Cancer Research 2006 12 4702-4713. 20 Mitaqishi M Taira K. U6-promoter-driven sirnas with four uridine 3 overhangs efficiently suppress targeted gene expression in mammalian cells J. Nature Biotechnology 2002 20 5 497-500. 21 Brummelkamp TR Bernards R Aqami R. A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science 2002 296 5567 550-553 22 Chen ZY He CY Kay MA. Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Human Gene Therapy 2005 16 1 126-131. 23 Kay MA He CY Chen ZY. A robust system for production of minicircle DNA vectors. Nature Biotechnology 2010 28 12 1287-1289. 24 Tang H Mclachan A. Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is critical determinant of viral tropism. Proceedings of the National Academy of Sciences of the United States of America 2001 98 4 1841-1846. A minicircle DNA vector-mediated sirna to stably suppress hepatitis B virus replication and expression Xiaoman Liu 1 Zhuo Yang 2 Tao Feng 1* 1 Department of Biochemistry and Molecular Biology College of Basic Medicine Chongqing Medical University Chongqing 400016 China 2 School of Life Sciences Tsinghua University Beijing 100084 China 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 DNAbased 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. Keywords minicircle DNA vector hepatitis B virus HBV sirna Huh-7 cell Supported by the National Science Foundation of China 81071770 * Corresponding author. Tel + 86-23-68485627 E-mail fengtao9_9@ yahoo. com Received 5 September 2011 /Revised 14 December 2011