The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-strande...The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-stranded RNA.It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.Since hepatitis G virus(HGV)genome is a positive-sense single-stranded RNA,the replication of HGV does not lead to an integrated DNA genome,suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells.The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus(ECMV)internal ribosome entry site(IRES)was constructed and transfected into human hepatoma cell Huh-7.The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods.Two specific HGV E2 siRNAs(1-E2siRNA,2-E2 siRNA)synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells.With the analyses of Western blot and the formation of hygromycin-resistant colonies,the inhibitions of expression of HGV structural protein by two HGV E2siRNAs were detected and found lasting at least one week.The inhibition of 2-E2 siRNA was stronger and only 1%of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies.These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells.In conclusion,the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist,in mammalian cells.展开更多
Lean body mass （LBM） and age at menarche （AAM） are two important complex traits for human health. The aim of this study was to identify pleiotropic genes for both traits using a powerful bivariate genome-wide asso...Lean body mass （LBM） and age at menarche （AAM） are two important complex traits for human health. The aim of this study was to identify pleiotropic genes for both traits using a powerful bivariate genome-wide association study （GWAS）. Two stud- ies, a discovery study and a replication study, were performed. In the discovery study, 909622 single nucleotide polymor- phisms （SNPs） were genotyped in 801 unrelated female Han Chinese subjects using the Affymetrix human genome-wide SNP array 6.0 platform. Then, a bivariate GWAS was performed to identify the SNPs that may be important for LBM and AAM. In the replication study, significant findings from the discovery study were validated in 1692 unrelated Caucasian female subjects One SNP rs3027009 that was bivafiately associated with left arm lean mass and AAM in the discovery samples （P=7.26x10-6） and in the replication samples （P=0.005） was identified. The SNP is located at the upstream of DARC （Duffy antigen receptor for chemokines） gene, suggesting that DARC may play an important role in regulating the metabolisms of both LBM and AAM.展开更多
基金This work was supported by the National Basic Research(973)Progrum of China(No.2003CB514129)the National INatural Science Foundation of China(Grant No.30170514).
文摘The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-stranded RNA.It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.Since hepatitis G virus(HGV)genome is a positive-sense single-stranded RNA,the replication of HGV does not lead to an integrated DNA genome,suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells.The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus(ECMV)internal ribosome entry site(IRES)was constructed and transfected into human hepatoma cell Huh-7.The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods.Two specific HGV E2 siRNAs(1-E2siRNA,2-E2 siRNA)synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells.With the analyses of Western blot and the formation of hygromycin-resistant colonies,the inhibitions of expression of HGV structural protein by two HGV E2siRNAs were detected and found lasting at least one week.The inhibition of 2-E2 siRNA was stronger and only 1%of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies.These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells.In conclusion,the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist,in mammalian cells.
基金supported by the Shanghai Leading Academic Discipline Project(Grant No.S30501)a start-up fund from the Shanghai University of Science and Technology,China+1 种基金supported by grants from National Institutes of Health(Grant Nos.P50AR055081, R01AG026564,R01AR050496,RC2DE020756,R01AR057049,and R03TW008221)supported by the National Natural Science Foundation of China(Grant No.31100902)
文摘Lean body mass （LBM） and age at menarche （AAM） are two important complex traits for human health. The aim of this study was to identify pleiotropic genes for both traits using a powerful bivariate genome-wide association study （GWAS）. Two stud- ies, a discovery study and a replication study, were performed. In the discovery study, 909622 single nucleotide polymor- phisms （SNPs） were genotyped in 801 unrelated female Han Chinese subjects using the Affymetrix human genome-wide SNP array 6.0 platform. Then, a bivariate GWAS was performed to identify the SNPs that may be important for LBM and AAM. In the replication study, significant findings from the discovery study were validated in 1692 unrelated Caucasian female subjects One SNP rs3027009 that was bivafiately associated with left arm lean mass and AAM in the discovery samples （P=7.26x10-6） and in the replication samples （P=0.005） was identified. The SNP is located at the upstream of DARC （Duffy antigen receptor for chemokines） gene, suggesting that DARC may play an important role in regulating the metabolisms of both LBM and AAM.
