目的观察miRNA-146a对血管平滑肌细胞增殖的作用并研究其机制。方法原代培养大鼠血管平滑肌细胞,分成抑制组、对照组和正常组,采用脂质体2000分别转染miRNA-146a抑制剂(50nmol/L)、错义链(50nmol/L)、PBS,使用real time PCR方法测定转染...目的观察miRNA-146a对血管平滑肌细胞增殖的作用并研究其机制。方法原代培养大鼠血管平滑肌细胞,分成抑制组、对照组和正常组,采用脂质体2000分别转染miRNA-146a抑制剂(50nmol/L)、错义链(50nmol/L)、PBS,使用real time PCR方法测定转染后miRNA-146a水平,CCK8法检测转染后血管平滑肌细胞增殖,transwell法检测转染后血管平滑肌细胞迁移程度,western blot检测转染后血管平滑肌细胞核因子κBp65(NF-κBp65)与增殖细胞核抗原蛋白(PCNA)水平。结果转染48 h后,抑制组血管平滑肌细胞的miRNA-146a水平明显低于对照组和正常组(P<0.01);其增殖和迁移比例显著低于对照组和正常组组(P<0.01);其NF-κBp65、PCNA蛋白表达水平降低(P<0.05)。结论 miRNA-146a可以促进血管平滑肌细胞的增殖和迁移,其机制与增加NF-κBp65表达相关。展开更多
Post-transcriptional gene silencing mediated by microRNAs(miRNAs)modulates numerous developmental and stress response pathways.For the last two decades,HASTY(HST),the ortholog of human EXPORTIN 5,was considered to be ...Post-transcriptional gene silencing mediated by microRNAs(miRNAs)modulates numerous developmental and stress response pathways.For the last two decades,HASTY(HST),the ortholog of human EXPORTIN 5,was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm.Here,we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis.We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs.Interestingly,protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain.Moreover,mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37.Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex,which in turn promotes the transcription and proper processing of primary miRNA transcripts(primiRNAs).Taken together,these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes,promoting the transcription and processing of primiRNAs rather than the direct export of processed miRNAs from the nucleus.展开更多
基金This work was supported by grants from the ANPCyT(Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica,Argentina)the HFSP(Human Frontier Science Program)+1 种基金the ICGEB(International Center for Genetic Engineering and Biotechnology)to P.A.M.and from the Max Planck Society to P.A.M.and D.W.P.A.M.and A.L.A.are members of CONICETD.A.C.,A.J.G.,and D.G.are fellows of the same institution.
文摘Post-transcriptional gene silencing mediated by microRNAs(miRNAs)modulates numerous developmental and stress response pathways.For the last two decades,HASTY(HST),the ortholog of human EXPORTIN 5,was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm.Here,we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis.We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs.Interestingly,protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain.Moreover,mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37.Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex,which in turn promotes the transcription and proper processing of primary miRNA transcripts(primiRNAs).Taken together,these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes,promoting the transcription and processing of primiRNAs rather than the direct export of processed miRNAs from the nucleus.
