摘要
目的:研究miR-29b对肝星状细胞(HSC)中TGF-βⅠ型受体(TGF-βⅠR)表达的调节作用。方法:生物信息学方法预测miR-29b靶基因为TGF-βⅠR。将靶基因3'端非编码区片段克隆至pMIR-re-porter载体构建重组质粒,然后和pRL-TK、miRNA前体共转染入HSC,检测荧光素酶的表达。将miRNA前体转染HSC后检测TGF-βⅠR的mRNA和蛋白质水平。结果:酶切和测序证实重组质粒构建成功;过表达miR-29b时,共转染pMIR-TGF-βⅠR质粒的细胞相对荧光素酶活性较相应过表达miR-NC组明显降低(P<0.05);而共转染pMIR-空载体质粒的细胞相对荧光素酶活性较相应过表达miR-NC组无显著差异(P>0.05);上调miR-29b水平后,TGF-βⅠR mRNA和蛋白的相对表达较转染miR-NC组差异有统计学意义(P<0.05)。结论:TGF-βⅠR是miR-29b的靶基因。miR-29b通过负向调控TGF-βⅠR表达而起抗肝纤维化作用。
Objective:To study the effect of miR -29b on the expression of TGF -β I R in rat HSC. Methods: Bioinformatics analysis indicated that TGF - βI R was a potential target gene of miR - 29b. The fragment of TGF -β I R mRNA 3' - UTR was cloned into pMIR - reporter luciferase vector. Recombinant plasmid, pRL - TK and miRNA precursors were co - transfected into HSC. The luciferase andβ - galactosidase activity was detected by using dual - light luciferase assay. Real - time RT - PCR and Western blot were performed to examine the effect of miR - 29b on the expression of TGF - βI R. Results :The recombinant plasmid was confirmed by restriction enzyme diges- tion and sequencing. As compared with miR - NC mimics, miR - 29b precursors reduced the relative luciferase ac- tivity in ceils co- transfected with pMIR -TGF- β I R vector( P 〈 0.05 ). In contrast, there is no change in the relative luciferase activity in cells co - transfected with pMIR - Luc vector( P 〉 0.05 ). Overexpression of miR - 29b dramatically suppressed the expression of TGF - β 1 R at mRNA and protein levels in HSC (P 〈 0.05 ). Conclu- sion: TGF - β I R is miR - 29b target gene. MiR - 29b can act as a antifibrotic agent by negatively regulating the expression of TGF- β I R in HSC.
出处
《中国卫生检验杂志》
北大核心
2013年第2期268-271,共4页
Chinese Journal of Health Laboratory Technology
基金
国家自然科学基金(81000176
81100292)
浙江省自然科学基金(Y2110634
Y2090326)
王宝恩肝纤维化研究基金(20100002)
温州市科技局(Y20110033)