Let-7a-5p靶向整合素αV调节气道平滑肌细胞转化生长因子β1表达和活化

Let-7a-5p targets integrinαV to regulate TGF-β1 expression and activation in airway smooth muscle cells

为探究微小RNA(microRNA,miRNA)let-7a-5p对气道平滑肌细胞(airway smooth muscle cells,ASMCs)转化生长因子β1(transformation growth factorβ1,TGFβ1)表达和活化的影响,本研究使用生物信息学方法预测let-7a-5p靶基因,构建含靶基因3′-UTR段的双荧光素酶报告载体,验证其活性;随后采用let-7a-5p模拟物和抑制剂分别增加和降低ASMCs胞内let-7a-5p表达,检测TGFβ1的表达以及分泌。预测显示,let-7a-5p靶向整合素αV(integrinαV,ITGAV)基因3′-UTR。构建含ITGAV基因3′-UTR序列双荧光素酶报告质粒、凝胶电泳和基因测序。结果表明,质粒重组成功。Let-7a-5p模拟物显著降低荧光素酶活性和ITGAV基因mRNA表达,提示let-7a-5p靶向ITGAV基因3′-UTR。Let-7a-5p模拟物显著降低ASMCs胞内TGFβ1的mRNA表达,但增加胞外TGFβ1含量。Let-7a-5p通过靶向ITGAV基因降低ASMCs胞内TGFβ1表达,但促进胞外TGFβ1活化,提示let-7a-5p可能通过靶向ITGAV促进TGFβ1活化参与气道结构和功能的调控。

To investigate the effect of microRNA(miNRA)let-7a-5p on transformation growth factorβ1(TGFβ1)expression and activation in airway smooth muscle cells,bioinformatics approach was used to predict the target gene of let-7a-5p,and a dual-luciferase reporter vector containing the 3'-UTR segment of the target gene integrinαV(ITGAV)was constructed to verify its activity.Subsequently,let-7a-5p mimics and inhibitors were used to regulate the intracellular let-7a-5p expression in ASMCs,and the mRNA expression of the target gene as well as the expression and secretion of TGFβ1 were examined.Bioinformatics analysis predicted a regulatory target of let-7a-5p on the 3'-UTR of the ITGAV gene.A dual-luciferase reporter recombinant plasmid containing the 3'-UTR(102 bp)sequence of the ITGAV gene was constructed and validated by gel electrophoresis and gene sequencing.The luciferase activity in HEK293T cells treated with let-7a-5p mimics was lower than that of HEK293T cells treated with negative controls.In addition,the mimics of let-7a-5p significantly decreased mRNA expression of ITGAV gene and TGFβ1 mRNA expression in ASMCs cells,but enhanced extracellular TGFβ1 activation.Let-7a-5p inhibited the mRNA expression and enhanced the activation of TGFβ1 of ASMCs by targeting the ITGAV gene,suggesting that let-7a-5p may regulate the structure and functions of airways via TGFβ1.