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Title:: Let-7a-5p targets integrin αV to regulate TGF-β1 expression and activation in airway smooth muscle cells

Author(s):: WEN Kang; GUO Jia; NI Kai; YANG Chongxin; LUO Mingzhi; DENG Linhong; Institute of Biomedical Engineering and Health Sciences, School of Pharmacy & School of Medicine, Changzhou University, Changzhou 213164, China

Keywords:: MicroRNAs; Airway inflammation; Airway cells; Inflammation factor; Integrin signaling; Dual luciferase reporter assay

摘要:: 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 factor β1, 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（integrin αV, 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。

Abstract:: To investigate the effect of microRNA (miNRA) let-7a-5p on transformation growth factor β1 (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 integrin αV (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.