miR-127-3p对气道瘢痕成纤维细胞的增殖和Ⅰ、Ⅲ型胶原蛋白、α-平滑肌肌动蛋白分泌的影响

Effects of miR-127-3p on proliferation of airway scar fibroblasts and secretion of collagenⅠ,collagen Ⅲ and smooth muscle actin-α

目的研究miR-127-3p对气道瘢痕成纤维细胞增殖和Ⅰ、Ⅲ型胶原蛋白、α-平滑肌肌动蛋白(α-SMA)分泌的影响。方法组织样本均来源于2018年1月至2020年1月成都医学院第一附属医院呼吸与危重症医学科就诊的良性气道瘢痕狭窄的患者8例。对气道瘢痕组织标本应用基因芯片筛选出和正常气道黏膜组织差异表达的miRNA,并借助反转录-聚合酶链反应技术验证miR-127-3p的表达;采用公共数据库网站TargetScanHuman预测miR-127-3p的下游靶基因,选择预测分值最高的靶基因进行研究,采用荧光素酶报告基因实验分析miR-127-3p对MAPK4的靶向作用。提取气道瘢痕成纤维细胞分别应用miR-127-3p的模拟物和抑制物,以检测miR-127-3p对靶基因MAPK4的调控作用,通过反转录-聚合酶链反应及蛋白质印迹技术检测Ⅰ、Ⅲ型胶原蛋白及α-SMA水平,分析miR-127-3p对成纤维细胞胶原蛋白和α-SMA分泌的影响;采用MTT法检测过表达miR-127-3p后成纤维细胞增殖的情况。结果miR-127-3p在气道狭窄的瘢痕组织中呈低表达(0.512±0.014),可与MAPK4的3′UTR区域直接结合,并抑制瘢痕成纤维细胞的生长。过表达miR-127-3p导致下游靶基因MAPK4和Ⅰ、Ⅲ型胶原蛋白及α-SMA水平显著下降,可能影响成纤维细胞向肌成纤维细胞的分化进程。结论miR-127-3p可以减少Ⅰ、Ⅲ型胶原蛋白、α-SMA的分泌,抑制瘢痕成纤维细胞的增殖。

Objective To study the effects of miR-127-3p on the proliferation of airway scar fibroblasts and the secretion of collagenⅠ,collagenⅢand smooth muscle actin-α(α-SMA).Methods Tissue samples were collected from eight patients with benign airway scar stenosis in the Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital of Chengdu Medical College from January 2018 to January 2020.The differentially expressed miRNA in airway scar tissue compared with normal airway mucosa tissue was screened by gene chip.The expression of miR-127-3p was verified by reverse transcription-polymerase chain reaction.The downstream target genes of miR-127-3p were predicted by TargetScanHuman.The target gene with the highest predictive score was selected for the study,and the luciferase reporter gene experiment was used to analyze the targeting effect of miR-127-3p on mitogen-activated protein kinase 4(MAPK4).The airway scar fibroblasts were extracted,mimics and inhibitors of miR-127-3p were used to detect the regulatory effect of miR-127-3p on target gene MAPK4.The expressions of collagenⅠ,collagenⅢandα-SMA were detected by reverse transcription-polymerase chain reaction and Western blot,to analyze the effects of miR-127-3p on secretion of collagen andα-SMA.The effect of overexpression of miR-127-3p on fibroblast proliferation was detected by MTT assay.Results miR-127-3p was downexpressed in airway stenosis scar tissue(0.512±0.014),could directly bind to the 3′UTR region of MAPK4 and inhibit the growth of scar fibroblasts.Overexpression of miR-127-3p could result in a significant decrease of target genes MAPK4,collagenⅠ,collagenⅢandα-SMA,inhibiting the process of fibroblast differentiation into myofibroblast.Conclusions miR-127-3p can reduce the secretion of collagenⅠ,collagenⅢandα-SMA,and inhibit the proliferation of scar fibroblasts.