miRNA-143-3p通过调控ITGB5表达影响人瘢痕疙瘩成纤维细胞增殖、迁移和侵袭机制的研究

Study on the mechanism of miRNA-143-3p-mediated proliferation,migration and invasion of human keloid fibroblasts through regulating ITGB5 expression

目的探讨miRNA-143-3p对瘢痕疙瘩成纤维细胞(keloid fibroblasts,KFB)增殖、迁移和侵袭的影响及作用机制。方法分离培养KFB和正常皮肤成纤维细胞(normal skin fibroblast,NFB),RT-qPCR法检测KFB和NFB中miRNA-143-3p和整合素β5(integrinβ5,ITGB5)的mRNA表达水平,Western blot检测ITGB5的蛋白质表达水平。MTT法、Transwell法、RT-qPCR和Western blot检测过表达miRNA-143-3p或抑制ITGB5表达对KFB细胞增殖、迁移和侵袭及相关蛋白质表达的影响。双荧光素酶报告基因试验验证miRNA-143-3p和ITGB5的靶向关系。结果与NFB相比较,KFB中miRNA-143-3p表达下调,ITGB5的mRNA和蛋白质表达水平上调。过表达miRNA-143-3p或抑制ITGB5表达均抑制了KFB的增殖、迁移和侵袭。miRNA-143-3p可负向调控ITGB5表达,ITGB5过表达逆转了miRNA-143-3p过表达对KFB增殖、迁移和侵袭的作用。结论miRNA-143-3p通过下调ITGB5表达抑制KFB的增殖、迁移和侵袭。

Objective To investigate the effects of miRNA-143-3p on the proliferation,migration and invasion of keloid fibroblasts(KFB)and the possible mechanism.Methods KFB and normal skin fibroblasts(NFB)were isolated and cultured.The expression of miRNA-143-3p and integrinβ5(ITGB5)at mRNA level in KFB and NFB were detected by RT-qPCR.Western blot was performed to measure the expression of ITGB5 at protein level.After overexpressing miRNA-143-3p or suppressing ITGB5 expression in KFB cells,MTT assay,Transwell assay,RT-qPCR and Western blot were used to detect the effects on the proliferation,migration and invasion of KFB and the expression of related proteins.Dual-luciferase reporter gene assay was used to validate the targeting relationship between miRNA-143-3p and ITGB5.Results Compared with NFB,KFB showed down-regulated miRNA-143-3p expression and up-regulated expression of ITGB5 at both mRNA and protein levels.The proliferation,migration and invasion of KFB were inhibited after overexpressing miRNA-143-3p or suppressing ITGB5 expression.Moreover,miRNA-143-3p could negatively regulate ITGB5 expression and overexpression of ITGB5 reversed the effects of miRNA-143-3p overexpression on KFB proliferation,migration and invasion.Conclusions This study suggested that miRNA-143-3p could inhibit the proliferation,migration and invasion of KFB through down-regulating ITGB5 expression.