Circ_0057452 sponges miR-7-5p to promote keloid progression through upregulating GAB1

Increasing evidence has shown that circular RNAs (circRNAs) play critical roles in various diseases, including keloid. The purpose of this study was to investigate the role of circ_0057452 and related action mechanisms during the development of keloid. The expression levels of circ_0057452, microRNA-7-5p (miR-7-5p) and GRB2 associated binding protein 1 (GAB1) mRNA were determined by quantitative real-time PCR (qRT-PCR). Cell proliferation was evaluated using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and 5-Ethynyl-2’-deoxyuridine (Edu) assays. Flow cytometry analysis was utilized to determine cell cycle distribution and cell apoptosis. Western blot assay was used to measure apoptosis-related, collagen synthesis-related, and GAB1 protein levels. Cell migration and invasion were detected by wound healing assay and transwell assay. The interaction between miR-7-5p and circ_0057452 or GAB1 was confirmed by dual-luciferase reporter, RNA pull-down, and RNA Immunoprecipitation (RIP) assays. Circ_0057452 and GAB1 were upregulated in keloid tissues and keloid fibroblasts (KFs), while miR-7-5p was downregulated. Circ_0057452 knockdown or miR-7-5p overexpression inhibited the proliferation, migration, invasion, and collagen synthesis and induced cell cycle arrest and apoptosis of KFs. MiR-7-5p was targeted by circ_0057452, and its inhibition overturned the effects of circ_0057452 knockdown. In addition, GAB1 was a target of miR-7-5p, and GAB1 upregulation abolished the role of miR-7-5p overexpression and circ_0057452 knockdown in KFs. Circ_0057452 regulated the expression of GAB1 by adsorbing miR-7-5p in KFs. Circ_0057452 knockdown suppressed keloid development by regulating miR-7-5p/GAB1 axis, which might provide a promising therapeutic target for keloid.

越来越多的研究证据表明，环状RNA（circular RNAs，circRNAs）在包括瘢痕疙瘩在内的多种疾病中发挥关键调控作用。本研究旨在探讨circ_0057452及其相关作用机制在瘢痕疙瘩发生发展过程中的功能。采用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测circ_0057452、微小RNA-7-5p（microRNA-7-5p，miR-7-5p）及GRB2结合蛋白1（GRB2 associated binding protein 1，GAB1）的mRNA表达水平；通过噻唑蓝（methylthiazolyldiphenyl-tetrazolium bromide，MTT）实验与5-乙炔基-2’-脱氧尿苷（5-Ethynyl-2’-deoxyuridine，Edu）实验评估细胞增殖能力；利用流式细胞术分析细胞周期分布与细胞凋亡水平；采用蛋白质印迹实验检测凋亡相关蛋白、胶原合成相关蛋白及GAB1的表达水平；通过划痕愈合实验与Transwell实验检测细胞迁移与侵袭能力；借助双荧光素酶报告基因实验、RNA下拉实验及RNA免疫沉淀（RNA Immunoprecipitation，RIP）实验验证miR-7-5p与circ_0057452或GAB1之间的靶向结合关系。实验结果显示，circ_0057452与GAB1在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞（keloid fibroblasts，KFs）中呈高表达，而miR-7-5p表达水平显著下调。敲低circ_0057452或过表达miR-7-5p均可抑制瘢痕疙瘩成纤维细胞的增殖、迁移、侵袭及胶原合成，并诱导细胞周期阻滞与细胞凋亡。miR-7-5p是circ_0057452的靶基因，抑制miR-7-5p的表达可逆转敲低circ_0057452所产生的生物学效应。此外，GAB1是miR-7-5p的靶基因，上调GAB1的表达可抵消过表达miR-7-5p及敲低circ_0057452对瘢痕疙瘩成纤维细胞的调控作用。进一步机制研究表明，circ_0057452可通过吸附miR-7-5p调控瘢痕疙瘩成纤维细胞中GAB1的表达水平。综上，敲低circ_0057452可通过调控miR-7-5p/GAB1轴抑制瘢痕疙瘩的发展进程，该研究结果可为瘢痕疙瘩的临床治疗提供潜在的新型靶点。