miR-192-5p在增生性瘢痕组织及成纤维细胞中的表达及调控

Expression and regulation of miR-192-5p in hypertrophic scar tissue and fibroblasts

背景:研究表明,miRNAs的表达与肝纤维化、肾纤维化及皮肤纤维化发生有关;并证实在痛风性关节炎中miR-192-5p与表皮调节素存在靶向调控关系。目的:探讨miR-192-5p在增生性瘢痕中的表达及调控作用,并验证miR-192-5p与表皮调节素之间存在靶向调控关系。方法:①在新疆医科大学第一附属医院收集6例增生性瘢痕组织及6例正常皮肤组织,采用qRT-PCR法检测miR-192-5p与表皮调节素mRNA表达。②组织块法获取原代增生性瘢痕成纤维细胞,传代培养至3-6代用于后续实验,实验分为3个组:阴性对照组、miR-192-5p模拟物组和miR-192-5p抑制物组,分别转染对应的序列。采用CCK-8法和EdU试剂盒检测细胞增殖活力,划痕实验检测细胞迁移能力,流式细胞术检测细胞凋亡,qRT-PCR和Western blot法检测表皮调节素、Ⅰ型胶原、Ⅲ型胶原和α-SMA的基因和蛋白表达,生物信息学网站预测miR-192-5p靶点,双荧光素酶实验验证靶向结合。结果与结论:①与正常皮肤组织及其成纤维细胞相比,miR-192-5p和表皮调节素在增生性瘢痕和增生性瘢痕成纤维细胞中均呈高表达(P<0.05或P<0.01);②过表达miR-192-5p后,与阴性对照组比较,细胞增殖能力增强(P<0.05),EdU阳性细胞率增加(P<0.01);抑制miR-192-5p后细胞活力降低(P<0.05),EdU阳性细胞率减少(P<0.05);③过表达miR-192-5p 24 h后,与阴性对照组比较,模拟物组细胞划痕间面积、细胞凋亡率减小(P<0.05),miR-192-5p抑制物组细胞划痕间面积、细胞凋亡率增加(P<0.01);④转染48 h后,miR-192-5p模拟物组表皮调节素mRNA及蛋白水平显著降低、Ⅰ型胶原、Ⅲ型胶原和α-平滑肌肌动蛋白mRNA及蛋白水平显著增高(P<0.05或P<0.01);miR-192-5p抑制物组上述4项指标呈现相反的变化(P<0.05或P<0.01);⑤Targetscan网站预测表皮调节素与miR-192-5p有潜在结合位点;⑥双荧光素酶实验显示,miR-192-5p能够与表皮调节素靶向结合。结果说明:miR-192-5p过表达可降低表皮调节素的表达,二者可能存在负向调控;提示通过调控表皮调节素可能起到抑制增生性瘢痕成纤维细胞增殖的作用。

BACKGROUND:miRNAs expression has been reported to be associated with hepatic and renal fibrosis,and dermal fibrogenesis.Moreover,a targeted regulatory relationship between miR-192-5p and epidermal regulators has been demonstrated in gouty arthritis.OBJECTIVE:To investigate the expression and regulatory role of miR-192-5p in hypertrophic scar and to verify whether there is a targeted regulatory relationship between miR-192-5p and epidermal regulators.METHODS:(1)Six cases of hypertrophic scar tissue and six cases of normal skin tissue were collected from the First Affiliated Hospital of Xinjiang Medical University.And miR-192-5p and epidermal regulator mRNA expression were detected by qRT-PCR.(2)The primary hypertrophic scar fibroblasts were obtained using tissue explant method and cultured to 3-6 generations for subsequent experiments.There were three groups in the experiment:negative control group,miR-192-5p mimic group and miR-192-5p inhibitor group.The latter two groups were transfected with the corresponding sequences.Cell proliferation viability was detected by the cell counting kit-8 assay and EdU kit;and the migration ability was detected by the cell scratch test.Cell apoptosis was detected by flow cytometry.The gene and protein expressions of epidermal regulator,type I collagen,type III collagen andα-smooth muscle actin were detected by qRT-PCR and western blot,respectively.miR-192-5p targets were predicted by a bioinformatics website,and target binding was validated by dual luciferase assay.RESULTS AND CONCLUSION:(1)Compared with normal skin tissues and their fibroblasts,miR-192-5p and epidermal regulator were highly expressed in hypertrophic scar and hypertrophic scar fibroblasts(P<0.05 or P<0.01).(2)After overexpression of miR-192-5p,cell proliferation was enhanced(P<0.05)and EdU positive cell rate increased(P<0.01)when compared with the negative control group;after inhibition of miR-192-5p,cell viability(P<0.05)and EdU positive rate decreased(P<0.05).(3)At 24 hours after overexpression of miR-192-5p,compared with the negative control group,the area between cell scratches and apoptosis rate decreased in the miR-192-5p mimic group(P<0.05)but increased in the miR-192-5p inhibitor group(P<0.01).(4)At 48 hours after transfection,the mRNA and protein levels of epidermal regulator were significantly decreased in the miR-192-5p mimic group,while the mRNA and protein levels of type I collagen,type III collagen andα-smooth muscle actin were significantly increased(P<0.05 or P<0.01).The miR-192-5p inhibitor group showed opposite changes in the above four indicators(P<0.05 or P<0.01).(5)The Targetscan website predicted that epidermal regulator had a potential binding site for miR-192-5p.(6)Dual luciferase assays showed that miR-192-5p could bind to epidermal regulator in a targeted manner.To conclude,overexpression of miR-192-5p can decrease the expression of epidermal regulator,and the two may be negatively regulated,suggesting that regulation of epidermal regulator may play a role in inhibiting the proliferation of hypertrophic scar fibroblasts.