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微RNA-133a-3p靶向调控转化生长因子-β1/Smad3对瘢痕疙瘩成纤维细胞增殖和凋亡的影响 被引量:4

Effect of microRNA-133a-3p on proliferation and apoptosis of keloid fibroblasts by targeting transforming growth factor-β_(1)/Smad3
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摘要 目的探讨微RNA(miR)-133a-3p靶向调控转化生长因子-β_(1)(TGF-β_(1))/Smad3对瘢痕疙瘩成纤维细胞增殖和凋亡的影响及机制。方法选择2020年2月至2021年2月焦作市人民医院皮肤科收治的瘢痕疙瘩患者29例为研究对象,手术切取瘢痕疙瘩组织及其周围正常皮肤组织,分离培养瘢痕疙瘩成纤维细胞(KF)和正常皮肤组织成纤维细胞(NF)。取对数生长期KF细胞,随机分为空白对照组、miR-阴性对照(NC)组、miR-133a-3p组、miR-133a-3p+pcDNA3.1组、miR-133a-3p+pcDNA3.1-TGF-β_(1)组;空白对照组细胞不做任何转染,miR-NC组细胞转染miR-NC,miR-133a-3p组细胞转染miR-133a-3p mimic,miR-133a-3p+pcDNA3.1组细胞共转染miR-133a-3p mimic和pcDNA3.1,miR-133a-3p+pcDNA3.1-TGF-β_(1)组细胞共转染miR-133a-3p mimic和pcDNA3.1-TGF-β_(1)。实时荧光定量聚合酶链反应法检测组织和细胞中miR-133a-3p和Ⅰ型胶原蛋白(collagen Ⅰ)、Ⅲ型胶原蛋白(collagen Ⅲ)、α-平滑肌肌动蛋白(α-SMA)mRNA的表达,细胞计数试剂盒(CCK-8)法检测细胞增殖活力,流式细胞术检测细胞凋亡情况。应用TargetScan数据库通过生物信息分析预测miR-133a-3p与TGF-β_(1)的3′非翻译区(3′-UTR)存在结合位点,构建含有预测结合位点的野生型(Wt)和突变型(Mt)TGF-β_(1)3′-UTR片段的荧光素酶报告基因载体,分别将用脂质体2000包裹的Wt TGF-β_(1)或Mt TGF-β_(1)报告基因载体与miR-133a-3p mimics或miR-NC共转染入KF细胞,将转染后的细胞设为miR-NC+Wt TGF-β_(1)组、miR-133a-3p+Wt TGF-β_(1)组、miR-NC+Mt TGF-β_(1)组、miR-133a-3p+Mt TGF-β_(1)组,双荧光素酶检测试剂盒检测4组细胞双荧光素酶活性。Western blot检测空白对照组、miR-NC组、miR-133a-3p组KF细胞中TGF-β_(1)、Smad3蛋白的相对表达量。结果瘢痕疙瘩组织中miR-133a-3p相对表达量显著低于正常皮肤组织(t=18.988,P<0.05)。KF细胞中miR-133a-3p相对表达量显著低于NF细胞(t=18.679,P<0.05)。miR-133a-3p组细胞中miR Objective To investigate the effect and mechanism of microRNA(miR)-133a-3p on the proliferation and apoptosis of keloid fibroblasts by targeting transforming growth factor-β_(1)(TGF-β_(1))/Smad3.Methods A total of 29 patients with keloid admitted to Department of Dermatology,Jiaozuo People's Hospital from February 2020 to February 2021 were selected as the research subjects.The keloid tissue and its surrounding normal skin tissues were surgically cut,and keloid fibroblasts(KF)and normal skin fibroblasts(NF)were isolated and cultured.KF cells in logarithmic growth phase were randomly divided into blank control group,miR-negative control(NC)group,miR-133a-3p group,miR-133a-3p+pcDNA3.1 group,miR-133a-3p+pcDNA3.1-TGF-β_(1) group;the cells in the blank control group were not transfected,the cells in the miR-NC group were transfected with miR-NC;the cells in the miR-133a-3p group were transfected with miR-133a-3p mimic;the cells in the miR-133a-3p+pcDNA3.1 group were co-transfected with miR-133a-3p mimic and pcDNA3.1;the cells in the miR-133a-3p+pcDNA3.1-TGF-β_(1) group were co-transfected with miR-133a-3p mimic and pcDNA3.1-TGF-β_(1).The expressions of miR-133a-3p and Collagen Ⅰ,Collagen Ⅲ,α-smooth muscle actin(α-SMA)mRNA in tissues and cells were detected by real-time fluorescence quantitative polymerase chain reaction.The cell proliferation activity was detected by cell counting kit-8(CCK-8),and the cell apoptosis was detected by flow cytometry.TargetScan database was used to predict the existence of binding sites of miR-133a-3p and 3′-untranslated region(3'-UTR)of TGF-β1 through bioinformation analysis,and the fragment lucifase reporter gene vectors containing the predicted binding sites of wild type(Wt)and mutant type(Mt)TGF-β_(1)3'-UTR were constructed,Wt TGF-β_(1) or Mt TGF-β_(1) reporter gene vector coated with liposome 2000 and miR-133a-3p mimics or miR-NC were co-transfected into KF cells,respectively.The transfected cells were divided into miR-NC+Wt TGF-β_(1) group,miR-133a-3p+Wt TGF-β_(1)
作者 卫艳萍 谢彤阳 王廷廷 张宁宁 WEI Yanping;XIE Tongyang;WANG Tingting;ZHANG Ningning(Department of Dermatology,Jiaozuo People's Hospital,Jiaozuo 454002,Henan Province,China;Graduate School of Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处 《新乡医学院学报》 CAS 2022年第4期330-336,340,共8页 Journal of Xinxiang Medical University
基金 河南省重点科技攻关项目(编号:192102310361)。
关键词 瘢痕疙瘩 微RNA-133a-3p 转化生长因子-β_(1) 增殖 凋亡 keloid microRNA-133a-3p transforming growth factor-β_(1) proliferation apoptosis
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