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

Effect of microRNA-133a-3p on proliferation and apoptosis of keloid fibroblasts by targeting transforming growth factor-β_(1)/Smad3

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-133a-3p相对表达量显著高于空白对照组和miR-NC组,CollagenⅠ、CollagenⅢ、α-SMA mRNA相对表达量显著低于空白对照组和miR-NC组(P<0.05);空白对照组与miR-NC组细胞中miR-133a-3p和CollagenⅠ、CollagenⅢ、α-SMA mRNA相对表达量比较差异无统计学意义(P>0.05)。miR-133a-3p组细胞凋亡率显著高于空白对照组和miR-NC组,细胞增殖活力显著低于空白对照组和miR-NC组(t=13.912、14.227、-8.020、-6.947,P<0.05);空白对照组与miR-NC组细胞凋亡率、细胞增殖活力比较差异无统计学意义(t=-0.607、0.448,P>0.05)。miR-133a-3p+Wt TGF-β_(1)组细胞相对荧光素酶活性显著低于miR-NC+Wt TGF-β_(1)组、miR-NC+Mt TGF-β_(1)组、miR-133a-3p+Mt TGF-β_(1)组(t=-15.729、-19.490、-16.186,P<0.05)。miR-NC+Wt TGF-β_(1)组、miR-NC+Mt TGF-β_(1)组、miR-133a-3p+Mt TGF-β_(1)组细胞相对荧光素酶活性两两比较差异均无统计学意义(t=-0.596、0.847、0.842,P>0.05)。KF细胞中Smad3和TGF-β_(1)蛋白相对表达量显著高于NF细胞(t=5.758、9.027,P<0.05)。miR-133a-3p组细胞中Smad3和TGF-β_(1)蛋白相对表达量显著高于miR-NC组(t=-4.913、-8.314,P<0.05)。miR-133a-3p+pcDNA3.1-TGF-β_(1)组细胞中Collagen Ⅰ、Collagen Ⅲ、α-SMA mRNA相对表达量和细胞增殖活力显著高于miR-133a-3p组和miR-133a-3p+pcDNA3.1组,细胞凋亡率显著低于miR-133a-3p转染组和miR-133a-3p+pcDNA3.1组(P<0.05)。miR-133a-3p组与miR-133a-3p+pcDNA3.1组细胞中Collagen Ⅰ、Collagen Ⅲ、α-SMA mRNA相对表达量及细胞凋亡率和细胞增殖活力比较差异无统计学意义(P>0.05)。结论过表达miR-133a-3p可通过负调控TGF-β_(1)抑制瘢痕疙瘩成纤维细胞增殖,促进细胞凋亡,提示miR-133a-3p/TGF-β_(1)通路可能是瘢痕疙瘩治疗的一个新的靶点。

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) group,miR-NC+Mt TGF-β1 group and miR-133a-3p+Mt TGF-β_(1) group,and the double luciferase activity of cells in each group was detected by double luciferase assay kit.The relative expression levels of TGF-β_(1) and Smad3 proteins in KF cells in the blank control group,miR-NC group and miR-133a-3p group were detected by Western blot.Results The relative expression level of miR-133a-3p in keloid tissue was significantly lower than that in normal skin tissue(t=18.988,P<0.05).The relative expression level of miR-133a-3p in KF cells was significantly lower than that in NF cells(t=18.679,P<0.05).The relative expression level of miR-133a-3p in cells in the miR-133a-3p group was significantly higher than that in the blank control group and miR-NC group,and the relative expression levels of Collagen Ⅰ,Collagen Ⅲ and α-SMA mRNA were significantly lower than those in the blank control group and miR-NC group(P<0.05).There was no significant difference in the relative expression levels of miR-133a-3p and Collagen Ⅰ,Collagen Ⅲ,α-SMA mRNA in cells between the blank control group and miR-NC group(P>0.05).The apoptosis rate of cells in the miR-133a-3p group was significantly higher than that in the blank control group and miR-NC group,and the cell proliferation viability was significantly lower than that in the blank control group and miR-NC group(t=13.912,14.227,-8.020,-6.947;P<0.05).There were no significant differences in apoptosis rate of cells and cell proliferation viability between the blank control group and miR-NC group(t=-0.607,0.448;P>0.05).The relative luciferase activity of cells in the miR-133a-3p+Wt TGF-β_(1) group was significantly lower than that in the miR-NC+Wt TGF-β_(1) group,miR-NC+Mt TGF-β_(1) group and miR-133a-3p+Mt TGF-β_(1) group(t=-15.729,-19.490,-16.186;P<0.05).There was no significant difference in the relative luciferase activity of cells among the miR-NC+Wt TGF-β_(1) group,miR-NC+Mt TGF-β_(1) group and miR-133a-3p+Mt TGF-β_(1) group(t=-0.596,0.847,0.842;P>0.05).The relative expression levels of Smad3 and TGF-β_(1) proteins in KF cells were significantly higher than those in NF cells(t=5.758,9.027;P<0.05).The relative expression levels of Smad3 and TGF-β_(1) proteins in KF cells in the miR-133a-3p group were significantly higher than those in the miR-NC group(t=-4.913,-8.314;P<0.05).The relative expression levels of Collagen Ⅰ,Collagen Ⅲ,α-SMA mRNA and cell proliferation viability in the miR-133a-3p+pcDNA3.1-TGF-β_(1) group were significantly higher than those in the miR-133a-3p group and miR-133a-3p+pcDNA3.1 group,and the apoptosis rate of cells was significantly lower than that in the miR-133a-3p group and miR-133a-3p+pcDNA3.1 group(P<0.05).There were no significant differences in the relative expression levels of Collagen Ⅰ,Collagen Ⅲ,α-SMA mRNA and apoptosis rate of cells and cell proliferation viability between the miR-133a-3p group and miR-133a-3p+pcDNA3.1 group(P>0.05).Conclusion Overexpres sion of miR-133a-3p can inhibit the proliferation and promote apoptosis of keloid fibroblasts by negatively regulating TGF-β_(1),which suggests that miR-133a-3p/TGF-β_(1) pathway may be a new therapeutic target for keloid.