miR-582-5p靶向DNMT1对人瘢痕疙瘩成纤维细胞增殖和凋亡的影响  

Effects of miR-582-5p targeting DNMT1 on the proliferation and apoptosis of human keloid fibroblasts

作　　者：高栋梁[1] 高东东[1] 白翠翠[1] 杨波 杨喜明[1] GAO Dongliang;GAO Dongdong;BAI Cuicui(Department of Burn Plastic Hand Surgery,Affiliated Hospital of Yan’an University,Shaanxi,Yan’an 716000,China)

机构地区：[1]延安大学附属医院烧伤整形手外科,陕西省延安市716000

出　　处：《河北医药》2021年第9期1302-1306,1311,共6页Hebei Medical Journal

摘　　要：目的探讨微小RNA-582-5p(miR-582-5p)对DNA甲基转移酶1(DNMT1)的靶向作用及对人瘢痕疙瘩成纤维细胞增殖和凋亡的影响。方法实时荧光定量PCR(qPCR)和蛋白质印迹法(Western Blot)检测瘢痕疙瘩成纤维细胞(KFB)和正常皮肤成纤维细胞(NFB)miR-582-5p、DNMT1 mRNA、DNMT1蛋白表达。starBase网站与双荧光素酶报告实验分析miR-582-5p与DNMT1的靶向关系。瘢痕疙瘩成纤维细胞中转染miR-582-5p或si-DNMT1,噻唑蓝(MTT)检测细胞增殖,流式细胞术检测细胞凋亡,Western Blot检测DNMT1、细胞周期蛋白D1(CyclinD1)、p21、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达。miR-582-5p和pcDNA-DNMT1共转染,观察DNMT1过表达对miR-582-5p过表达诱导的瘢痕疙瘩成纤维细胞增殖、凋亡的影响。结果与NFB组比较,KFB中miR-582-5p表达量明显减少(P<0.05),DNMT1 mRNA和DNMT1蛋白表达量显著增加(P<0.05)。miR-582-5p靶向调控DNMT1的表达。miR-582-5p过表达或抑制DNMT1表达明显抑制瘢痕疙瘩成纤维细胞的增殖、CyclinD1蛋白、Bcl-2蛋白表达(P<0.05),显著促进细胞凋亡、p21蛋白、Bax蛋白表达(P<0.05)。DNMT1过表达逆转miR-582-5p过表达对瘢痕疙瘩成纤维细胞增殖、CyclinD1蛋白、Bcl-2蛋白表达的抑制作用,以及对细胞凋亡、p21蛋白、Bax蛋白表达的促进作用。结论miR-582-5p通过靶向调控DNMT1的表达抑制人瘢痕疙瘩成纤维细胞增殖,并诱导其凋亡。Objective To investigate the effects of miR-582-5p targeting DNMT1 on the proliferation and apoptosis of human keloid fibroblasts.Methods The qPCR and Western Blot were used to detect the expression levels of miR-582-5p,DNMT1 mRNA and DNMT1 protein in keloid fibroblasts(KFB)and normal skin fibroblasts(NFB).The starBase website and the dual luciferase reporter assay were performed to analyze the targeting correlation between miR-582-5p and DNMT1.The keloid fibroblasts were transfected with miR-582-5p or si-DNMT1,then the cell proliferation was detected by MTT,and cell apoptosis was detected by flow cytometry.Moreover the expression levels of DNMT1,CyclinD1,p21,Bcl-2 and Bax were detected by Western Blot.The miR-582-5p and pcDNA-DNMT1 were co-transfected to observe the effects of DNMT1 overexpression on the proliferation and apoptosis of keloid fibroblasts induced by miR-582-5p overexpression.Results As compared with those in NFB group,the expression levels of miR-582-5p were significantly decreased in KFB group(P<0.05),however,the expression levels of DNMT1 mRNA and DNMT1 protein were significantly increased(P<0.05).The miR-582-5p targetedly regulated the expression of DNMT1,and the overexpression of miR-582-5p or the inhibition of DNMT1 expression obviously inhibited the proliferation of keloid fibroblasts,and the expressions of CyclinD1 protein and Bcl-2 protein(P<0.05),which significantly promoted cell apoptosis and the expressions of p21 protein and Bax protein(P<0.05).In addition the overexpression of DNMT1 reversed the inhibitory effects of miR-582-5p overexpression on the proliferation of keloid fibroblasts and the expressions of CyclinD1 protein and Bcl-2 protein,which reversed the promotive effects on cell apoptosis,and the expressions of p21 protein and Bax protein.Conclusion The miR-582-5p inhibits the proliferation of human keloid fibroblasts and induces apoptosis by targetedly regulate the DNMT1 expression.

关 键 词：miR-582-5p DNMT1 人瘢痕疙瘩成纤维细胞 增殖 凋亡 

分 类 号：R619.6[医药卫生—外科学]