miR-497-5p靶向WWP1调控人瘢痕疙瘩成纤维细胞的增殖和凋亡  

miR-497-5p affects proliferation and apoptosis of human keloid fibroblasts through targeted regulation of WWP1

作　　者：高栋梁[1] 高东东[1] 池凯 杨喜明[1] GAO Dong-liang;GAO Dong-dong;CHI Kai(Department of Burn and Plastic Surgery.Affiliated Hospital of Yan'an University,Yan'an,716000,China;Zhengzhou Noah's Medical Aestheic Clinic,Zhengzhou,450000,Chima)

机构地区：[1]延安大学附属医院烧伤整形手外科,陕西延安716000 [2]郑州郑东诺亚一家医疗美容门诊部整形外科,河南郑州450000

出　　处：《中国实验诊断学》2020年第6期983-989,共7页Chinese Journal of Laboratory Diagnosis

摘　　要：目的探讨微小RNA-497-5p(miR-497-5p)是否通过靶向含有WW结构域的E3泛素蛋白连接酶1(WWP1)调控人瘢痕疙瘩成纤维细胞的增殖和凋亡。方法实时荧光定量PCR(qPCR)和蛋白质印迹法(Western blot)测定瘢痕疙瘩成纤维细胞(KFB)和正常皮肤成纤维细胞(NFB)中miR-497-5p、WWP1 mRNA和蛋白表达。TargetScan预测miR-497-5p和WWP1的靶向关系,双荧光素酶报告和Western blot进一步验证。构建miR-497-5p过表达或抑制WWP1表达的细胞,Western blot检测WWP1、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,噻唑蓝(MTT)法检测细胞增殖,流式细胞术检测细胞凋亡。共转染miR-497-5p和pcDNA-WWP1,观察WWP1过表达对miR-497-5p过表达诱导的KFB细胞增殖和凋亡的影响。结果与NFB相比,KFB内miR-497-5p表达量明显下调(P<0.05),WWP1mRNA和蛋白表达量显著上调(P<0.05)。miR-497-5p靶向调控WWP1的表达。miR-497-5p过表达明显降低KFB 48h、72h的细胞活性、CyclinD1、Bcl-2蛋白表达,提高细胞凋亡率、p21、Bax蛋白水平(P<0.05),与抑制WWP1表达结果相同。WWP1过表达逆转了miR-497-5p过表达对KFB增殖、WWP1、CyclinD1、Bcl-2蛋白表达的抑制作用,以及对细胞凋亡、p21、Bax蛋白表达的促进作用。结论过表达miR-497-5p通过靶向调控WWP1抑制瘢痕疙瘩成纤维细胞的增殖,并诱导其凋亡。Objective To investigate whether miR-497-5 p regulates the proliferation and apoptosis of human keloid fibroblasts by targeting WWP1.Methods qPCR and Western blot were used to detect the expression of miR-497-5 p and WWP1 mRNA and protein in keloid fibroblasts(KFB)and normal skin fibroblasts(NFB).TargetScan predicted the targeting relationship between miR-497-5 p and WWP1,which was further verified by dual luciferase reporter and Western blot.miR-497-5 p overexpressing or inhibiting WWP1 expression of cells was constructed.Expression of WWP1,CyclinD1,p21,Bcl-2,Bax protein was determined by Western blot.Cell proliferation was detected by MTT assay,and apoptosis was detected by flow cytometry.The effects of WWP1 overexpression on proliferation and apoptosis of KFB cells induced by miR-497-5 p overexpression were observed by co-transfection of miR-497-5 p and pcDNA-WWP1.Results Compared with NFB,the expression of miR-497-5 p in KFB was significantly down-regulated(P<0.05),and the expression of WWP1 mRNA and protein was obviously up-regulated(P<0.05).miR-497-5 p targeteds to regulate the expression of WWP1.The overexpression of miR-497-5 p greatly decreased the cell viability,CyclinD1 and Bcl-2 protein expression of KFB at 48 hand 72 h,increased the apoptosis rate,p21 and Bax protein levels(P<0.05),which was the same results in the inhibition of WWP1 expression.WWP1 overexpression reversed the inhibitory effect of miR-497-5 p overexpression on KFB proliferation,WWP1,CyclinD1,Bcl-2 protein expression,and the promotion of apoptosis,p21 and Bax protein expression.Conclusion Overexpression of miR-497-5 p inhibits the proliferation of keloid fibroblasts and induces apoptosis by targeting WWP1.

关 键 词：miR-497-5p WWP1 瘢痕疙瘩成纤维细胞 增殖 凋亡 

分 类 号：R392[医药卫生—免疫学]