miR-425-5p靶向TGF-β1/Smad2信号通路减少小鼠增生性瘢痕组织胶原纤维沉积  

miR-425-5p targets TGF-β1/Smad2 signaling pathway to attenuate collagen fiber deposition in hypertrophic scar tissue in mice

作　　者：梁艳[1] 王丽娟[1] 吴惠林 LIANG Yan;WANG Lijuan;WU Huilin(Department of Dermatology and Venereology,the First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061;Department of Dermatology,the Second Affiliated Hospital of Xi'an Medical University,Xi'an 710038,China)

机构地区：[1]西安交通大学第一附属医院皮肤性病科,陕西西安710061 [2]西安医学院第二附属医院皮肤科,陕西西安710038

出　　处：《基础医学与临床》2023年第3期393-401,共9页Basic and Clinical Medicine

基　　金：陕西省自然科学基础研究计划(2021JM-267)。

摘　　要：目的探究miR-425-5p在增生性瘢痕(HS)形成中的作用及机制。方法通过RT-qPCR和Western blot检测了16对HS组织和正常皮肤组织中miR-425-5p、转化生长因子-β1(TGF-β1)和SMAD家庭成员2(Smad2)的水平。将成纤维细胞分为9组:对照组(control组)、miR-425-5p-mimic组、NC-mimic组、miR-425-5p-inhibitor组、NC-inhibitor组、TGF-β1-pcDNA3.1组、NC-pcDNA3.1组、miR-425-5p-mimic+NC-pcDNA3.1组和miR-425-5p-mimic+TGF-β1-pcDNA3.1组,并采用Lipofectamine 2000对细胞进行转染处理。通过双荧光素酶报告基因实验验证miR-425-5p和TGF-β1的靶向关系。通过博莱霉素(BLM)诱导小鼠增生性瘢痕,然后将小鼠随机分为3组,对照组(NC-agomir)、模型组(NC-agomir+BLM)和转染miR-425-5p干预模型组(miR-425-5p-agomir+BLM),小鼠均治疗21 d。通过Masson三色染色检测HS组织的胶原纤维沉积。通过RT-qPCR和Western blot检测组织和细胞中miR-425-5p、Ⅰ型胶原(Col-Ⅰ)、Ⅲ型胶原(Col-Ⅲ)、α-平滑肌肌动蛋白(α-SMA)、TGF-β1和Smad2的表达水平。结果与正常组相比,HS组的miR-425-5p表达水平降低(t=4.242,P<0.001),而TGF-β1 mRNA和蛋白表达水平均升高(P<0.001)。与对照组相比,miR-425-5p-mimic组的Col-Ⅰ、Col-Ⅲ、α-SMA、TGF-β1和Smad2的mRNA和蛋白表达水平均降低(P<0.05)。与control组相比,miR-425-5p-inhibitor组的Col-Ⅰ、Col-Ⅲ、α-SMA、TGF-β1和Smad2的mRNA和蛋白表达水平均升高(P<0.05)。与miR-425-5p-mimic共转染后,TGF-β1-3′-UTR-WT组的相对荧光素酶活性比TGF-β1-3′-UTR-MUT组降低(P<0.05)。与miR-425-5p-mimic+NC-pcDNA3.1组相比,miR-425-5p-mimic+TGF-β1-pcDNA3.1组的Col-Ⅰ、Col-Ⅲ、α-SMA、TGF-β1和Smad2的mRNA和蛋白表达水平均升高(P<0.05)。与NC-agomir+BLM组相比,miR-425-5p-agomir+BLM组的相对胶原蛋白含量降低(P<0.05),miR-425-5p表达水平升高,而TGF-β1的mRNA和蛋白表达水平均降低(P<0.05)。结论miR-425-5p在HS组织中表达下调,上调miR-425-5p通过抑制TGF-β1/Smad2信号通Objective To investigate the role and mechanism of miR-425-5p in the formation of hypertrophicscar(HS).Methods The levels of miR-425-5p,transforming growth factor-β1(TGF-β1)and SMAD family member 2(Smad2)in 16 pairs of HS tissue and normal skin tissue were detected by RT-qPCR and Western blot.The fibroblast cells were divided into nine groups:control group,miR-425-5p-mimic group,NC-mimic group,miR-425-5p-inhibitor group,NC-inhibitor group,TGF-β1-pcDNA3.1 group,NC-pcDNA3.1 group,miR-425-5p-mimic+NC-pcDNA3.1 group and miR-425-5p-mimic+TGF-β1-pcDNA3.1 group.The cells were transfected with Lipofectamine 2000.The targeting relationship between miR-425-5p and TGF-β1 was verified by dual-luciferase reporter gene assay.Hypertrophic scars in mice were induced by bleomycin(BLM),and then the mice were randomly divided into 3 groups,control group(NC-agomir),model group(NC-agomir+BLM)and intervention model group transfected with miR-425-5p(miR-425-5p-agomir+BLM),mice were treated for 21 days.Collagen fiber deposition in HS tissue was detected by Masson’s trichrome staining.The expressions of miR-425-5p,typeⅠcollagen(Col-Ⅰ),typeⅢcollagen(Col-Ⅲ),α-smooth muscle actin(α-SMA),TGF-β1 and Smad2 in tissues and cells were detected by RT-qPCR and Western blot.Results Compared with normal group,the expression of miR-425-5p in HS group was decreased(t=4.242,P<0.001),while the mRNA and protein expression of TGF-β1 was increased(P<0.001).Compared with control group,the mRNA and protein expression of Col-Ⅰ,Col-Ⅲ,α-SMA,TGF-β1 and Smad2 in miR-425-5p-mimic group were all decreased(P<0.05).Compared with control group,mRNA and protein expression of Col-Ⅰ,Col-Ⅲ,α-SMA,TGF-β1 and Smad2 in miR-425-5p-inhibitor group was all increased(P<0.05).After co-transfection with miR-425-5p-mimic,the relative luciferase activity of TGF-β1-3′-UTR-WT group was reduced as compared with TGF-β1-3′-UTR-MUT group(P<0.05).Compared with miR-425-5p-mimic+NC-pcDNA3.1 group,mRNA and protein expression of Col-Ⅰ,Col-Ⅲ,α-SMA,TGF-β1

关 键 词：增生性瘢痕 miR-425-5p 胶原沉积 转化生长因子-β1 SMAD2 

分 类 号：R619.6[医药卫生—外科学]