微小RNA-199a-3p对小鼠皮肤瘢痕疙瘩成纤维细胞的抑制作用及其机制  

Inhibition of microRNA-199a-3p on mouse skin keloid formation and its mechanism

作　　者：冼文娇 梁景南 卢巍 洪跃辉 XIAN Wenjiao;LIANG Jingnan;LU Wei;HONG Yuehui(School of Basic Medicine,Guangdong Jiangmen Chinese Medicine College,Jiangmen 529000;Jiangmen Key Laboratory of Traditional Chinese Medicine Ingredients and Their Mechanisms of Action,Jiangmen 529000;Department of Pharmacy,Jiangmen Central Hospital,Jiangmen 529030,China)

机构地区：[1]广东江门中医药职业学院基础医学院,广东江门529000 [2]江门市中药成分及其作用机制重点实验室,广东江门529000 [3]江门市中心医院药学部,广东江门529030

出　　处：《西安交通大学学报（医学版）》2024年第6期934-940,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：广东省中医药局科研项目(No.20241368);广东省普通高校青年创新人才类项目(No.2023KQNCX252)。

摘　　要：目的 探讨微小RNA-199a-3p(miR-199a-3p)对小鼠皮肤瘢痕疙瘩成纤维细胞的调控作用及其作用靶基因。方法 构建小鼠皮肤瘢痕疙瘩模型,采用实时荧光定量PCR(RT-qPCR)检测miR-199a-3p、瘢痕疙瘩相关基因和Smad1的mRNA表达。原代分离和体外培养成体C57BL/6小鼠皮肤成纤维细胞;利用脂质体将miR-199a-3p模拟物和Smad1 siRNA转染至小鼠皮肤成纤维细胞中;双荧光素酶报告实验验证miR-199a-3p的靶基因;Cell Counting Kit-8(CCK8)方法验证miR-199a-3p和沉默Smad1对皮肤成纤维细胞增殖的影响;RT-qPCR和Western blot法分别检测过表达miR-199a-3p皮肤成纤维细胞的瘢痕疙瘩相关基因和Smad1的mRNA和蛋白表达;Western blot检测小鼠皮肤成纤维细胞分别转染Smad1 siRNA和miR-199a-3p模拟物后瘢痕疙瘩相关基因、Smad1的蛋白表达。结果 疤痕疙瘩组织中瘢痕疙瘩相关基因Col1a1(t=-3.334,P=0.016)、Col3a1(t=-5.927,P=0.001)和ACTA2(t=-3.673,P=0.010)mRNA表达和Smad1(t=-4.403,P=0.010)表达显著高于正常小鼠皮肤组织,而miR-199a-3p(t=7.059,P<0.001)表达显著下降。在皮肤成纤维细胞中过表达miR-199a-3p可以抑制瘢痕疙瘩的相关基因Col1a1(t=5.514,P=0.005)、Col3a1(t=5.132,P=0.014)和ACTA2(t=4.136,P=0.026)mRNA表达和相关蛋白Col1a1(t=4.643,P=0.001)、Col3a1(t=6.554,P=0.003)和α-SMA(t=4.681,P=0.008)表达。miR-199a-3p与Smad1 3′-UTR有结合作用。过表达miR-199a-3p抑制Smad1 mRNA(t=3.556,P=0.024)和蛋白(t=3.781,P=0.019)的表达。分别转染miR-199a-3p模拟物和Smad1 siRNA后均能同时抑制小鼠皮肤成纤维细胞的增殖(F=18.622,P<0.001、<0.001)和瘢痕疙瘩的相关蛋白Col1a1(F=18.804,P=0.003、0.022)、Col3a1(F=33.212,P=0.001、0.001)和α-SMA(F=10.181,P=0.020、0.028)表达。结论 miR-199a-3p通过靶向Smad1抑制瘢痕疙瘩的形成。Objective To investigate the role of miR-199a-3p on mouse skin scar fibroblasts and the potential target of miR-199a-3p.Methods A mouse skin keloid model was established.The mRNA levels of miR-199a-3p,Smad1 and keloid related genes in keloid tissues and normal skin tissues were detected by Real-time quantitative PCR.C57BL/6 mouse skin fibroblasts were isolated and cultured for the cellular experimental study.miR-199a-3p mimic and Smad1 siRNA matter were transiently transfected into mouse skin fibroblasts by liposome reagent.the interaction between miR-199a-3p and the 3′-UTR of Smad1 was confirmed by the dual luciferase reporter assay.The expressions of Smad1 and keloid-related genes at mRNA and protein levels after transfection of miR-199a-3p mimic were determined.The expressions of Smad1 and keloid-related genes at protein level after transfection of miR-199a-3p mimic and Smad1 siRNA were determined by Western blot assay.Results Compared with normal skin tissues,the expressions of Smad1(t=-4.403,P=0.010)and keloid related genes,Col1a1(t=-3.334,P=0.016),Col3a1(t=-5.927,P=0.001)and ACTA2(t=-3.673,P=0.010),were significantly increased in keloid tissues,while miR-199a-3p(t=7.059,P<0.001)expression was significantly decreased.Over-expression of miR-199a-3p could significantly decrease the expressions of keloid-related genes,Col1a1(t=5.514,P=0.005),Col3a1(t=5.132,P=0.014)and ACTA2(t=4.136,P=0.026),in mouse skin fibroblasts.Moreover,the dual luciferase reporter assay revealed that miR-199a-3p could interact with the 3′-UTR of Smad1.miR-199a-3p was observed to inhibit Smad1 at mRNA expression level(t=3.556,P=0.024),and at the post-transcriptional level(t=3.781,P=0.019).Meanwhile,miR-199a-3p mimic,in parallel to Smad1 siRNA,decreased the expressions of keloid-related genes,Col1a1(F=18.804;P=0.003,0.022),Col3a1(F=33.212;P=0.001,0.001)andα-SMA(F=10.181;P=0.020,0.028),and decreased the proliferation of skin fibroblasts(F=18.622;P=<0.001,<0.001).Conclusion miR-199a-3p inhibits the formation of keloid by targeting Smad1.

关 键 词：皮肤瘢痕疙瘩 皮肤成纤维细胞 微小RNA SMAD1 

分 类 号：R751[医药卫生—皮肤病学与性病学]