miR-129-3p靶向Smad3在TGF-β诱导NIH3T3细胞转分化为肌成纤维细胞过程中抑制细胞活力与迁移  

miR-129-3p targets Smad3 to inhibit viability and migration of NIH3T3 cells during TGF-β-induced transformation into myofibroblasts

作　　者：郝洲华 卢晓明[2] 孟浦[3] 钟严艳[4] 李晓南[4] 李盛[1] HAO Zhou-hua;LU Xiao-ming;MENG Pu;ZHONG Yan-yan;LI Xiao-nan;LI Sheng(Department of Surgery, Hospital of Huazhong University of Science and Technology, Wuhan 430074 , China;Department of Digestive Tumor Surgery, Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022 , China;Department of General Practice, Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022 , China;Department of General Practice, Huazhong University of Science and Technology Hospital, Wuhan 430074 , China)

机构地区：[1]华中科技大学医院外科,湖北武汉430074 [2]华中科技大学同济医学院附属协和医院消化肿瘤外科,湖北武汉430022 [3]华中科技大学同济医学院附属协和医院全科,湖北武汉430022 [4]华中科技大学医院全科,湖北武汉430074

出　　处：《中国病理生理杂志》2019年第9期1668-1675,共8页Chinese Journal of Pathophysiology

基　　金：湖北省自然科学基金资助项目(No.2015CFB621)

摘　　要：目的:探讨微小RNA-129-3p(miR-129-3p)在转化生长因子β(TGF-β)诱导NIH3T3细胞转分化为肌成纤维细胞的过程中对细胞活力与迁移的作用和相关机制。方法: RT-qPCR法检测肾癌患者癌旁组织和肾纤维化组织中miR-129-3p的表达量。建立TGF-β诱导NIH3T3细胞转分化为肌成纤维细胞的模型,RT-qPCR法确定miR-129-3p的表达模式。在细胞中给予miR-129-3p模拟物转染48 h,利用MTT实验检测细胞活力,Western blot法检测Ki-67的蛋白表达水平,利用划痕实验观察其对细胞迁移的影响。利用miRNA靶点分析数据库TargetScan和双萤光素酶报告基因实验对miR-129-3p的靶点进行分析,并通过Western blot实验对其靶点蛋白表达水平的检测进行确证。结果:和肾癌患者癌旁组织相比较,在肾纤维化组织中miR-129-3p的表达明显下调( P <0.01)。同时,在TGF-β诱导NIH3T3细胞转分化为肌成纤维细胞的模型中,miR-129-3p的表达水平也有下调( P <0.01)。在给予TGF-β刺激的同时给予miR-129-3p的模拟物导致细胞活力下降,Ki-67表达降低,细胞迁移行为也受到抑制。TargetScan预测Smad3的3’-UTR与miR-129-3p存在结合位点,并被双萤光素酶报告基因实验确认;Western blot实验结果也进一步证实了miR-129-3p影响Smad3表达。结论: miR-129-3p靶向Smad3在TGF-β诱导NIH3T3细胞转分化为肌成纤维细胞的模型中抑制细胞活力与迁移。AIM: To explore the effects of microRNA-129-3p (miR-129-3p) on the viability and migration of NIH3T3 cells during transforming growth factor-β(TGF-β)-induced transformation into myofibroblasts and the underlying molecular mechanisms. METHODS: RT-qPCR was used to examine the relative expression of miR-129-3p in renal cell carcinoma (RCC)-adjacent tissues and fibrotic renal tissue. NIH3T3 cells were stimulated with TGF-β to transform into myofibroblasts, and miR-129-3p expression level was detected. After transfection with miR-129-3p mimics for 48 h in vitro , the cell viability was measured by MTT assay, the protein expression level of Ki-67 was determined by Western blot, and the cell migration was observed by wound healing assay. The direct target of miR-129-3p was predicted by online database TargetScan and confirmed by dual-luciferase reporter assay. The expression level of target protein was further confirmed by Western blot. RESULTS: Compared with the RCC-adjacent tissues, the expression of miR-129-3p was down-regulated in fibrotic renal tissue ( P <0.01). In TGF-β-induced NIH3T3 cell transformation into myofibroblasts, the expression of miR-129-3p was also decreased ( P <0.01). Transfection with miR-129-3p mimics followed by TGF-β stimulation in the NIH3T3 cells inhibited the viability, Ki-67 expression and migration. TargetScan analysis showed miR-129-3p had binding sites in the 3’-UTR of Smad3, which was confirmed by dual-luciferase reporter assay. The results of Western blot further confirmed that miR-129-3p affected the expression of Smad3. CONCLUSION: miR-129-3p inhibits the viability and migration ability of NIH3T3 cells during TGF-β-induced transformation into myofibroblasts by directly targeting Smad3.

关 键 词：微小RNA-129-3p 肌成纤维细胞 细胞活力 细胞迁移 SMAD3蛋白 

分 类 号：R692[医药卫生—泌尿科学] R363.2[医药卫生—外科学]