MiR-199a-5p对肝星状细胞活化增殖的影响  

Effect of MiR-199a-5p on Activity and Proliferation of Hepatic Stellate Cells

作　　者：王源 刘章心怡 苏静慧 王密斯 张荣花 熊亚南 王梅梅 甄永占 章广玲 Wang Yuan;Liu Zhangxinyi;Su Jinghui;Wang Misi;Zhang Ronghua;Xiong Yanan;Wang Meimei;Zhen Yongzhan;Zhang Guangling(Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases,Hebei Key Laboratory for Chronic Diseases,School of Basic Medical Sciences,North China University of Science and Technology,Tangshan,063000;Xiangya School of Medicine,Central South University,Changsha,410013)

机构地区：[1]华北理工大学基础医学院,河北省慢性疾病重点实验室,唐山市慢性病临床基础研究重点实验室,唐山063000 [2]中南大学湘雅医学院,长沙410013

出　　处：《基因组学与应用生物学》2021年第3期1324-1330,共7页Genomics and Applied Biology

基　　金：国家自然科学青年基金资助项目(81201281);河北省自然科学基金资助项目(H2013209180,C2012401037,H2013209040);河北省唐山市科技计划资助项目(12140209A-4);华北理工大学研究生创新项目(2015S16);国家级省级大学生创新项目(201410081021)共同资助。

摘　　要：miR-199a-5p是miRNAs家族的一员。为探讨对人肝星状细胞活化增殖和迁移的影响,为临床肝纤维化治疗提供新的思路,本研究构建miR-199a-5p过表达载体、合成miR-199a-5p反义寡聚核苷酸(antisense oligonucleotide,ASO)经脂质体转染LX-2细胞。采用CCK-8法和Transwell分别检测细胞增殖和迁移能力;集落形成实验检测LX-2细胞的集落形成能力;实时荧光定量PCR技术检测细胞中转染miR-199a-5p后纤维化相关基因α-SMA和CollagenⅠ的mRNA表达水平;Western blot检测各组细胞中α-SMA表达水平。结果表明miR-199a-5p可以促进LX-2细胞增殖(P<0.01)、迁移(P<0.01)和集落形成(P<0.01);而ASO-199a-5p-5p组细胞增殖(P<0.01)、迁移能力(P<0.01)和集落形成能力(P<0.01)受到抑制。RTqPCR结果显示miR-199a-5p在受TGF-β1刺激后的LX-2细胞中的miRNA表达水平高于未受刺激组的(P<0.05),转染miR-199a组细胞中α-SMA的mRNA(P<0.01)和蛋白(P<0.05)表达水平升高。以上结论表明miR-199a-5p过表达能促进肝星状细胞活化、增殖。miR-199 a-5 p is a member of miRNAs family. To explore the effect on activation, proliferation and migration of human hepatic stellate cells and provide a new idea for clinical treatment of hepatic fibrosis, in this study, the overexpression vector of miR-199 a-5 p was constructed and the antisense oligonucleotide(ASO) of miR-199 a-5 p was synthesized and transfected into LX-2 cells by liposome. The Cell Counting Kit-8 kit and Transwell were used to detect cell proliferation and migration ability;the colony formation experiment was used to detect the colony forming ability of LX-2 cells;real-time quantitative PCR was used to detect the mRNA expression levels of the fibrosis-related genes α-SMA and CollagenⅠafter miR-199 a-5 p transfection in the cells;Western blot was used to detect the α-SMA and Collagen I protein levels in each group. The results showed that miR-199 a-5 p can promote the proliferation(P<0.01), migration ability(P<0.01) and colony formation ability(P<0.01) of LX-2 cells,while inhibiting the expression of miR-199 a-5 p can inhibit cell proliferation, cell migra tion abilities and colony formation ability(P<0.01). The RT-qPCR results showed that the expression level of miR-199 a-5 p in LX-2 cells that were treated with TGF-β1 were higher than that without TGF-β1 treated(P<0.05), over-expression of miR-199 a-5 p could make both the α-SMA mRNA(P<0.01) and protein(P<0.05) expression levels increased. These conclusions indicated that miR-199 a-5 p could promote the cells activity and proliferation ability of LX-2 cells.

关 键 词：miR-199a-5p 肝星状细胞 细胞增殖 迁移 

分 类 号：R575[医药卫生—消化系统]