circZDHHC4通过miR-208/NLK通路调控心肌成纤维细胞活化增殖  

circZDHHC4 Regulates the Proliferation and Activation of Cardiac Fibroblasts Through miR-208/NLK Pathway

作　　者：徐熙媛 李溪 别自东 穆彬 XU Xiyuan;LI Xi;BIE Zidong;MU Bin(Department of Cardiovascular,Affiliated Hospital of Shanxi University of Chinese Medicine,Xianyang 712000,China;Department of Cardiovascular Medicine,Hospital of Cardio Cerebrovascular Disease,General Hospital of Ningxia Medical University,Yinchuan 750001,China;Department of Cardiovascular Medicine,Fei County People’s Hospital Affiliated to Shandong Medical College,Fei County 273400,China)

机构地区：[1]陕西中医药大学附属医院心血管三科,咸阳712000 [2]宁夏医科大学总医院心脑血管病医院心内科,银川750001 [3]山东医学高等专科学校附属费县人民医院心内科,费县273400

出　　处：《宁夏医科大学学报》2020年第8期776-781,786,共7页Journal of Ningxia Medical University

基　　金：山东省医药卫生科技发展计划项目(2019WS119);宁夏医科大学科学研究基金资助项目(XY2017113)。

摘　　要：目的探讨circZDHHC4/miR-208/NLK通路在心肌成纤维细胞活化增殖进程中的作用。方法核质分离检测circZDHHC4在细胞中的表达定位。双荧光素酶报告基因实验检测miR-208与circZDHHC4、NLK的结合能力。心肌成纤维细胞分别转染circZDHHC4过表达载体、miR-208抑制物和NLK过表达载体后,再加入Ang 2诱导,然后用荧光定量PCR检测circZDHHC4、miR-208、NLK、α-SMA、Collagen 1和Collagen 3的RNA表达,CCK-8检测细胞增殖,Western blot检测NLK蛋白水平,免疫荧光检测α-SMA表达。结果 Ang 2能抑制circZDHHC4和NLK在心肌成纤维细胞中的表达,并促进miR-208、α-SMA、Collagen 1和Collagen 3的表达,同时促进细胞增殖。circZDHHC4主要表达定位于细胞质,能竞争性结合miR-208。双荧光素酶报告基因实验同时证实,NLK是miR-208的一个靶基因。circZDHHC4过表达或miR-208表达抑制均能阻断Ang 2抑NLK表达的作用,进而减少α-SMA、Collagen 1和Collagen 3的表达。circZDHHC4还能降低Ang 2促细胞增殖的作用,NLK过表达也能抑制Ang 2促细胞活化的作用。结论 circZDHHC4能通过miR-208/NLK通路抑制Ang 2在心肌成纤维细胞活化增殖中的作用,可能与心肌纤维化密切相关。Objective To investigate the role of circZDHHC4/miR-208/NLK pathway in the activation and proliferation of cardiac fibroblasts. Methods The expression and localization of circZDHHC4 in cells were detected by nuclear and cytoplasmic separation. The binding ability of miR-208 with circZDHHC4 or NLK was detected by dual luciferase reporter gene assay. Cardiac fibroblasts were transfected with circZDHHC4 overexpression vector,miR-208 inhibitors and NLK overexpression vector respectively,and then induced by Ang 2. The RNA expression of circZDHHC4,miR-208,NLK,α-SMA,Collagen 1 and Collagen 3 were detected by fluorescence quantitative PCR,the cell viability was detected by CCK-8,the protein level of NLK was detected by western blot,and α-SMA was detected by immunofluorescence. Results Ang 2 inhibited the expression of circ ZDHHC4 and NLK in cardiac fibroblasts,promoted the expression of miR-208,α-SMA,Collagen 1 and Collagen 3,and promoted cell viability. circZDHHC4 was mainly expressed in cytoplasm and competitively bound miR-208. The dual luciferase reporter gene assay also confirmed that NLK was a target gene of miR-208. The overexpression of circZDHHC4 or the inhibition of miR-208 expression blocked the inhibition of NLK expression by Ang 2,and then reduced the expression of α-SMA,Collagen 1 and Collagen 3. circZDHHC4 reduced the effect of Ang 2 on promoting cell viability,and NLK overexpression also inhibited Ang 2 induced-cell activation. Conclusion circZDHHC4 inhibits the role of Ang 2 in the activation and proliferation of cardiac fibroblasts through miR-208/NLK pathway,which may be closely related to myocardial fibrosis.

关 键 词：心肌纤维化 心肌成纤维细胞 环状RNA miR-208 NLK 

分 类 号：R542.22[医药卫生—心血管疾病]