环状RNA circ_0036176结合miR-218-5p发挥抑制心肌纤维化的作用  

Circular RNA circ_0036176 Inhibiting Myocardial Fibrosis Via Sponging miRNA-218-5p

作　　者：黄智琪 严钰敏 郭晶 郭继深 朱杰宁[5] 方咸宏[5] 徐金东[5] 单志新 HUANG Zhi-qi;YAN Yu-min;GUO Jing;GUO Ji-shen;ZHU Jie-ning;FANG Xian-hong;XU Jin-dong;SHAN Zhi-xin(School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China;School of Pharmacy,Southern Medical University,Guangzhou 510515,China;School of Medicine,South China University of Technology,Guangzhou 510006,China;The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510280,China;Guangdong Cardiovascular Institute,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,China)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]南方医科大学药学院,广东广州510515 [3]华南理工大学医学院,广东广州510006 [4]南方医科大学第二临床医学院,广东广州510280 [5]广东省心血管病研究所,广东省人民医院//广东省医学科学院,广东广州510080

出　　处：《中山大学学报（医学科学版）》2022年第1期61-69,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82070254,81770264);广州市科技计划项目(202002030039,202102080093);广东省自然科学基金(2021A1515011554);广东省人民医院心血管专项(2020XXG003)。

摘　　要：【目的】探究环形RNAcirc_0036176对心肌纤维化表型的调控作用。【方法】通过RT-qPCR检测人心肌组织(包括18例器官捐赠健康志愿者和28例心力衰竭患者)中circ_0036176及其宿主基因MYO9A的表达水平。利用血管紧张素Ⅱ(Ang-Ⅱ)处理人心房肌成纤维细胞(HAFs),RT-qPCR检测circ_0036176和MYO9A的表达水平。放线菌素D和RNaseR处理,鉴定circ_0036176的典型环状结构。使用circ_0036176腺病毒在HAFs中实现充分过表达,mRNA及蛋白水平检测纤维化相关基团的表达差异。利用双萤光素酶报告基因实验和RNA反义纯化技术(RAP)筛选可与circ_0036176有效结合的miRNA。利用C57BL/6乳小鼠心肌成纤维细胞(mCFs)研究miR-218-5p是否介导circ_0036176对心肌纤维化表型的调节作用。【结果】Circ_0036176在心衰心肌中表达增加(P<0.001),但在Ang-Ⅱ诱导的HAFs纤维化细胞模型中,宿主基因及该circRNA的表达一致下调(P<0.01)。放线菌素D和RNaseR实验证实circ_0036176具有典型的环形RNA稳定性。在HAFs中充分过表达circ_0036176后,纤维化相关基因表达均有所下降(P<0.05)。双萤光素酶报告基因实验与RAP实验证实miR-218-5p与circ_0036176间存在结合作用。mCFs中转染miR-218-5p促进了纤维化相关基团的表达,并可有效抵抗circ_0036176的纤维化抑制作用。【结论】Circ_0036176在心衰病人的心肌组织中表达上调,并可通过吸附miR-218-5p的方式来抑制心肌纤维化。【Objective】To investigate the biological effect of circ_0036176 on myocardial fibrosis.【Methods】Levels of circ_0036176 and its host gene Myo9a were determined by real-time quantitative polymerase chain reaction(RT-qPCR)assay in human myocardial tissue,including 22 healthy organ donors and 26 patients with heart failure(HF). A cell model of angiotensin Ⅱ(Ang-Ⅱ)-induced fibrosis in human atrial fibroblasts(HAFs)was achieved. To test the typical ring structure of circ_0036176,actinomycin D treatment and RNase R exonuclease digestion were performed. The expression of fibrosis-related gene in HAFs with overexpression of circ_0036176 was detected at mRNA and protein level. To select miRNAs that can effectively bind to circ_0036176,dual luciferase reporter gene assay and RNA antisense purification assay(RAP)were conducted,respectively. The neonatal mouse cardiac fibroblasts(mCFs)were used to study whether mir-218-5p mediates the effect of circ_0036176 on myocardial fibrosis phenotype.【Results】The expression of circ_0036176 was up-regulated in the myocardium of HF patients(P<0.001),and the expression of circ_0036176 and the host gene Myo9a was down-regulated in Ang-Ⅱ-induced HAFs(P<0.01). In response to actinomycin D treatment and RNase R exonuclease digestion,circ_0036176 was more stable than Myo9a mRNA. The expression of COL1A1,COL3A1,TGF-β1 and ACTA2 was down-regulated in HAFs with overexpression of circ_0036176(P<0.05). Results of dual luciferase reporter gene assay and RAP assay confirmed the interaction between miR-218-5p and circ_0036176. Overexpression of miR-218-5p could promote the expression of fibrosis-related genes,and attenuate the inhibitory effect of circ_0036176 on cardiac fibrosis.【Conclusions】Circ_0036176 is up-regulated in the myocardium of HF patients,and circ_0036176 inhibits the expression of fibrosis-related gene through sponging miR-218-5p in CFs.

关 键 词：环状RNA 心肌纤维化 心肌成纤维细胞 微小RNA 

分 类 号：R363.2[医药卫生—病理学]