南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (11): 1312-1319.doi: 10.12122/j.issn.1673-4254.2019.11.08

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CircRNA_005647通过结合 miR-27b-3p抑制小鼠心肌成纤维细胞中纤维化相关基因表达

袁淑菁，梁景南，张 铭，朱杰宁，潘 蓉，李 晖，曾 妮，温艺红，易芷瑶，单志新   

  • 出版日期:2019-12-05 发布日期:2019-11-20
  • 基金资助:

CircRNA_005647 inhibits expressions of fibrosis-related genes in mouse cardiac fibroblasts via sponging miR-27b-3p

  

  • Online:2019-12-05 Published:2019-11-20

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摘要： 目的 研究环形RNA circRNA_005647对小鼠心肌成纤维细胞（CFs）纤维化表型的影响及分子机制。方法 利用血管紧张素Ⅱ 缓释泵[1.46 mg/ （kg·d），2周]诱导建立小鼠高血压心肌纤维化模型，采用Masson染色观察心肌纤维化程度。实时荧光定量PCR检测发生纤维化的小鼠心肌及血管紧张素Ⅱ诱导的小鼠CFs中circRNA_005647的表达。利用放线菌素D和RNase R实验检测circRNA_005647的稳定性。利用重组circRNA_005647 腺病毒（rAd-circRNA_005647）感染小鼠CFs，检测CFs中纤维化相关基因Col1a1、Col3a1、Acta2 的mRNA和蛋白表达变化。双荧光素酶报告基因实验、RNA反义纯化实验及RNA Pulldown实验验证circRNA_005647与微小RNA miR-27b-3p的结合作用。结果 RT-qPCR结果显示，circRNA_005647在高血压诱导纤维化的小鼠心肌和Ang-II处理的小鼠CFs中表达增强（P<0.01）。放线菌素D和RNase R实验证实circRNA_005647比其宿主基因 Myo9a mRNA具有更好的稳定性（P<0.01）和抵抗 RNase R的降解作用。过表达 circRNA_005647可显著抑制小鼠CFs中纤维化相关基因 Col1a1、Col3a1和 Acta2表达（P<0.05）。双荧光素酶报告基因实验、RNA反义纯化实验及 RNA Pull down实验一致性地证实了circRNA_005647与miR-27b-3p之间存在特异结合作用，miR-27b-3p具有促进小鼠CFs的纤维化表型作用（P<0.05），并可减弱circRNA_005647对小鼠CFs中纤维化相关基因表达的抑制作用。结论 CircRNA_005647在心肌纤维化中表达增强，并通过结合miR-27b-3p发挥抑制心肌纤维化作用。

Abstract: Objective To investigate the effect of circRNA_005647 on fibrotic phenotype of mouse cardiac fibroblasts (CFs) and explore its mechanism. Methods We used an angiotensin II (Ang-II) capsule pump (daily dose of 1.46 mg/kg for 2 weeks) to establish a mouse model of myocardial fibrosis in C57BL/6 mice. Masson staining was used to detect myocardial fibrosis in the myocardium. The expression of circRNA_005647 in the myocardium of Ang-II-infused mice and in Ang-II-treated CFs were detected with real-time PCR. Actinomycin D and RNase R exonuclease digestion were used to test the stability of circRNA_005647 in mouse CFs. Over-expression of circRNA_005647 was achieved in the CFs by infecting the cells with a recombinant circRNA_005647 adenovirus (rAd-circRNA_005647), and the expressions of Col1a1, Col3a1 and Acta2 were detected in the cells with real-time PCR and Western blotting. Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay were performed to identify the interaction between circRNA_005647 and miR-27b-3p. Results CircRNA_005647 was up-regulated in the myocardium of Ang-II- infused mice and in Ang-II-treated mouse CFs (P<0.01). CircRNA_005647 was more stable than its host gene Myosin IXA (Myo9a) in response to actinomycin D (P<0.01) and RNase R exonuclease treatment. The expressions of fibrosis-associated genes was down-regulated in the CFs over-expressing circRNA_005647 (P<0.05). Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay revealed the interaction between miR-27b-3p and circRNA_005647. MiR-27b-3p obviously enhanced the fibrotic phenotype but reversed the inhibitory effect of circRNA_005647 on the expression of fibrosis associated genes in the CFs (P<0.05). Conclusion CircRNA_005647 is upregulated in cardiac fibrosis and inhibits the expression of fibrosis-related genes through sponging miR-27b-3p in mouse CFs.