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

CircRNA＿005647 inhibits expressions of fibrosis?related genes in mouse cardiac fibroblasts via sponging miR-27b-3p

目的研究环形RNA circRNA005647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circRNA005647的表达。利用放线菌素D和RNase R实验检测circRNA005647的稳定性。利用重组circRNA005647腺病毒(rAd-circRNA005647)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circRNA005647与微小RNA miR-27b-3p的结合作用。结果 RT-qPCR结果显示,circRNA005647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circRNA005647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circRNA005647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circRNA005647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circRNA005647对小鼠CFs中纤维化相关基因表达的抑制作用。结论 CircRNA005647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。

Objective To investigate the effect of circRNA005647 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 C57 BL/6 mice. Masson staining was used to detect myocardial fibrosis in the myocardium. The expression of circRNA005647 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 circRNA005647 in mouse CFs. Over-expression of circRNA005647 was achieved in the CFs by infecting the cells with a recombinant circRNA005647 adenovirus(rAd-circRNA005647), and the expressions of Col1 a1, Col3 a1 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 circRNA005647 and miR-27 b-3 p. Results CircRNA005647 was up-regulated in the myocardium of Ang-II-infused mice and in Ang-II-treated mouse CFs(P<0.01).CircRNA005647 was more stable than its host gene Myosin IXA(Myo9 a) 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 circRNA005647(P<0.05). Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay revealed the interaction between miR-27 b-3 p and circRNA005647. MiR-27 b-3 p obviously enhanced the fibrotic phenotype but reversed the inhibitory effect of circRNA005647 on the expression of fibrosis associated genes in the CFs(P<0.05). Conclusion CircRNA005647 is upregulated in cardiac fibrosis and inhibits the expression of fibrosis-related genes through sponging miR-27 b-3 p in mouse CFs.