【摘要】 【目的】研究环形RNA circRNA₁00395调节心肌成纤维细胞中纤维化相关基因表达的作用机制。【方法】CircRNAs表达谱芯片分析结合实时荧光定量PCR验证circRNA₁00395在长期持续性房颤（AF）病人左心耳组织中的表达。检测血管紧张素Ⅱ（Ang-Ⅱ）诱导人心房肌成纤维细胞（HAFs）中circRNA₁00395的表达及在细胞核质中分布情况。利用放线菌素D实验检测circRNA₁00395的RNA稳定性。制备重组circRNA₁00395腺病毒（rAd-circRNA₁00395）并感染HAFs,检测HAFs中纤维化相关基因Col1a1、Col3a1和Acta2的m RNA和蛋白表达。通过双荧光素酶报告基因实验和RNA pull-down实验分别鉴定circRNA₁00395与微小RNA miR-144-3p的结合作用。【结果】Masson染色结果显示AF病人心耳组织的纤维化明显加重（P <0.01）。CircRNA₁00395在AF患者心耳组织中表达降低（P <0.05）,但在Ang-Ⅱ诱导的HAFs中表达明显升高（P <0.05）,且主要分布于HAFs细胞质内。放线菌素D实验证明circRNA₁00395的RNA稳定性明显高于其宿主基因KLHL20 mRNA。过表达circRNA₁00395抑制HAFs中纤维化相关基因表达。双荧光素酶报告基因实验和RNA Pull-down实验均证实circRNA₁00395与miR-144-3p存在特异性的结合作用。CircRNA₁00395可抑制miR-144-3p促HAFs中纤维化相关基因表达的作用。【结论】CircRNA₁00395通过特异结合miR-144-3p来发挥抑制纤维化相关基因表达的作用。更多还原

【Abstract】 【Objective】To investigate the effect of circRNA₁00395 on myocardial fibrosis and explore its mechanism.【Methods】Circular RNA（circRNA）microarray was performed to show the differential expression of circRNAs in the left atrial appendage between patients with long-standing persistent atrial fibrillation（AF）and the healthy controls,and the expression of circRNA₁00395 was detected by RT-qPCR,as well as its distribution in the cytoplasm and nucleus of human atrial fibroblasts（HAFs）. Expression of circRNA₁00395 and its host gene KLHL20 was detected in angiotensin Ⅱ（Ang-Ⅱ）-induced HAFs by RT-qPCR assay. Actinomycin D treatment was performed to test the RNA stability of circRNA₁00395 in HAFs. Over-expression of circRNA₁00395 was achieved in HAFs with infection of the recombinant circRNA₁00395 adenovirus（rAd-circRNA₁00395）. The expression of Col1a1,Col3a1 and Acta2 was detected in HAFs by RT-qPCR and Western blot assay,respectively. Dual luciferase reporter assay and RNA pull-down assay were performed to identify the interaction between circRNA₁00395 and miR-144-3p.【Results】Masson staining revealed that myocardial fibrosis was markedly increased in the left atrial appendage of AF patients（P < 0.01）. CircRNA₁00395 was decreased in the left atrial appendage of AF patients（P < 0.05）,but was increased in Ang-II-induced HAFs（P < 0.05）,with high enrichment in the cytoplasm of HAFs. CircRNA₁00395 was more stable than mRNA of its host gene of KLHL20 when it was subjected to actinomycin D treatment. Overexpression of circRNA₁00395 significantly inhibited the expression of fibrosis-related genes in HAFs. Bioinformatics predicted the potential binding site of miR-144-3p in circRNA₁00395,and the specific interaction between circRNA₁00395 and miR-144-3p was confirmed by the dual luciferase reporter assay and RNA pull-down assay,respectively. Overexpression of miR-144-3p contributed to the expression of fibrosis-related genes in HAFs,while circRNA₁00395 could abolish the pro-fibrosis effect of miR-144-3p.【Conclusion】CircRNA₁00395 exhibits an anti-fibrosis effect in HAFs through sponging miR-144-3p.更多还原