circRNA001131通过结合miR-25-3p抑制心肌成纤维细胞中纤维化相关基因的表达

目的:研究环形RNA(circRNA)001131对大鼠心肌成纤维细胞(CFs)纤维化表型的影响及分子机制。方法:采用Masson三色染色法对腹主动脉缩窄(AAC)手术诱导的心肌重构大鼠心肌进行胶原纤维的染色观察。利用芯片检测AAC手术诱导的大鼠心肌中circRNA表达谱的变化。RT-qPCR检测AAC大鼠心肌及血管紧张素Ⅱ(Ang-Ⅱ)诱导的大鼠CFs中circRNA001131的表达。利用放线菌素D和RNase R实验检测circRNA001131的稳定性。利用重组circRNA001131腺病毒(rAd-circRNA001131)感染大鼠CFs,检测CFs中纤维化相关基因Ⅰ型胶原α1链(Col1a1)、Ⅲ型胶原α1链(Col3a1)和肌动蛋白α2(Acta2)的mRNA和蛋白表达。双萤光素酶报告基因实验验证circRNA001131与miR-25-3p的结合作用。结果:RT-qPCR结果证实,circRNA001131在AAC手术诱导的大鼠心肌和Ang-Ⅱ处理的大鼠CFs中表达增强。放线菌素D和RNase R实验结果显示,circRNA001131与其宿主基因--第10号染色体缺失的磷酸酶及张力蛋白同源基因(Pten)mRNA相比,降解水平明显降低。过表达circRNA001131可显著抑制大鼠CFs中纤维化相关基因Col1a1和Col3a1的表达。双萤光素酶报告基因实验证实circRNA001131与miR-25-3p间存在结合作用,miR-25-3p可以减弱circRNA001131对大鼠CFs中纤维化相关基因表达的抑制作用。结论:circRNA001131在心肌纤维化中表达增强,并通过结合miR-25-3p发挥抑制心肌纤维化的作用。

circRNA001131 inhibits expression of fibrosis-related genes in cardiac fibroblasts via sponging miR-25-3p

AIM:To investigate the effect of circular RNA(circRNA)001131 on the fibrotic phenotype of rat cardiac fibroblasts(CFs)and its mechanism.METHODS:Masson trichrome staining was performed on the myocardium of a rat model of abdominal aorta coarctation(AAC)-induced myocardial remodeling.The profile of circRNA expression in the myocardium of AAC-induced rats was detected by circRNA microarray.The expression of circRNA001131 in the myocardium of AAC-induced rats and angiotensin-Ⅱ(Ang-Ⅱ)-treated CFs was confirmed by RT-qPCR.Actinomycin D treatment and RNase R exonuclease digestion were used to test the stability of circRNA001131 in rat CFs.Over-expression of circRNA001131 was achieved in CFs with infection of recombinant circRNA001131 adenovirus(rAd-circRNA001131).The mRNA and protein expression of collagen typeⅠα1 chain(Col1 a1),collagen typeⅢα1 chain(Col3 a1)and actinα2(Acta2)was determined in rat CFs by RT-qPCR and Western blot,respectively.Dual-luciferase reporter assay was performed to identify the interaction between circRNA001131 and miR-25-3 p.RESULTS:circRNA001131 was up-regulated in the myocardium of AAC-induced rats and in Ang-Ⅱ-treated rat CFs.circRNA001131 was more stable than its host gene,phosphatase and tension homolog deleted on chromosome ten(Pten),when it was subjected to actinomycin D and RNase R exonuclease treatment.The expression of fibrosis-associated genes was down-regulated in CFs with over-expression of circRNA001131.Dual-luciferase reporter assay revealed the interaction between miR-25-3 p and circRNA001131,and miR-25-3 p reversed the inhibitory effect of circRNA001131 on the expression of fibrosis-associated genes in rat CFs.CONCLUSION:circRNA001131 is up-regulated in cardiac fibrosis,and inhibits the expression of fibrosis-related genes in CFs through sponging miR-25-3 p.