【摘要】 【目的】研究长链非编码RNA(lncRNA)RP11-879F14.2调控心肌成纤维细胞纤维化表型的作用及机制。【方法】对心衰患者及健康对照者心肌组织进行Masson染色检测心肌胶原水平。lncRNA表达谱芯片检测心衰患者及健康对照者心肌中lncRNAs的表达变化,实时定量荧光PCR(RT-qPCR)验证RP11-879F14.2在心衰患者心肌中的表达。利用重组RP11-879F14.2腺病毒(rAd-RP11-879F14.2)感染人心房肌成纤维细胞(HAFs),检测纤维化相关基因Col1a1,Col3a1和Acta2表达。RT-qPCR检测HAFs的核/质组分中RP11-879F14.2的水平。基于生物信息学预测和双荧光素酶报告基因实验鉴定RP11-879F14.2与多聚嘧啶区结合蛋白(PTBP1)的结合作用。检测敲低HAFs中PTBP1表达对RP11-879F14.2调控心肌纤维化相关基因表达的影响。【结果】Masson染色结果显示,心衰病人心肌组织发生明显纤维化。RT-qPCR结果证实RP11-879F14.2在心衰患者心肌组织中表达增加(P<0.01)。过表达RP11-879F14.2可在RNA及蛋白水平显著抑制心肌纤维化相关基因表达。核质分离及RT-qPCR检测结果证实RP11-879F14.2主要分布于细胞核中。RP11-879F14.2可与PTBP1结合,并促进HAFs中PTBP1表达,而敲低PTBP1可逆转RP11-879F14.2抑制HAFs中纤维化相关基因表达的作用。【结论】PTBP1可介导RP11-879F14.2发挥抑制心肌纤维化的作用。更多还原

【Abstract】 【Objective】To investigate the effect of lncRNA RP11-879F14.2 on fibrotic phenotype of cardiac fibroblasts(CFs)and the mechanism involved.【Methods】Masson’s trichrome staining was performed to detect the level of myocardial fibrosis in the myocardium of patients with heart failure(HF)and the healthy controls. LncRNAs micro-array was used to detect the expression of lncRNAs in human myocardium samples. Real-time quantitative PCR(RT-qPCR)was performed to verify RP11-879F14.2 expression in the myocardium of HF patients and the healthy controls. The recombinant RP11-879F14.2 adenovirus(rAd-RP11-879F14.2)was used to infect human atrial myofibroblasts(HAFs). Expression of Col1a1,Col3a1 and Acta2 was detected by RT-qPCR and Western blot assay,respectively. Distribution of RP11-879F14.2 in the nucleus and cytoplasma of HAFs was determined by RT-qPCR assay. According to the results of bio-informatic prediction,dual-luciferase reporter assay was performed to confirm the interaction between RP11-879F14.2 and polypyrimidine tract binding protein 1(PTBP1). Effect of PTBP1 knock-down on fibrosis-related genes expression modulated by RP11-879F14.2 in HAFs was determined.【Results】Masson’s trichrome staining showed that the myocardial fibrosis was significantly increased in the myocardium of HF patients. Consistent with lncRNA microarray results,RP11-879F14.2 was found obviously up-regulated in the myocardium of HF patients. Over-expression of RP11-879F14.2 inhibited mRNA and protein expression of myocardial fibrosis-related genes in HAFs. Results of nucleocytoplasmic separation and RT-qPCR assay showed that RP11-879F14.2 mainly distributed in the nucleus of HAFs. Dual-luciferase reporter assay revealed the interaction between RP11-879F14.2 and PTBP1. Over-expression of PTBP1 enhanced PTBP1 expression in HAFs,but knock-down of PTBP1 could reverse the anti-fibrotic effect of RP11-879F14.2 in HAFs.【Conclusion】PTBP1 mediates the anti-fibrotic effect of RP11-879F14.2 in HAFs.更多还原