【摘要】 【目的】探究环状RNA circ＿0018478调控心肌成纤维细胞纤维化表型的作用和可能机制。【方法】RTqPCR检测健康器官捐献者（n=18）和心衰（n=28）患者心肌中circ＿0018478及其宿主基因含E3泛素蛋白连接酶4的HECT和RLD结构域（HERC4）的表达水平。荧光原位杂交（FISH）实验和核质RNA定量检测circ＿0018478的细胞分布情况，放线菌素D干预实验和核糖核酸外切酶（RNase R）消化实验检测circ＿0018478的RNA稳定性。过表达腺病毒介导的circ＿0018478时，分别在RNA和蛋白水平检测乳小鼠心肌成纤维细胞（mCFs）中如Ⅰ型和Ⅲ型胶原等纤维化相关基因表达的影响。利用EdU染色和Trans-well细胞迁移实验鉴定circ＿0018478对mCFs增殖和迁移能力的影响。质谱shot-gun分析circ＿0018478可能翻译蛋白的肽段序列。利用小干扰RNA(siRNA)抑制HERC4-193表达，检测对circ＿0018478调控mCFs纤维化表型的影响。【结果】在心衰病人心肌中，相较于无显著表达差异的宿主基因HERC4，环形RNA circ＿0018478表达显著增加。FISH和核质分离实验结果证实circ＿0018478主要定位于心肌细胞胞质中。放线菌素D和RNase R消化实验证实circ＿0018478具有典型的RNA稳定性。过表达circ＿0018478可抑制mCFs的增殖、迁移和纤维化相关基因的表达。质谱shot-gun和Western blot检测结果提示circ＿0018478可翻译预期的HERC4-193蛋白。过表达circ＿0018478和HERC4-193可一致地抑制mCFs的纤维化表型，而抑制HERC4-193表达可有效减弱circ＿0018478抑制mCFs中纤维化相关基因表达的作用（P<0.05）。【结论】Circ＿0018478通过翻译蛋白HERC4-193发挥抑制心肌成纤维细胞纤维化表型的作用。更多还原

【Abstract】 【Objective】To investigate the effect of circ＿0018478 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved.【Methods】The expression of circ＿0018478 and its host gene of HECT and RLD domain containing E3 ubiquitin protein ligase 4(HERC4)in the myocardium of patients with heart failure(HF)(n=28)and healthy donors(n=18)was analyzed by real-time quantitative polymerase chain reaction(RT-qPCR)assay. The distribution of circ＿0018478 was identified by fluorescence in situ hybridization(FISH)assay and RT-qPCR assay based on nucleocytoplasmic RNA in human AC16 cardiomyocytes. Actinomycin D and RNase R exonuclease digestion were used to test the stability of circ＿0018478 in AC16 cells. RNA and protein expression of fibrosis-related genes was detected in mouse cardiac fibroblasts(mCFs)with adenovirus-mediated over-expression of circ＿0018478. EdU staining and transwell migration assay were performed to detect the effects of circ＿0018478 on mCFs proliferation and migration activities. The potential circ＿0018478-translated protein in mCFs was identified by mass spectrometry(MS)shot-gun assay. HERC4-193was inhibited by small interfering RNA(siRNA),and the effect of HERC4-193 knock-down on the fibrotic phenotype of mCFs with over-expression of circ＿0018478 was studied.【Results】The expression of circ＿0018478 was up-regulated in the myocardium of HF patients,with no significant difference in its host gene of HERC4. The results of FISH and RT-qPCR assay showed that circ＿0018478 was mainly in the cytoplasm of AC16 cardiomyocytes. The characteristic RNA stability of circ＿0018478 was verified by Actinomycin D and RNase R assay,respectively. The enforced expression of circ＿0018478 suppressed proliferation and migration of mCFs,and inhibited the expression of fibrosis-related genes in mCFs.The results of MS shot-gun assay and Western blotting showed that circ＿0018478 could translate protein HERC4-193.Overexpression of the circ＿0018478 and protein HERC4-193 could consistently inhibit the fibrotic phenotype of mCFs.Knock-down of HERC4-193 could attenuate the inhibitory effect of circ＿0018478 on fibrosis-related gene expression in mCFs(P<0.05).【Conclusions】Circ＿0018478 inhibits the fibrotic phenotype of cardiac fibroblasts via translating HERC4-193 protein.更多还原