Zhang Lu, Yin Hongli, Jiao Lei, Liu Tianyi, Gao Yuqiu, Shao Yingchun, Zhang Yuanyuan, Shan Hongli, Zhang Ying, Yang Baofeng
Department of Pharmacology (State-Province Key Laboratories of Biomedicine- Pharmaceutics of China, Key Laboratory of Cardiovascular Medicine Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China.
Department of Pharmaceutics, Dalian Children's Hospital, Dalian, China.
Cell Physiol Biochem. 2018;45(4):1641-1653. doi: 10.1159/000487732. Epub 2018 Feb 21.
BACKGROUND/AIMS: Cardiac fibrosis is an important cardiac remodeling event that can ultimately lead to the development of severe arrhythmia and heart failure. MicroRNAs (miRNAs) are involved in the pathogenesis of many cardiovascular diseases. Here, we aimed to investigate the effects of caveolin-3 (Cav3) on the pathogenesis of cardiac fibrosis and the underlying molecular mechanisms.
Cav3 expression was decreased in cardiac fibrosis in vivo and in vitro model. To investigate the role of Cav3 in cardiac fibrosis, we transfected cardiac fibroblasts (CFs) with the siRNA of Cav3 and Cav3-overexpressing plasmid. The collagen content and proliferation of CFs were detected by qRT-PCR, western blot, MTT, and immunofluorescence. A luciferase reporter gene assay and gain/loss of function were used to detect the relationship between miR-22 and Cav3.
Cav3 depletion in CFs induced an increase in collagen content, cell proliferation, and phenotypic conversion of fibroblasts to myofibroblasts. Conversely, Cav3 overexpression in CFs was shown to inhibit angiotensin II-mediated excessive collagen deposition through protein kinase C (PKC)ε inactivation. Cav3 was experimentally confirmed as a direct target of miR-22, containing two seed binding sites in its 3'-untranslated region, and miR-22 was demonstrated to be significantly upregulated in the ischemic border zone in mice after myocardial infarction and in neonatal rat CFs pretreated with angiotensin II. miR-22 overexpression increased CFs proliferation, and collagen and α-smooth muscle actin levels in CFs, while the knockdown of endogenous miR-22 decreased CFs numbers.
Our findings demonstrate that miR-22 accelerates cardiac fibrosis through the miR-22-Cav3-PKCε pathway, which, therefore, may represent a new therapeutic target for treatment of excessive fibrosis-associated cardiac diseases.
背景/目的：心脏纤维化是一种重要的心脏重塑事件，最终可导致严重心律失常和心力衰竭的发生。微小RNA（miRNA）参与多种心血管疾病的发病机制。在此，我们旨在研究小窝蛋白-3（Cav3）对心脏纤维化发病机制的影响及其潜在分子机制。
在体内和体外心脏纤维化模型中，Cav3表达均降低。为研究Cav3在心脏纤维化中的作用，我们用Cav3的小干扰RNA（siRNA）和Cav3过表达质粒转染心脏成纤维细胞（CFs）。通过定量逆转录聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法、MTT法和免疫荧光法检测CFs的胶原蛋白含量和增殖情况。采用荧光素酶报告基因检测法和功能获得/缺失实验检测miR-22与Cav3之间的关系。
CFs中Cav3缺失导致胶原蛋白含量增加、细胞增殖以及成纤维细胞向肌成纤维细胞的表型转化。相反，CFs中Cav3过表达通过蛋白激酶C（PKC）ε失活抑制血管紧张素II介导的过度胶原蛋白沉积。实验证实Cav3是miR-22的直接靶点，在其3'非翻译区含有两个种子结合位点，且miR-22在心肌梗死后小鼠的缺血边缘区和用血管紧张素II预处理的新生大鼠CFs中显著上调。miR-22过表达增加CFs增殖以及CFs中的胶原蛋白和α-平滑肌肌动蛋白水平，而内源性miR-22的敲低减少CFs数量。
我们的研究结果表明，miR-22通过miR-22-Cav3-PKCε途径加速心脏纤维化，因此这可能代表了治疗与过度纤维化相关心脏病的新治疗靶点。
