†School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K.
‡Division of Cardiovascular and Diabetes Research, Leeds Institute of Genetics, Health and Therapeutics (LIGHT), University of Leeds, Leeds LS2 9JT, U.K.
Clin Sci (Lond). 2014 Aug;127(4):243-9. doi: 10.1042/CS20130420.
ACE2 (angiotensin converting enzyme 2) plays a critical role in the local tissue RAS (renin-angiotensin system) by hydrolysing the potent hypertensive and mitogenic peptide AngII (angiotensin II). Changes in the levels of ACE2 have been observed in a number of pathologies, including cardiovascular disease, but little is known of the mechanisms regulating its expression. In the present study, therefore, the potential role of miRNAs in the regulation of ACE2 expression in primary human cardiac myofibroblasts was examined. Putative miRNA-binding sites were identified in the 3'-UTR of the ACE2 transcript using online prediction algorithms. Two of these, miR-200b and miR-421, were selected for further analysis. A reporter system using the 3'-UTR of ACE2 fused to the coding region of firefly luciferase was used to determine the functionality of the identified binding sites in vitro. This identified miR-421, but not miR-200b, as a potential regulator of ACE2. The ability of miR-421, an miRNA implicated in the development of thrombosis, to down-regulate ACE2 expression was subsequently confirmed by Western blot analysis of both primary cardiac myofibroblasts and transformed cells transfected with a synthetic miR-421 precursor. Real-time PCR analysis of miR-421 revealed widespread expression in human tissues. miR-421 levels in cardiac myofibroblasts showed significant inter-patient variability, in keeping with the variability of ACE2 expression we have observed previously. In conclusion, the present study is the first to demonstrate that ACE2 may be subject to post-transcriptional regulation and reveals a novel potential therapeutic target, miR-421, which could be exploited to modulate ACE2 expression in disease.
ACE2（血管紧张素转换酶 2）通过水解强效高血压和有丝分裂原肽 AngII（血管紧张素 II）在局部组织 RAS（肾素-血管紧张素系统）中发挥关键作用。在包括心血管疾病在内的许多病理中都观察到 ACE2 水平的变化，但对调节其表达的机制知之甚少。因此，在本研究中，研究了 microRNA 在调节原代人心肌成纤维细胞中 ACE2 表达中的潜在作用。使用在线预测算法在 ACE2 转录本的 3'-UTR 中鉴定推定的 microRNA 结合位点。选择其中两个，miR-200b 和 miR-421，进行进一步分析。使用融合了萤火虫荧光素酶编码区的 ACE2 3'-UTR 的报告系统，在体外确定鉴定的结合位点的功能。该系统鉴定出 miR-421 而不是 miR-200b 是 ACE2 的潜在调节剂。miR-421 能够下调 ACE2 表达，这一点随后通过 Western blot 分析原代人心肌成纤维细胞和转染合成 miR-421 前体的转化细胞得到了证实。miR-421 的实时 PCR 分析显示其在人体组织中广泛表达。心肌成纤维细胞中的 miR-421 水平显示出显著的患者间变异性，与我们之前观察到的 ACE2 表达变异性一致。总之，本研究首次证明 ACE2 可能受到转录后调节，并揭示了一种新的潜在治疗靶点 miR-421，可用于调节疾病中的 ACE2 表达。
