Abstract

Purpose

MicroRNA (miRNA) is known to be involved in the pathological process of congenital heart disease (CHD), and nodal modulator1 (NOMO1) is a critical determinant of heart formation. The present study aims to discover the effect of miR-33a-5p and NOMO1 on CHD.

Methods

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expressions of miR-33a-5p mimic or inhibitor and overexpressed NOMO1 plasmid orNOMO1 knockdown. Human cardiomyocyte progenitor cells (hCMPCs) proliferation was measured by cell counting kit-8 (CCK-8) at 24, 48 and 72 h. Flow cytometry was applied to investigate hCMPCs cell cycle progression and apoptosis. Expressions of cell apoptotic proteins Bax, Cleaved(C) caspase-3 and Bcl-2, and expressions of cardiomyocyte differentiation markers GATA4, troponin T (cTnT) and myocyte enhancer factor2C (MEF2C) in hCMPCs were identified by qRT-PCR and western blot. Target genes and potential binding sites of NOMO1 and miR-33a-5p were predicted with Targetscan 7.2, and was confirmed through dual-luciferase reporter assay.

Results

Up-regulation of miR-33a-5p inhibited hCMPCs proliferation, cell cycle G0/S transition but promoted hCMPCs apoptosis, which was partially mitigated by overexpressed NOMO1. NOMO1 was the target gene of miR-33a-5p. Expressions of Bax and C caspase-3 were enhanced but expressions of Bcl-2, GATA4, cTnT and MEF2C were reduced by up-regulation of miR-33a-5p, which was partially mitigated by overexpressed NOMO1.

Conclusion

Up-regulation of miR-33a-5p inhibited hCMPCs proliferation, cell cycle G0/S transition and differentiation into cardiomyocytes but promoted apoptosis via targeting NOMO1.

中文翻译：

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MiR-33a-5p 靶向 NOMO1 调节人心肌祖细胞增殖、分化和凋亡

摘要

目的

已知 MicroRNA (miRNA) 参与先天性心脏病 (CHD) 的病理过程，而淋巴结调节剂 1 (NOMO1) 是心脏形成的关键决定因素。本研究旨在发现 miR-33a-5p 和 NOMO1 对 CHD 的影响。

方法

定量实时聚合酶链反应 (qRT-PCR) 用于检测 miR-33a-5p 模拟物或抑制剂的表达以及过表达的 NOMO1 质粒或 NOMO1 敲低。通过细胞计数试剂盒 8 (CCK-8) 在 24、48 和 72 小时测量人心肌祖细胞 (hCMPC) 增殖。流式细胞术用于研究 hCMPCs 细胞周期进程和细胞凋亡。qRT-PCR和western blot检测hCMPCs中细胞凋亡蛋白Bax、Cleaved(C) caspase-3和Bcl-2的表达，以及心肌细胞分化标志物GATA4、肌钙蛋白T(cTnT)和肌细胞增强因子2C(MEF2C)的表达。 . 使用 Targetscan 7.2 预测 NOMO1 和 miR-33a-5p 的靶基因和潜在结合位点，并通过双荧光素酶报告基因分析确认。

结果

上调 miR-33a-5p 抑制 hCMPCs 增殖、细胞周期 G0/S 转换，但促进 hCMPCs 凋亡，过表达 NOMO1 可部分缓解这种情况。NOMO1是miR-33a-5p的靶基因。Bax 和 C caspase-3 的表达增强，但 Bcl-2、GATA4、cTnT 和 MEF2C 的表达因 miR-33a-5p 的上调而降低，过表达的 NOMO1 可部分减轻这种情况。

结论

miR-33a-5p 的上调抑制 hCMPCs 增殖、细胞周期 G0/S 转变和分化为心肌细胞，但通过靶向 NOMO1 促进细胞凋亡。