Cardiovascular Center, Children's Hospital of Nanjing Medical University, Nanjing 210036, PR China.
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, PR China.
J Mol Cell Cardiol. 2018 Jan;114:300-308. doi: 10.1016/j.yjmcc.2017.11.015. Epub 2017 Nov 22.
The objective of the study was to elucidate the mechanism by which microRNA-34a (miR-34a) influences heart development and participates in the pathogenesis of congenital heart disease (CHD) by targeting NOTCH-1 through the Notch signaling pathway. Forty D7 pregnant mice were recruited for the purposes of the study and served as the CHD (n=20, successfully established as CHD model) and normal (n=20) groups. The positive expression of the NOTCH-1 protein was evaluated by means of immunohistochemistry. Embryonic endocardial cells (ECCs) were assigned into the normal, blank, negative control (NC), miR-34a mimics, miR-34a inhibitors, miR-34a inhibitors+siRNA-NOTCH-1, siRNA-NOTCH-1, miR-34a mimics+NOTCH-1 OE and miR-34a mimics+crispr/cas9 (mutant NOTCH-1) groups. The expressions of miR-34a, NOTCH-1, Jagged1, Hes1, Hey2 and Csx in cardiac tissues and ECCs were determined by both RT-qPCR and western blotting methods. MTT assay and flow cytometry were conducted for cell proliferation and apoptosis measurement. A dual luciferase reporter assay was applied to demonstrate that NOTCH-1 was the target gene of miR-34a. In comparison to the normal group, the expressions of miR-34a, Jagged1, Hes1 and Hey2 displayed up-regulated levels, while the expressions of NOTCH-1 and Csx were down-regulated in the CHD group. Compared with the blank and NC groups, the miR-34a mimics and siRNA-NOTCH-1 groups displayed reduced expressions of NOTCH-1 and Csx as well as a decreased proliferation rate, higher miR-34a, Jagged1, Hes1 and Hey2 expressions and an increased rate of apoptosis; while an reverse trend was observed in the miR-34a inhibitors group. The expressions of MiR-34a recorded increased levels in the miR-34a mimics+NOTCH-1 OE and miR-34a mimics+crispr/cas9 (mutant NOTCH-1) groups, however no changes in the expressions of NOTCH-1, Jagged1, Hes1, Hey2, Csx, as well as cell proliferation and apoptosis were observed when compared to the blank and NC groups. The results of our study demonstrated that miR-34a increases the risk of CHD through its downregulation of NOTCH-1 by modulating the Notch signaling pathway.
本研究的目的是通过 Notch 信号通路，阐明 microRNA-34a（miR-34a）通过靶向 NOTCH-1 影响心脏发育并参与先天性心脏病（CHD）发病机制的机制。招募了 40 只 D7 只怀孕小鼠用于该研究，分为 CHD（n=20，成功建立 CHD 模型）和正常（n=20）组。通过免疫组织化学评估 NOTCH-1 蛋白的阳性表达。将胚胎心内膜细胞（ECCs）分为正常组、空白组、阴性对照组（NC）、miR-34a 模拟物组、miR-34a 抑制剂组、miR-34a 抑制剂+siRNA-NOTCH-1 组、siRNA-NOTCH-1 组、miR-34a 模拟物+NOTCH-1 OE 组和 miR-34a 模拟物+crispr/cas9（突变 NOTCH-1）组。通过 RT-qPCR 和 Western blot 方法测定心脏组织和 ECCs 中 miR-34a、NOTCH-1、Jagged1、Hes1、Hey2 和 Csx 的表达。通过 MTT 测定法和流式细胞术测量细胞增殖和凋亡。应用双荧光素酶报告基因实验证明 NOTCH-1 是 miR-34a 的靶基因。与正常组相比，CHD 组 miR-34a、Jagged1、Hes1 和 Hey2 的表达水平上调，而 NOTCH-1 和 Csx 的表达水平下调。与空白组和 NC 组相比，miR-34a 模拟物和 siRNA-NOTCH-1 组 NOTCH-1 和 Csx 的表达降低，增殖率降低，miR-34a、Jagged1、Hes1 和 Hey2 的表达升高，凋亡率升高；而 miR-34a 抑制剂组则呈现相反的趋势。miR-34a 模拟物+NOTCH-1 OE 和 miR-34a 模拟物+crispr/cas9（突变 NOTCH-1）组的 miR-34a 表达水平升高，但与空白组和 NC 组相比，NOTCH-1、Jagged1、Hes1、Hey2、Csx 的表达以及细胞增殖和凋亡均无变化。本研究结果表明，miR-34a 通过下调 Notch 信号通路下调 NOTCH-1 增加 CHD 的发病风险。
