目的: 探究硝酸钕[Nd(NO3)3]对小于胎龄儿(small for gestational age,SGA)绒毛膜细胞凋亡及标志性microRNA表达变化的影响,揭示钕致SGA的潜在分子作用机制。方法: 选取人绒毛膜细胞系(JEG-3),通过CCK-8确定Nd(NO3)3最佳染毒剂量、时间;流式细胞术检测细胞凋亡情况;提取对照组(0 μg/mL)、实验组[低、中、高剂量组(0.2 、2、20 μg/mL)]细胞的miRNA进行qPCR,检测miR-15a、miR-466b-3p、miR-328-3p、miR-144-5p的表达水平。转染miR-15a、miR-144-5p抑制剂后检测细胞转染情况,再次进行CCK-8、流式细胞术验证细胞凋亡情况。结果: CCK-8结果显示,中剂量组细胞存活率下降至75.53%,流式细胞检测同样发现在中剂量组出现了更多凋亡。对JEG-3细胞染毒24 h提取细胞miRNA检测其miR-15a、miR-466b-3p、miR-328-3p、miR-144-5p表达水平,与对照组相比均出现不同程度上调。JEG-3细胞系转染上述microRNA抑制剂后,CCK-8中剂量组miR-144-5p、miR-15a抑制剂转染组其存活率由原75.53%回升至76.71%、83.03%,而凋亡水平则由原18.70%下降至18.44%、15.34%。结论: 硝酸钕对JEG-3毒性影响呈现剂量依赖性,miR-15a、miR-466b-3p、miR-328-3p、miR-144-5p表达水平出现上调,激活下游信号通路调节细胞凋亡,对硝酸钕引起的细胞应激反应具有重要影响,为探究SGA的分子作用机制提供了新的研究方向。
Objective: To investigate the effects of neodymium nitrate [Nd(NO3)3] on chorionic cell apoptosis and the changes of microRNA expression in small for gestational age (SGA) infants, aiming to explore the molecular mechanisms of neodymium inducing SGA. Methods: The human trophoblast cell line (JEG-3) was selected. The optimal dose and time of Nd(NO3)3 exposure were determined by CCK-8 assay. Apoptosis was measured using flow cytometry. MicroRNA (miRNA) was extracted from the control group (0 μg/mL) and experimental group treated with low, medium, and high doses (0.2, 2, 20 μg/mL) of Nd(NO3)3. The expression levels of miR-15a, miR-466b-3p, miR-328-3p, and miR-144-5p were measured by qPCR. After transfection with miR-15a and miR-144-5p inhibitors, transfection efficiency was evaluated, and CCK-8 and flow cytometry assays were repeated to confirm their effects on apoptosis. Results: The CCK-8 assay showed that cell viability in the medium-dose group decreased to 75.53%, and Flow cytometry also indicated increased apoptosis in this group. After 24 hours of Nd(NO3)3 exposure, qPCR analysis showed that miR-15a, miR-466b-3p, miR-328-3p, and miR-144-5p were upregulated compared to the control group. After JEG-3 cell line transfected with the above microRNA inhibitors, the survival rate of miR-144-5p and miR-15a inhibitor transfected group in CCK-8 medium-dose group increased from 75.53% to 76.71% and 83.03%, respectively, while the apoptosis level decreased from 18.70% to 18.44% and 15.34% respectively. Conclusion: The toxic effect of [Nd(NO3)3] on cells is dose-dependent. With increased expression levels of miR-15a, miR-466b-3p, miR-328-3p, and miR-144-5p, downstream signaling pathway is activated and apoptosis mechanism is regulated, which plays an important role in cell stress response induced by (Nd(NO3)3). This study provides a new research direction for exploring the molecular mechanism of SGA.
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