5-氮杂胞苷上调miR-27b-5p表达对模拟失重下血管内皮细胞凋亡的影响

目的 探究5-氮杂胞苷（5-azacytidine, 5-AZA）对模拟失重效应下血管内皮细胞miR-27b-5p表达及细胞凋亡的影响。 方法 体外培养人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs），利用MTT试剂盒检测不同浓度5-AZA（0.1 μmol/L、1 μmol/L、5 μmol/L、10 μmol/L、50 μmol/L和100 μmol/L）对HUVECs的毒性作用。采用qRT-PCR技术观察模拟失重环境下miR-27b-5p表达的时程变化及5-AZA药物处理对miR-27b-5p表达的影响；Western blot检测凋亡相关蛋白Bcl-2、Bax和Cleaved Caspase-3的表达，观察5-AZA在回转模拟失重效应下对HUVECs凋亡的影响。 结果 5-AZA对HUVECs的细胞毒性呈浓度依赖性。与对照组相比，0.1 μmol/L、1 μmol/L、5 μmol/L和10 μmol/L组的细胞存活率无统计学差异，50 μmol/L 5-AZA组和100 μmol/L5-AZA组的细胞存活率显著降低（P<0.05）。因此，我们采用10 μmol/L作为后续实验5-AZA的加药浓度。在回转模拟失重效应下，HUVECs的miR-27b-5p表达显著下降（P<0.05），且随回转时间的延长而下降程度增大（P<0.05）。qRT-PCR检测结果提示，5-AZA可显著上调模拟失重效应下HUVECs中miR-27b-5p表达（P<0.05）。Western blot检测结果提示，5-AZA可显著下调模拟失重效应下HUVECs中Bax和Cleaved Caspase-3蛋白表达（P<0.05），上调Bcl-2蛋白表达（P<0.05）。 结论 5-AZA通过上调miR-27b-5p表达，抑制模拟失重效应下HUVECs凋亡的发生。

Effect of 5-azacytidine on apoptosis of vascular endothelial cells induced by simulated microgravity via promoting expression of miR-27b-5p

AIM To investigate the effect of 5-azacytidine (5-AZA) on expression of miR-27b-5p and apoptosis of vascular endothelial cells under simulated microgravity environment. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and MTT kit was used to detected the toxicity of 5-AZA in different concentrations (0.1 μmol/L, 1 μmol/L, 5 μmol/L, 10 μmol/L, 50 μmol/L and 100 μmol/L) on HUVECs. qRT-PCR assay was conducted to validate the time-course changes of miR-27b-5p and the effect of 5-AZA on expression level of miR-27b-5p under simulated microgravity condition. The protein expressions of Bcl-2, Bax and Cleaved Caspase-3 were assessed using Western blot method to observe the effect of 5-AZA on apoptosis of vascular endothelial cells induced by simulated microgravity. RESULTS The cytotoxic effect of 5-AZA on HUVECs was concentration-dependent. There were no statistical differences in the cell viability between 0.1, 1, 5 and 10 μmol/L groups, compared with control group. However, the cell viability in 50 μmol/L and 100 μmol/L groups were decreased significantly (P<0.05). Thus, the concentration of 5-AZA in subsequent experiments is 10 μmol/L. The expression level of miR-27b-5p was down-regulated (P<0.05) and continuously decreased over time (P<0.05) in response to simulated microgravity. The results of qRT-PCR indicated that 5-AZA could induce up-regulation of miR-27b-5p in HUVECs under simulated microgravity (P<0.05). Western blot assay showed that 5-AZA could reduce the protein expressions of Bax and Cleaved Caspase-3 (P<0.05) and enhance the protein expression of Bcl-2 (P<0.05). CONCLUSION 5-AZA protect HUVECs from apoptosis under simulated microgravity via promoting expression of miR-27b-5p.