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

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

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(0.1μmol/L、1μmol/L、5μmol/L、10μmol/L、50μmol/L和100μmol/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,0.1μmol/L、1μmol/L、5μmol/L和10μmol/L组的细胞存活率无统计学差异,50μmol/L 5-AZA组和100μmol/L5-AZA组的细胞存活率显著降低(P<0.05)。因此,我们采用10μmol/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。

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(0.1μmol/L,1μmol/L,5μmol/L,10μmol/L,50μmol/L and 100μmol/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 simu-lated 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 10μmol/L groups,compared with control group.However,the cell viability in 50μmol/L and 100μmol/L groups were decreased significantly(P<0.05).Thus,the concentration of 5-AZA in subsequent experiments is 10μmol/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.