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.
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