Possible Mechanism Of MiR-24Inhibits Hypothermia-induced Apoptosis Of Cardiaomyocytes

Objective：To investigate whether the hypothermia environment (25°C) can induce theapoptosis of cardiaomyocytes, and to identify whether miR-24can inhibithypothermia-induced apoptosis of cardiaomyocytes and the possible mechanism.Methods：H9C2cardiaomyocytes were used for all experiments.(1)H9C2cardiaomyocyteswere respectively treated for4h、8h、16h、24h、32h、40h and48hours at25°C inthe low temperature incubator, and normal group as control, then flow cytometricassay was used for measuring the effect of apoptosis, western blot was used to detectthe level of caspase-3and Bim protein, and Quantitative real-time polymerase chainreaction(qRT-PCR) was used to detect the expression level of miR-24. According tothe apoptosis rate we choose24hours as the hypothermia stress time in the nexttransfection experiment.(2)Using Lipofectarmine-2000to mediate transfection, theartificial synthetic miR-24mimic and miR-24inhibitor were transfected tocardiaomyocytes, then fluorescence microscope was used to observe whethertransfection is successful and the qRT-PCR detect the expression level of miR-24and estimate the transfection efficiency.（3）After the transfected cardiaomyocyteswere stressed by low temperature for24hours, we also use flow cytometry to detectthe apoptosis rate, western blot to detect the expression of caspase-3and Bim proteinand the qRT-PCR to detect the expression level of miR-24.Results：（1）After the cardiaomyocytes were stressed by low temperature respectivelyfor4h、8h、16h、24h、32h、40h and48hours, compared with normal control group,the apoptosis rate was gradually increasing and reached a peak at24hours (P＜0.05);the expression of apoptosis market protein caspase-3was also gradually increasingand reached a peak at24hours(P＜0.005); the expression level of miR-24wasgradually decreasing and reached a slack at24hours(P＜0.01); the expression level of Bimprotein was also gradually increasing after16hours (when the miR-24level droppedsignificantly) and reached a peak at24hours(P＜0.005).(2) Transfection concentration for100nm, the level of miR-24was highlyexpressed for8times by miR-24mimic and was low expressed for79%by miR-24inhibitor (P＜0.005).(3) Compared with the hypothermia control group, in the miR-24mimic groupafter hypothermia stress the apoptosis rate was significangtly decreased(P＜0.01),and also the expression level of caspase-3(P＜0.05) and Bim protein(P＜0.05); Onthe contrary in the miR-24inhibitor group after hypothermia stress the apoptosisrate was significangtly increased, and also the expression level of caspase-3and Bimprotein.Conclusion：The25C low temperature can induce cardiaomyocyte apoptosis, the miR-24caninhibit the hypothermia-induced apoptosis in cardiaomyocytes, and its mechanismmay be associated with inhibiting the expression of promote apoptosis protein Bim.