Effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit+ cardiac stem cells apoptosis / 中华心血管病杂志

ABSTRACT

Objective@#To explored the effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit+ cardiac stem cells apoptosis.@*Methods@#C-kit+ cardiac stem cells were isolated from SD rats by the methods of enzyme digestion and magnetic bead. Cells were divided into the following experimental groups (1) negative control mimics (NCM)group cells were transfected with negative control miRNA-21 mimics for 48 hours; (2)mimics group cells were transfected with miRNA-21 mimics for 48 hours; (3) NCM+ H2O2 group negative control miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H2O2 for 2 hours; (4)mimics+ H2O2 group miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H2O2 for 2 hours. mRNA of miRNA-21 was detected by RT-PCR. The apoptosis rate of C-kit+ cardiac stem cells was determined using the annexin V-FITC/PI staining assay. Western blot was employed to measure the expression of apoptosis related proteins(Caspase-3, Bax, and Bcl-2).@*Results@#(1) Compared with the NCM group, the mRNA expression level of miRNA-21 was significantly up-regulated in mimics group, while obviously down-regulated in NCM+ H2O2 group(all P<0.05). Compared with the mimics group, the mRNA expression levels of miRNA-21 in mimics+ H2O2 group was significantly downregulated (P<0.05), but remarkably upregulated compared with the NCM+ H2O2 group(P<0.05). (2) Flow cytometry results indicated that the early apoptosis rates were similar between the NCM group and mimics group ((4.57±3.45)% vs. (5.13±3.21)%, P>0.05). Compared with the NCM group, the early apoptosis rates were remarkably increased ((79.07±5.75)% vs.(4.57±3.45)%, P<0.05) in NCM+ H2O2 group. Compared with the mimics group, the early apoptosis was significantly up-regulated in the mimics+ H2O2 group ((30.27±1.36)% vs.(5.13±3.21)%, P<0.05), which were further down-regulated in mimics+ H2O2 group compared with the NCM+ H2O2 group ((30.27±1.36)% vs.(79.07±5.75)%, P<0.05). (3) Western blot results showed similar protein expression of Caspase-3, Bax and Bcl-2 between NCM group and mimics group(all P>0.05). Compared with the NCM group, the Caspase-3 and Bax protein expression was significantly increased in NCM+ H2O2 group (all P<0.05), but the protein expression level of Bcl-2 was similar between the 2 groups(P>0.05). The Caspase-3 and Bax protein expression was markedly decreased, while Bcl-2 apparently increased in the mimics+ H2O2 group compared with the NCM+ H2O2 group(all P<0.05).@*Conclusion@#Overexpression of miRNA-21 protects the C-kit+ cardiac stem cells from apoptosis caused by oxidative stress through downregulating proapoptotic and upregulating the antiapoptotic proteins.