Effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit+ cardiac stem cells apoptosis
10.3760/cma.j.issn.0253-3758.2017.11.014
- VernacularTitle: 微小核糖核酸－21对过氧化氢诱导C－kit＋心脏干细胞凋亡的作用及机制
- Author:
Ranzun ZHAO
1
;
Yan WANG
;
Wenwen DENG
;
Bei SHI
;
Xianping LONG
;
Zhenglong WANG
;
Wenming CHEN
Author Information
1. Department of Cardiology, Affiliated Hospital of Zunyi Medical College, Zunyi 563000, China
- Publication Type:Journal Article
- Keywords:
Stem cells;
Apoptosis;
Oxidative stress
- From:
Chinese Journal of Cardiology
2017;45(11):971-977
- CountryChina
- Language:Chinese
-
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.
