Effect of RNAi targetingp38MAPK on high glucose-induced apoptosis of osteoblast MC3T3-E1 cell line
10.3760/cma.j.issn.1000-6699.2011.06.019
- VernacularTitle:慢病毒介导p38MAPK基因沉默对高糖诱导成骨细胞凋亡的影响
- Author:
Zhengping FENG
;
Huacong DENG
;
Rong JIANG
;
Jia DU
;
Danyan CHEN
;
Xiaoyan LIANG
- Publication Type:Journal Article
- Keywords:
p38 mitogen-activated protein kinase;
MC3T3-E1 cell line;
RNA interference;
High glucose;
Apoptosis
- From:
Chinese Journal of Endocrinology and Metabolism
2011;27(6):518-522
- CountryChina
- Language:Chinese
-
Abstract:
Objective To examine the role of p38MAPK in high glucose-induced apoptosis of osteoblast MC3T3-E1 cell line, and to investigate its effect on the expressions of apoptosis-related molecules including caspase3, bax, and bcl-2. Methods The lentiviral vector containing short hairpin RNA targeting p38MAPK was constructed. The cultured osteoblast MC3T3-E1 cell were divided into 5 groups:normal control group(A group), high glucose group(B group), p38MAPK-shRNA transfection group(C group), signal transduction inhibitor group(D group), and transfection with negative control siRNA group(E group). RT-PCR was used to determine the p38MAPK mRNA expression levels in MC3T3-E1 cells. Flow cytometry(FCM)was employed to detect the cell apoptotic percentage. The protein levels of apoptosis-related molecules p38MAPK, p-p38MAPK, caspase-3, bax, and bcl-2 were assayed by Western blot. Ultrastructural alternation of MC3T3-E1 cell was observed under transmission electron microscopy(TEM). Results The lentiviral vector containing short hairp in RNA targeting p38MAPK was successfully constructed and transfected into MC3T3-E1 cells. RT-PCR result suggested that the siRNA targeting p38MAPK could effectively reduce the p38MAPK mRNA expression level induced by high glucose in MC3T3-E1 cell line. FCM showed siRNA significantly decreased high glucose-induced apoptosis percentage of MC3T3-E1 cells(P＜0.01). Meanwhile, we also found the siRNA significantly attenuated the proteins levels of p38MAPK, p-p38MAPK, caspase-3, and gene bax induced by high glucose in MC3T3-E1 cells, whereas the protein level of gene bcl-2 was enhanced remarkably when compared with high glucose group and negative control siRNA group(P＜0.01, P＜0.05).Conclusion The iRNA targeting p38MAPK suppressed high glucose-induced MC3T3-E1 cell apoptosis via inhibiting the activation of p38MAPK signaling pathway, thereby reducing the expressions levels of p-p38MAPK, caspase-3 and gene bax, and up-regulating the level of gene bcl-2.
