Inhibitory effect of transgiutaminase 2 on apoptosis of osteosarcoma cell line MG-63 and its mechanism
10.13481/j.1671-587x.20140418
- VernacularTitle:Ⅱ型谷氨酰胺转氨酶对骨肉瘤MG-63细胞凋亡的抑制作用及其机制
- Author:
Wentao CAI
;
Hong XIA
;
Ningjiang SHENG
;
Mingxia LIN
- Publication Type:Journal Article
- Keywords:
osteosarcoma;
transgiutaminase 2;
bcl-2-associated x protein;
cytochrome C;
apoptosis
- From:
Journal of Jilin University(Medicine Edition)
2014;(4):782-789
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the anti-apoptosis effect of transgiutaminase 2 (TG2)in osteosarcoma cell line MG-63 and to explore the mechanism of inhibiting apoptosis of tumor cells.Methods The TG2-tgrgeted siRNA was designed,and the hypoxia culture model of MG-63 cells was established by a hypoxia incubator and the cells were divided into four groups:normal oxygen group,the cells were cultured under normal oxygen;hypoxia group, the cells were cultured in hypoxic incubators;control siRNA hypoxia group,the cells were cultured in hypoxic incubators after transfection of control siRNA;TG2 siRNA hypoxia group , the cells were cultured in hypoxic incubators after transfection of TG2 siRNA.The expressions of Bax and cytochrome C (Cyt C)and the apoptotic rates were observed at different time (6,12,24,48,and 72 h)after hypoxia culture.Microtiter plate assay was performed to monitor the intracellular TG2 activity.RT-PCR was used to detect the mRNA expressions of TG2 and Bax.The expressions levels of TG2,Bax and Cyt C were observed by immunohistochemical staining and Western blotting method.The apoptotic rates were analyzed using flow cytometry.Results Compared with normal oxygen group,the activity of TG2,the mRNA and protein expression levels of TG2 in hypoxia group and control siRNA hypoxia group were increased remarkably with the prolongation of the hypoxia time (P<0.01);the expression level of Bax protein was decreased significantly (P<0.01 ), but the expression level of Bax mRNA had no significant change (P>0.05);the expression levels of Cyt C protein in cytoplasm and mitochondria and the apoptotic rates had no markedly changes (P>0.05).Compared with hypoxia group and control siRNA hypoxia group,the expression levels of mRNA and protein of TG2 in TG2 siRNA hypoxia group were decreased significantly at different time points (P<0.01);the protein expression levels of Bax and Cyt C in cytoplasm and the apoptotic rates were increased markedly (P<0.01 );the expression level of Cyt C in mitochondria was decreased (P<0.01).Conclusion TG2 can inhibit the apoptosis of tumor cells through down-regulating the Bax expression and preventing Cyt C releasing into the cytoplasm.
