Effect of hypoxia on the expression of TG2 and apoptosis of osteosarcoma cell line MG-63

Objective To investigate the effect of hypoxia on the expression of TG2 and the relationship between TG2 and apoptosis of osteosarcoma cell line MG-63 under hypoxia environment. Methods The hypoxia culture model was established by a hypoxia incubator and divided into four groups. The expression of TG2,cytochrome C,caspase-3 and the rate of apoptosis were observed at different hypoxia culture phases. Microtiter plate assay was performed to monitor intracellular TG2 activity. Caspase-3 activities were measured using chromogenic substrates and determined by measuring the absorbance at 405 nm according to the protocol. The reverse transcriotion PCR was used to detect the mRNA expression of TG2. The protein level of TG2,cytochrome C and Caspase-3 was observed by Western blot analysis. The rate of apoptosis were analyzed using flow cytometry. Results Compared with the oxygen group,the activity,the mRNA level and protein expression of TG2 were increased remarkably with correspondence to the hypoxia time in the pure hypoxia group and the control siRNA hypoxia group. But the activity and protein expression of Caspase-3 didn't change significantly. Similarly,the protein level of cytochrome C in cytosol and the apoptosis rate didn't increase markedly. In TG2 siRNA hypoxia group,the Caspase-3 activity and the protein expression of Caspase-3 were all increased significantly( P 0. 01). Besides,the protein expression of cytochrome C in cytosol and the apoptosis rate rose up significantly( P 0. 01). Conclusion The activity, the mRNA level and protein expression of TG2 increases remarkably with correspondence to the hypoxia time in the hypoxia condition. Overexpression of TG2 can inhibite the hypoxia-induced apoptosis through preventing cytochrome C releasing into the cytosol and suppressing caspase-3 activities.