Influence of caspase-3 silencing on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells under hypoxia.

AIMS

To investigate the effects of caspase-3 silencing on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells (MSCs) under hypoxia.

METHODS

Rat bone marrow MSCs were transfected with a recombinant shRNA lentivirus targeting caspase-3 expression. Protein expression of caspase-3 was measured by western blotting. Cell proliferation was measured with MTS, and the cell cycle was analyzed by flow cytometry. The apoptosis rate was measured at various time points under hypoxia. Apoptotic morphology was assessed by Hoechst 33258 staining. mRNA levels of caspase-3, Bcl-2, and Bax were measured by real-time PCR.

RESULTS

Western blotting showed that the rat MSCs were stably transfected with the shRNA targeting caspase-3 by a significant reduction of caspase-3 expression. Silencing of caspase-3 expression resulted in a significant increase of MSC proliferation (P < 0.05), an increase of cells in S-phase (52.66 ± 0.30%), and a significant decrease of apoptotic MSCs (P < 0.05). These effects exhibited a slow increase during hypoxic culture. Furthermore, caspase-3 silencing significantly down-regulated mRNA expression of caspase-3 (P < 0.01) and Bax (P < 0.01), and up-regulated Bcl-2 mRNA expression (p < 0.01), thereby increasing the ratio of Bcl-2/Bax (P < 0.05).

CONCLUSION

Caspase-3 silencing modulates the cell cycle of MSCs, promotes cell proliferation, and enhances the anti-apoptotic capacity of MSCs under hypoxia in vitro.