Regulation of expression of nestin in osteosarcoma MG63 cells induced by rapamycin and its mechanism / 肿瘤

ABSTRACT

Objective: To investigate the effect of rapamycin (RAPA) on expression of nestin in osteosarcoma MG63 cells, and to explore its possible mechanism. Methods: The relative proliferation rate of MG63 cells treated with RAPA was determined by MTT assay. The expression levels of nestin mRNA and protein in MG63 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The MG63 cells were pre-treated with extracellular signal-regulated kinase (ERK) inhibitor U0126, protein kinase B (PKB, Akt) inhibitor wortmannin, glycogen synthase kinase-3β (GSK-3β) inhibitor lithium chloride (LiCl) or transient transfection of small interfering RNA (siRNA) targeting GSK-3β and protein kinase A (PKA) inhibitor H89, and then treated with RAPA. The effects of the activities of ERK, Akt, GSK-3β and PKA on the expression of nestin induced by RAPA were examined by Western blotting. The effects of RAPA treatment on the activities of Akt and Gsk-3β [the ratio of phospho-Akt (p-Akt) to Akt and the ratio of p-Gsk-3β to Gsk-3β] were detected by Western blotting. Results: RAPA had no effect on relative proliferation rate of MG63 cells. The protein expression levels of nestin in MG63 cells treated with RAPA (100 nmol/L) for 48 h was 1.93 folds as high as that of the control group (P < 0.05). The mRNA expression levels of nestin in MG63 cells treated with RAPA (100 nmol/L) for 24 and 48 h were 1.89 and 1.84 folds as high as those of the control group, respectively (both P < 0.05). Akt inhibitor wortmannin promote the upregulation of nestin expression induced by RAPA, while GSK-3β inhibitor LiCl or GSK-3β-siRNA inhibited the upregulation of nestin expression induced by RAPA through interfering the expression of GSK-3β. U0126 and H89 did not have significant effect on nestin expression. The ratios of p-Akt to Akt were decreased by 21% and 63% after treatment with RAPA for 24 and 48 h, respectively; while the ratios of p-GSK-3β to GSK-3β were decreased by 26% and 42%, respectively. Conclusion: RAPA increases the expression of nestin in MG63 cells by inhibiting the activity of Akt and increasing the activity of GSK-3β.