Effect of attenuated expression of neuraminidase 3 via RNA interference on the proliferation and apoptosis of osteosarcoma MG-63 cells

Abstract

To investigate the effect of attenuated expression of neuraminidase 3 (NEU3) via RNA interference on the proliferation and apoptosis in human osteosarcoma MG-63 cells. MG-63 cells were immunostained to observe the expression of NEU3. The cells were then divided into 5 groups: MG-63 cells in normal control group (group A) were not treated; MG-63 cells in 30, 50, and 100 nmol/L NEU3 RNA interference groups (groups B, C, and D) were transfected with 30, 50, and 100 nmol/L of NEU3 small interfering RNA (siRNA); negative control group (group E), MG-63 cells were transfected with different species negative siRNA (actin siRNA of mice, 50 nmol/L). The expression level of NEU3 mRNA was measured with real-time fluorescence quantitative PCR (qPCR). The proliferation of the cells was measured by cell counting kit 8 (CCK-8). The cell apoptosis rate was detected by flowcytometry (FCM). The expressions of cell apoptosis related proteins (Ras and Bcl-2) were detected by Western blot assay. NEU3 expressed in the cytoplasm of MG-63 cells under fluorescence microscope. The qPCR results showed that NEU3 mRNA levels were significantly lower in groups B, C, D than that in groups A and E ( P<0.05) after 24 hours of transfection; meanwhile, with the increase of siRNA concentration, NEU3 mRNA levels were significantly decreased ( P<0.05). The CCK-8 results showed that with the increase of siRNA concentration, the survival rate of MG-63 cells was significantly suppressed ( P<0.05) and the apoptosis rate of MG-63 cells was significantly accelerated ( P<0.05) after 48 hours of transfection. FCM results showed that after 24 hours of transfection, the number of live MG-63 cells decreased as that of the dead cells increased in groups B, C, D, and showing significant differences between 3 groups ( P<0.05). While the apoptosis rate in groups B, C, and D showed significant difference when compared with that of group A ( P<0.05); and when compared with group E, the apoptosis rate in groups C and D were significantly reduced ( P<0.05), but there was no significant difference between groups B and E ( P>0.05). The results of Western bolt assay showed that the protein levels of Ras and Bcl-2 in groups B and C were not significantly different from groups A and E ( P>0.05), while the protein levels of Ras and Bcl-2 were significantly decreased in group D ( P<0.05). Attenuated expression of NEU3 could inhibit the survival of MG-63 cells and accelerate its apoptosis. The results suggest that NEU3 could be a possible target for treating osteosarcoma.