The Effect Of MicroR-33 On Melanoma Cell Line Proliferation And Apoptosis

Objective:Based on preliminary study, to approach the effect of MicroR-33 to melanoma cells line proliferation and apoptosis.Methods:Construct targeted MicroR-33 over-expression mimics and inhibitor, then use gene transfer technology to transfer them into B16F10 melanoma cells.Let the expression of MicroR-33 up or down. The differences of MicroR-33 gene expression were validated by Real-time quantitative PCR; the cell growth curve were draw by MTT; the apoptosis rate and cell cycle were measured by flow cytometry. Finally, using statistical method to analyse the effect of MicroR-33 on B16F10 cell proliferation and apoptosis..Results:1. After transfection, cy3 fluorescent material can be observed in the transfected cells.2. Real-time PCR verified that the relative MicroR-33 gene expression of MicroR-33 mimics group (1.7733E3±2.4583E2)was significantly higher than the non-transfected group and the mimics control group (0.9340±0.2913) (t=12.487, P=0.006<0.05); The relative MicroR-33 gene expression in MicroR-33 inhibitor group (0.6973±0.1958)was partly lower than the non-transfected group and the inhibitor control group(1.0200±0.0848) but not reach the significant difference (P>0.05). There was also no significant difference between the non-transfected group, the mimics control group and the inhibitor control group (P>0.05).3. From visual field of microscope we found the cells density of MicroR-33 mimics group significant lower than other group after transfection 48h.Cell growth curve showed that compare with non-transfected group, the growth trendency of MicroR-33 mimics group declined.And after transfection 48h (1.1875±0.0502)(t=-14.5, P=0.044< 0.05) and72h (1.7500±0.0933) (t=-13.59, P=0.047<0.05), the trend showed significant difference.The growth trendency of the MicroR-33 inhibitor group, mimics control group and inhibitor control group was similar to the non-transfected group.4. Flow cytometry demonstrated the apoptotic ratio of MicroR-33 mimics group(1.8050±0.2050)%remarkably increased, higher than non-transfected group (1.13±0.1414)%(t=l5.000, P=0.042<0.05). The apoptotic ratio of mimics control group (1.3650±0.0212)%, MicroR-33 inhibitor group (1.4450±0.0212)%and inhibitor control group (1.3700±0.2546)%, was no significant difference, respectively, compared with the non-transfected group (P>0.05).5.Cell cycle result showed the proportion of Gl period in MicroR-33 mimics group (62.7±1.7321) compared with non-transfected group (53.5±1.9630) remarkably increased(t=4.98,P=0.016＜0.05), the proportion of S period (23.4±2.5044) compared with non-transfected group (32.6±2.5566) remarkably decreased (t=-4.998,P=0.004<0.05); the proportion of G2 period didn't change a lot compared with non-transfected group.Cell cycle of MicroR-33 inhibitor group,mimics control group and inhibitor control group didn't change a lot compared with non-transfected group (P>0.05)Conclusion:MicroR-33 over expression can restrain the proliferation of B16F10 cells, promote B16F10 cells apoptosis.