The Effect Of Let-7b And MiR-199a On B16F10 Cell Growth And Proliferation

Objective:Based on the preliminary study, to further explore the contribution of let-7b and miR-199a to malignant melanoma growth and proliferation.Methods:Construct an over-expression plasmid and an inhibitor, which targeted on let-7b and miR-199a. It were transferred to highly metastatic melanoma cells (B16F10) using gene transfer technology. So, the expression of let-7b and miR-199a is up or down. The gene expression was validated by Real-time quantitative PCR, the related proteins (cyclinD1 and Met) were detected using Western-blot, the cell growth rate were draw by MTT, and the apoptosis rates were measured using flow cytometry. Finally, using statistical method to analyze the effect of let-7b and miR-199a on B16F10 cell growth and proliferation.Results:1. After transfection, fluorescent cells were observed in the transfected group. The success of transfection has been hinted at.2. Real-time PCR verified that the relative let-7b gene expression of let-7b plasmid group was (3.8776±0.1372), which was significantly higher than the non-transfected group and the empty vector group (1.1400±0.2769) (P<0.05); In the let-7b inhibitor group, the relative let-7b gene expression was (0.2057±0.0263), which was significantly lower than the non-transfected group and the inhibitor control group (0.9760±0.2300) (P<0.05). Also real-time PCR detected that the relative miR-199a gene expression of miR-199a plasmid group was (2.8660±0.2821), which was significantly higher than the non-transfected group and the empty vector group (0.9808±0.1703) (P<0.05); In the miR-199a inhibitor group, the relative miR-199a gene expression was (0.2656±0.0253), which was significantly lower than the non-transfected group and the inhibitor control group (0.7512±0.0690) (P<0.05). Interestingly, there was no significant difference between the non-transfected group, the empty vector group and the inhibitor control group (P>0.05).3. Gray scale analysis indicated that cyclinDl band values in the let-7b plasmid group and let-7b inhibitor group were remarkably high, i.e, (2.023±0.315) and (1.857±0.377), which were significantly higher than the non-transfected group (0.997±0.041) (P<0.05). The miR-199a plasmid group (1.763±0.172), the empty vector group (1.490±0.292), the miR-199a inhibitor group (1.590±0.286), the inhibitor control group (1.443±0.099) and the non-transfected group showed no significant difference (P>0.05).Gray scale analysis indicated that Met band values in the miR-199a plasmid group and miR-199a inhibitor group were remarkably high, i.e, (5.19±0.309) and (4.87±0.044), which were significantly higher than the non-transfected group (2.2±0.198) (P<0.05). The let-7b plasmid group (3.24±0.340), the empty vector group (2.85±0.047), the let-7b inhibitor group (2.49±0.068), the inhibitor control group (2.73±0.033) and the non-transfected group showed no significant difference (P>0.05).4. Cell growth rate showed that the growth trend of the let-7b plasmid group and the miR-199a plasmid group declined.On the third day after transfection, the trend is up to the peak (P<0.05). The growth trend of the let-7b inhibitor group, miR-199a inhibitor group, inhibitor control group and the empty vector group was similar to the non-transfected group.5. Flow cytometry demonstrated that the let-7b plasmid group and the miR-199a plasmid group remarkably increased the apoptotic ratio, i,e, (11.8±1.19)% and (11.3±1.59)%, respectively, compared with the non-transfected group (P<0.05). The apoptotic ratio of the empty vector group (6.75±1.59)%, the let-7b inhibitor group (4.39±1.52)%, the miR-199a inhibitor group (4.97±1.47)%, the inhibitor control group (6.68±1.71)%, was no significant difference, respectively, compared with the non-transfected group (5.77±1.74)% (P>0.05). Conclusion:let-7b and miR-199a can be a negative regulator on the B16F10 cell growth and proliferation.