Role and mechanisms of lentivirus mediated MBP-1 overexpression in the regulation of proliferation and apoptosis of colon cancer HCT116 cells

Abstract

Objective To investigate the role and mechanisms of lentivirus-mediated overexpression of c-Myc promoter binding protein-1 (MBP-1) gene in the regulation of proliferation and apoptosis of colon cancer HCT116 cells and its mechanism. Method HCT116 cells cultured in vitro were divided into the three groups: no-load infection group, cells were not treated; control lentivirus group, empty vector control lentivirus (LV-GFP) infected HCT116 cells; MBP-1 overexpression lentivirus group (MBP-1 group) : Lentivirus overexpressing MBP-1 (LV-MBP-1-GFP) infected HCT116 cells. The expression of MBP-1 mRNA in each group was detected by RT-PCR, the proliferation rate of cells was observedby CCK-8 test, cell cycle was determined by flow cytometry, and the expression of MBP-1, NF-κB p65, c-Myc, Cyclin D1, Bcl-2, Bax and cleaved caspase-3 proteins was measured by Western blot. SPSS 22.0 was applied to conduct statistical analysis. The data collected from the three groups were analyzed by one-way ANOVA, while F-test was conducted to determinethe homogeneity of variance. If the variances were shown to be homogeneous, LSD test would be carried out for comparisonto be performed between different groups, otherwise Dunnett test would be conducted for comparison to be drawn between different groups. Results Compared with control lentivirus group, the expression of MBP-1 mRNA and protein was significantly increased in MBP-1 group (MBP-1 mRNA:1.02±0.15 vs 4.56±1.03; MBP-1 protein: 0.18±0.01 vs 0.72±0.10; P< 0.001) . Relative to the control lentivirus group [S phase (39.12±2.18) ﹪, G2/M phase (14.81±1.02) ﹪], the MBP-1 group was observed to be in the S phase (45.64±3.21) ﹪ and the G2/M phase (23.16±2.12) ﹪, where a significantly increase of percentage was discovered (P< 0.001) . The expression level of Bcl-2 protein showed declining trend (0.23±0.02, P< 0.001) . In comparison with the control lentivirus group [ (0.55±0.10) , (0.45±0.08) ], the levels of Bax (0.76±0.11) and cleaved caspase-3 protein expression (0.81±0.11) were improved in MBP-1 group (P< 0.001) . Nevertheless, the proliferation of cells (OD value) at 48 h (0.43±0.03) and 72 h (0.52±0.09) , as well as the percentage (31.36±2.02) ﹪ of cells at the G0/G1 phase in the MBP-1 group were reduced when compared to the control lentivirus group [ (46.06±1.89) ﹪, (0.61±0.13) , (0.79±0.15) , (0.62±0.09) ] (P< 0.001) , as were the levels of NF-κB p65 (0.16±0.03) (P< 0.001) , c-Myc (0.43±0.05) (P< 0.001) and CyclinD1 protein expression (0.32±0.001) (P< 0.001) . There was no significant difference observed in the indicators between the control group and the NC group. Conclusion Overexpression of MBP-1 can be effective in suppressing the proliferation of HCT116 cells and inducing cell cycle arrest in S phase. Moreover, its mechanism is speculated to be associated with the inhibited activation of NF-κB signaling pathway. Key words: Lentivirus; Colon cancer; c-Myc gene; Cell proliferation; Apoptosis; NF-κB signaling pathway