Lentivirus-Mediated NIBP Gene Silencing Suppresses Epithelial-Mesenchymal Transition Of Human Colon Cancer Cells

Objective To explore the migration and invasion of human colon cancer cell line HCT116, as well as its potential mechanism by observing the effect of Lentivirus-mediated NIBP gene silencing on epithelial-mesenchymal transition (EMT) of human colon cancer cell line HCT116 before and after using TNF-a.Methods Using recombination DNA technique, designed and builded NIBP gene slow virus expression vector Lenti-EGFP-NIBP-miR, and the other one has nothing to do at the same sequence as a negative control (negative control, NC) infected the target cells and the empty cells to be contrast. And joined BSD to screen a stable transfect NIBP lower expression of monoclonal HCT116 cells. QRT-PCR and Western blot analysis were used to examine the expression of NIBP to detect transfect efficiency. Cultured cells were classified into two groups. One treated with nothing which included NIBP-RNAi group、NC group and CON group. And the other one treated with 20 ng/mL of tumor necrosis factor-a (TNF-a) including NIBP-RNAi+ group. NC+ group and CON+ group. Four days later, the changes associated with EMT of cell morphology were observed under a phasecontrast microscope. The mRNA expressions of E-cadherin and N-cadherin were detected by quantitative real-time PCR, and the protein expressions of E-cadherin and N-cadherin were detected by Western blot.Results Stable transfection HCT116 cell lines were Established by NIBP-shRNA slow virus carrier. Green fluorescence of the vector transfect HCT116 cell lines was observed under the fluorescence inverted microscope. Quantitative real-time PCR and Western blot indicated that the NIBP expression of NIBP-RNAi group was significantly lower than CON group and NC group (P<0.05). Follows were the detailed results, the mRNA expressions of E-cadherin in NIBP-RNAi group (8.12±0.50) and NIBP-RNAi+group (4.11±1.65) were higher than CON group (1.00±0.00) and NC group (1.04±0.05) (P＜0.05), while CON+group (0.30±0.13) and NC+group (0.32±0.10) were lower than CON group and NC group (P<0.05). And the mRNA expressions of N-cadherin in NIBP-RNAi group (0.18±0.08) and NIBP-RNAi+group (0.48±0.18) were lower than CON group (1.00±0.00) and NC group (0.94±0.03) (P<0.05), however, CON+ group (6.55±2.31) and NC+ group (7.26±2.20) were larger than CON group and NC group (P<0.05). The protein expression of E-cadherin in NIBP-RNAi group (1.30±0.11) and NIBP-RNAi+ group (0.64±0.01) were more than CON group (0.37±0.01) and NC group (0.34±0.02) (P<0.05), while CON+ group (0.18±0.02) and NC+ group (0.18±0.01) were less than CON group and NC group (P＜0.05). In contrast, the protein expressions of N-cadherin in NIBP-RNAi group (0.18±0.06) and NIBP-RNAi+ group (0.34±0.01) were lower than CON group (0.56±0.05) and NC group (0.47±0.13) (P＜0.05), while CON+ group (0.67±0.06) and NC+ group (0.68±0.03) were higher than CON group and NC group (P<0.05). As we suspected, compared with CON group, the cells in CON+ group and NC+ group had a complete EMT phenotype which manifested as the formation of more filopodia-like processes and spindle-cell shape while the cell tight junctions were decreased. And the mRNA and protein levels of E-cadherin in CON+ group and NC+ group were significantly reduced, but the mRNA and protein levels of N-cadherin were up-regulated(P<0.05). The cells in NIBP-RNAi and NIBP-RNAi+ group were observed had a obvious morphological conversion from mesenchymal cells to epithelial cells which arranged more closely and filoreticulopodias were obviously decreased, and showed that increased E-cadherin mRNA and protein expression but decreased N-cadherin mRNA and protein expression (P<0.05). No statistically significant difference of the above indexes was observed between CON group and NC group, also CON+ group and NC+ group (P>0.05).Conclusion Targeted down-regulation of NIBP expression can inhibit the EMT of human colon cancer cell line HCT116, which may be relate with the inactivation of NF-κB classical way.