P16INK4a affects the malignant biological behavior of gallbladder cancer cells by regulating the Wnt/β-catenin pathway

Objective To investigate the effect of cyclin-dependent kinase inhibitor 2A (p16INK4a) on the malignant biological behavior of gallbladder carcinoma (GBC) cells and analyze the underlying mechanism. Methods GBC-SD cells were assigned into four groups: control, pc-NC, pc-p16INK4a, and pc-p16INK4a+SKL2001. GBC-SD cells overexpressing p16INK4a were constructed by lentiviral vector infection and treated with Wnt/β-catenin pathway agonist SKL2001. CCK-8 experiment and colony formation experiment were used to detect cell proliferation activity. Flow cytometry was used to detect cell cycle distribution. Transwell chamber was used to detect the migration and invasion abilities of cells. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA level of p16INK4a in cells. Western Blot was used to detect the expression of p16INK4a and Wnt/β-catenin pathway-related proteins in cells. The in vivo effect of overexpression of p16INK4a on the growth of transplanted tumors was analyzed using a nude mouse subcutaneous transplant tumor model. Results p16INK4a showed low expression in GBC cell lines. Compared with the control group and the pc-NC group, the mRNA and protein expressions of p16INK4a in pc-p16INK4a group were significantly higher with higher proportion of G0/G1 phase cells, while the OD value of cell proliferation, colony number, proportions of S and G2/M phase cells, numbers of cell migration and invasion, and protein levels of β-catenin, c-Myc, and Cyclin D1 were much lower ( P  < 0.05). SKL2001 could conspicuously weaken the effect of overexpression of p16INK4a on GBC-SD cells ( P  < 0.05). The results of in vivo experiments showed that compared with the control group and the pc-NC group, the volume and weight of transplanted tumors, and positive expression of β-catenin protein in the pc-p16INK4a group were lower, while the positive expression of p16INK4a protein was higher ( P  < 0.05), indicating that SKL2001 can attenuate the inhibitory effect of overexpression of p16INK4a on the growth of transplanted tumors in nude mice (P < 0.05). Conclusion Overexpression of p16INK4a may inhibit the malignant biological behavior of GBC-SD cells by suppressing the activation of the Wnt/β-catenin signaling pathway.