1.MUC3A promotes the proliferation of cholangiocarcinoma cells and inhibits cell cycle arrest.
Stable MUC3A knockout RBE cell lines and control RBE cell lines were established by infecting with MUC3A-KO1, KO2, and KONC-transfected viral fluid. The expression level of MUC3A in KO group cells was significantly reduced, as validated by western blotting(Fig. 1A). To clarify the effect of MUC3A on the proliferative ability of cholangiocarcinoma cells, CCK-8 assay and colony formation assay were performed. The results of the CCK-8 assay showed that MUC3A knockout significantly weakened the proliferation ability of RBE cells༈Fig. 1B༉. Furthermore, the growth inhibitory effect caused by MUC3A knockout in RBE cells was also confirmed by colony formation assay༈Fig. 1C༉.
Previous studies have shown that cell proliferation capacity is closely related to the cell cycle(21). Therefore, we detected the cell cycle using flow cytometry. The results showed that the G1 phase was significantly increased and the S phase was significantly decreased in the MUC3A knockout group. The RBE cells with MUC3A knockout were significantly arrested in the G1 phase(Fig. 1D). These results indicate the involvement of MUC3A in regulating the cell cycle process of cholangiocarcinoma cells. We further examined the expression of cell cycle-related proteins and found that MUC3A knockout induced a significant increase in the expression of cell cycle-related proteins P53 and P21༈Fig. 1E༉. In conclusion, our data suggest that the expression level of MUC3A is closely associated with the proliferative capacity of cholangiocarcinoma cells, and the proliferation ability of cholangiocarcinoma cells is significantly weakened by MUC3A knockout.
2.MUC3A promotes the invasion and metastasis of cholangiocarcinoma cells.
To further investigate the impact of MUC3A on the migration and invasion abilities of cholangiocarcinoma, Transwell experiments and wound healing assays were conducted. The results of the Transwell experiments revealed that knockout of MUC3A significantly inhibited the migration and invasion abilities of cholangiocarcinoma cells(Fig. 2A,2B). Consistent results were obtained from the wound healing assays༈Fig. 2C༉. Furthermore, we examined the expression of EMT-related proteins and found that knockout of MUC3A led to an increase in the expression of the EMT-related protein E-cadherin and a decrease in the expression of N-cadherin༈Fig. 1E༉. In conclusion, these findings indicate a close association between MUC3A and the invasion and metastasis of cholangiocarcinoma, as the knockout of MUC3A markedly reduces the invasion and migration abilities of cholangiocarcinoma cells.
3.MUC3A regulates the ERK signaling pathway in cholangiocarcinoma cells.
To elucidate the mechanism by which MUC3A regulates the occurrence and development of cholangiocarcinoma (CCA), we conducted western blot to validate common signaling pathways. The results indicated that knockout of MUC3A inhibits the ERK signaling pathway(Fig. 2D).
4.MUC3A regulates the proliferation and cell cycle of cholangiocarcinoma cells through the ERK signaling pathway.
To clarify whether MUC3A regulates the proliferation and cell cycle of cholangiocarcinoma cells through the ERK signaling pathway, we used the ERK pathway inhibitor SCH772984. The results showed that after SCH772984 intervention, the difference in proliferation ability between the knockout group and the control group disappeared(Fig. 3A,3B). At the same time, the differences between the cell cycle༈Fig. 3C༉ and cell cycle-related proteins༈Fig. 3D༉ also disappeared. These results indicate that MUC3A regulates the proliferation and cell cycle of cholangiocarcinoma cells through the ERK signaling pathway.
5.MUC3A regulates the invasion and metastasis of cholangiocarcinoma cells through the ERK signaling pathway.
To clarify whether MUC3A regulates the invasion and migration ability of cholangiocarcinoma cells through the ERK signaling pathway, we used the ERK pathway inhibitor SCH772984. The results showed that after SCH772984 intervention, the difference in invasion and migration ability between the knockout group and the control group disappeared(Fig. 4A-4C). At the same time, the expression differences between EMT-related proteins also disappeared༈Fig. 3D༉. These results indicate that MUC3A regulates the invasion and metastasis of cholangiocarcinoma cells through the ERK signaling pathway.
6.MUC3A promotes the growth of cholangiocarcinoma in vivo through the ERK signaling pathway.
To study the role of MUC3A in cholangiocarcinoma in vivo, we established a xenograft model. The results showed that compared to the control group, the tumor growth rate in the KO group was significantly reduced(Fig. 5A). However, this difference was significantly decreased under treatment with SCH772984༈Fig. 5A༉. All of these results indicate that MUC3A promotes the proliferation of cholangiocarcinoma and does so through the ERK signaling pathway.