EPB49 PROMOTES COLORECTAL CANCER CELL PROLIFERATION AND MIGRATION BY REGULATING WNT AND MAPK SIGNALING PATHWAYS THROUGH TIAMLRACL SIGNALING PATHWAY

Xiuhua Li, Limin Yang^*

First Affiliated Hospital of Jinzhou Medical University, Jinzhou 121001, Liaoning Province, China

Objective: To investigate the effect and mechanism of erythrocyte membrane protein band 4.9 (EPB49) regulating mitogen activated protein kinase (MAPK) and wingless MMTV family members (Wnt) signaling pathway on colorectal cancer cell proliferation and migration through T-lymphoma invasion and metastasis inducing factor (Tiam1)-Rac1 signaling pathway. 

Methods: Human colon cancer cell lines SW480 and HT29 were collected for routine culture. An EPB49 overexpression group was constructed using the pSin-EF2-puro vector along with a negative control (NC) group. HT29 was constructed by using PLTHM to create the interference lentivirus group (shRNA group) and its corresponding negative control group. The number of migration cells, Rac1-GTP, EPB49 white expression level, Wnt signaling pathway (p27 and cyclin D1), MAPK signaling pathway related proteins (c-Jun N-terminal kinase (JNK) and p-JNK) expression levels and cell proliferation rate (OD value) of each cell group at 1d, 3d, 5d, 7d were compared, and the interaction between EPB49 and Tiam1 was analyzed. 

Results: At 5d and 7d, the proliferation rate of SW480 cells in the EPB49 group was significantly lower than the NC group (P < 0.05). In HT29 cells, the proliferation rate of the shRNA group was significantly higher than that of the NC group (P<0.05). In SW480 cells, the number of migration cells in the EPB49 group was significantly lower compared to the NC group (P<0.05). In HT29 cells, cell migration in the shRNA group was significantly higher than that found in the NC group (P<0.05). In SW480 cells, the expression level of Rac1-GTP protein in the EPB49 group was significantly lower than that in the NC group (P<0.05). In HT29 cells, the expression level of Rac1-GTP protein in the shRNA group was significantly higher than the NC group (P<0.05). Interaction between EPB49 and Tiam1 was found using immunoprecipitation. In SW480 cells, the expression levels of EPB49 and p27 in the EPB49 group were significantly higher than those in the NC group, and the cyclin D1 protein expression level was significantly lower compared to the control. In HT29 cells, the expression levels of EPB49 and p27 in the shRNA group were significantly lower than those in the NC group, and cyclin D1 protein expression level was significantly higher than the NC group (P<0.05). In SW480 cells, the expression level of p-JNK protein in the EPB49 group was significantly lower compared to the NC group (P<0.05), and there was no significant difference in JNK protein expression among all groups (P>0.05). In HT29 cells, the expression level of p-JNK protein in the shRNA group was significantly higher than that in the NC group (P<0.05), but there was no significant difference in JNK protein expression level among different groups (P>0.05). 

Conclusion: EPB49 can regulate Wnt and MAPK signaling pathways through Tiam1-Rac1 signaling pathway to promote the proliferation and migration of colorectal cancer cells.