Abstract:
Objective To investigate the expression of AJUBA in esophageal squamous cell carcinoma (ESCC) and whether AJUBA affects the proliferation, invasion, and migration of ESCC by regulating MST1, YAP1 and TAZ factors.
Methods Western blot was used to detect the expression levels of the AJUBA protein in the esophageal cancer cell lines KYSE30, KYSE150 and KYSE450. A ShRNA interference vector for AJUBA was constructed and transfected into the KYSE150 esophageal cancer cell lines. The effects of AJUBA on the proliferation, cycle, migration and invasion of KYSE150 cells were investigated by in vitro cloning and formation assay, flow cytometry, scratch assay, and Transwell migration assay. The mRNA and protein expressions of MST1, YAP1 and TAZ were detected by RT-PCR and Western blot. The expression of the AJUBA, YAP1 and TAZ proteins were detected by nucleo-cytoplasmic separation assay.
Results The stable knockdown of AJUBA gene in KYSE150 cell line was established. The results of plate cloning experiment showed that the number of cell clones formed in shAJUBA group was significantly lower than that in the negative control group (P< 0.0001). Flow cytometry results showed that KYSE150 cells in shAJUBA group were mainly blocked in G0/G1 phase, and and the number of cells were significantly reduced in G2/M and S phases (P<0.05). The results of scratch test and Transwell invasion test showed that the migration ability and invasion ability of shAJUBA group were significantly decreased compared with control group (P< 0.001). RT-PCR and Western blot results showed that the mRNA and protein expression levels of MST1, YAP1 and TAZ in shAJUBA group were significantly decreased, the differences were statistically significant (P<0.05). The results of nucleo-cytoplasmic separation indicated that AJUBA was expressed in SHEE (Human esophagus epithelial cell) nucleus, and AJUBA protein was expressed in KYSE150 cells cytoplasm and nucleus. The proteins of YAP1and TAZ were not expressed in SHEE cells, but were expressed in cytoplasm and nucleus of KYSE150 cells. The above results showed statistically significant differences (P<0.05).
Conclusions The AJUBA protein can promote the proliferation, invasion, and migration of esophageal carcinoma cells. This may be related to the activation of MST1, YAP1, and TAZ which may consequently affect the progression of ESCC.