Molecular Mechanism And Clinical Significance Of AJUBA Mutation In Esophageal Squamous Cell

Background:Esophageal cancer is the eighth most frequently diagnosed cancer and the sixth leading cause of cancer death worldwide, most of which is esophageal squamous cell carcinoma (ESCC). The 5-year overall survival rate of ESCC remains below 15%; Few significant improvements in overall survival have been achieved due to the lack of effective therapies. As high-throughput sequencing technique advanced, researchers identified several AJUBA mutations in a few cohorts. However, we knew little about mutations of AJUBA. It may be important to study the molecular mechanism during the development of ESCC and the clinical significance of AJUBA mutations, thus providing new therapy target and improving patients’ prognosis.Methods:We collected 5 ESCC cohorts, including 545 cases, of which the clinical features and results of whole exome sequencing were accessible. We analyzed whether the mutation state of AJUBA was correlated to TNM staging. Moreover, we drew Kaplan-Meier curves of wildtype and mutant cases and identified whether there was significant difference via Log-Rank test, the result of which might guide mechanism exploration and clinical application. In addition, we conducted lentivirus package by 293T cells and infected targeted cells to establish stably transfected cell lines, including AJUBA knockdown cell lines and wild-type or mutant AJUBA-overexpressing cell lines, which were confirmed by reverse transcription PCR (RT-PCR) and western blot. We then investigated cell proliferation, migration and invasion by CCK-8 and transwell experiment in vitro.Results:There was no significant difference between mutation state of AJUBA and TNM staging (Fisher exact probability, p= 1.000). Based on Kaplan-Meier curves, the cases with mutant AJUBA exhibited a trend of longer survival (Log-Rank test, p=0.13). After screening by hygromycin B or FACS, we detected the effectiveness of knockdown by RT-PCR and western blot. However, the effectiveness of knockdown of AJUBA was not satisfactory. We used puromycin to screen the overexpressing cell lines, followed by confirmation using western blot. We could see the bands of wild-type and mutant AJUBA, which indicated that we established the overexpressing cell lines successfully. We discovered that the overexpressing of wild-type or mutant AJUBA could promote cell proliferation (t tests,48h:p<0.001, p<0.001, p<0.001; 72h:p=0.008, p<0.001, p<0.001). The results of transwell exhibited a stronger migration ability of the three overexpressing cell lines (t tests:p<0.001, p<0.001, p<0.001) and a stronger invasion ability of the cell lines overexpressing wild-type or mutant AJUBA (t tests:p<0.001, p=0.035, p=0.576). Cell line overexpressing wild-type AJUBA had the strongest abilities of migration and invasion.Conclusions:ESCC patients with mutant AJUBA had the trend of better prognosis. The stably transfected knockdown cell lines could serve as the control of further study and the basis of establishing mutant AJUBA-overexpressing cell lines from previously-knockdown wild-type AJUBA cells. Mutant AJUBA-overexpressing cell lines could serve as the materials for further studies in vitro and vivo. The overexpressing of mutant or wild-type AJUBA promoted proliferation, and the overexpressing cell lines had stronger abilities of migration and invasion (except the invasion ability of mutant 2-overexpressing cell line). The cell lines overexpressing wild-type AJUBA had the strongest abilities of migration and invasion.