Molecular Mechanism Of EPHA2 In Regulating Malignant Phenotypes Of Esophageal Cancer Cells

Objective(s): Esophageal cancer is the seventh most common malignancy in the world,and ranks sixth among all cancers in mortality.The mortality rate of advanced esophageal cancer is as high as 90%,but the mortality rate of early esophageal cancer is quite lower,therefore,early diagnosis and therapy are very important.EPHA2 is a receptor tyrosine kinase and highly expressed in many types of cancers indicative of poor prognosis.Literatures and our previous results indicated that EPHA2 was involved in the progression of esophageal cancer,however the underlying regulatory mechanism is still fairly unclear.Therefore,we explored the molecular mechanism of EPHA2 in promoting malignant phenotypes of esophageal cancer cells,and these findings will provide candidate molecular targets for the diagnosis and treatment of esophageal cancer.Methods: We first constructed cell models of EPHA2 knockdown and inactivation(ALW-II-41-27,ALW),and analyzed the effects of EPHA2 silence on the proliferation and migration of esophageal cancer cells.RNA-seq and IP-MS methods were applied to identify differentially expressed genes after EPHA knockdown and EPHA2-interacted proteins in esophageal cancer cells.We also examined the effects of EPHA2 knockdown on key molecules and important signaling pathways in tumor metastasis.In order to facilitate subsequent animal experiments,we also constructed EPHA2 CRISPR KO plasmids.Results: 1.We confirmed the efficiency of EPHA2 knockdown and found that ALW downregulated the protein level of EPHA2 in esophageal cancer cells using Western blotting assay.Functional experiments indicated that knockdown of EPHA2 inhibited the proliferation and migration of esophageal cancer cells.2.We identified 59 differentially expressed genes using RNA-seq method in EPHA2-silenced esophageal cancer cells,and verified the regulation of EPHA2 on selected genes including PI3,GLRX,EGR1 and ANXA10 using qRT-PCR technology.3.We identified 28 EPHA2-interacted proteins in esophageal cancer cells KYSE150 and KYSE510 using IP-MS assay.Based on analysis of databases and literatures,we selected PYCR1 for further verification,and found that PYCR1 bound with EPHA2,but there was no regulatory connection between EPHA2 and PYCR1 in protein level.4.Knockdown of EPHA2 did not regulate the protein expression of uPA,N-cadherin and Vimentin,but significantly suppressed the phosphorylation of STAT3 at Y705 using Western blotting assay.Conclusion(s): 1.Knockdown of EPHA2 inhibited proliferation and migration of ESCC cells.2.In EPHA2-silenced ESCC cells,we identified 59 differentially expressed genes,and verified downregulation mRNA levels of PI3,GLRX,EGR1,and ANXA10 after knocking EPHA2 using qRT-PCR.3.In ESCC cells,we identified 28 EPHA2-interacted proteins,and verified interaction between EPHA2 and PYCR1 using Co-IP assay.4.Silence of EPHA2 suppressed the phosphorylation of STAT3 at Y705.