GPX3 Suppresses Tumor Migration And Invasion Via FAK/AKT Pathway In Esophageal Squamous Cell Carcinoma

Esophageal cancer is the most common malignancy in China.The incidence of esophageal cancer in China is the highest in the world,and the mortality rate ranks second in China for malignant tumors.Although the prognosis of early esophageal cancer has improved significantly with the advancement of treatment methods and detection techniques,the early metastasis of esophageal cancer is a major cause of the deterioration of the disease and an important mechanism for the recurrence of esophageal cancer.Increasing clinical trials have confirmed that GPX3 as a tumor suppressor gene is downregulated in liver cancer,breast cancer,lung cancer,cervical cancer,and other malignant tumors,and has a statistically significant relationship with the distant metastasis of tumors.Furthermore,in the mechanism study,it was also confirmed that GPX3 can inhibit the invasion and migration of prostate cancer or liver cancer through the Erk-NFKB-SIP1orWnt/?-Catenin pathway.These studies all indicate that GPX3 may have a signal pathway to inhibit the invasion and migration of tumor cells in malignant tumors.Although in esophageal cancer,there have been clinically reported low expression of GPX3 methylation and the development and prognosis of esophageal cancer have certain statistical significance,but it has not been further studied in the molecular mechanism.Therefore,the purpose of this study was to investigate the mechanism of GPX3 in the invasion and metastasis of esophageal squamous cell carcinoma.Methods1?Bisulfite modification of genomic DNA and Methylation-Specific PCR(MSP)According to the instructions of tissue DNA extraction kit,DNA was extracted from the collected tissues,then the DNA was methylated by methylation modification kit,then the transformed DNA was amplified by MS-PCR.Finally,the 2%agarose gel was selected to be separated and analyzed in the UV Gel spectrophotometer according to the length of the DNA fragment.2?Transwell invasion assayTransfected KYSE-510 cells suspended with 100ul RPIM1640 containing 5%FBS were added into the upper chamber,RPIM1640 containing 15%FBS was put into the bottom,then allow the cells invading for 48h.finally,the transwell room was taken to dye and count the invasive cells.3?Tail vein metastasis assayStably transfected esophageal squamous cell carcinoma cells were injected from tail vein of nude mice after resuspended in PBS.The liver and lung of nude mice were taken out and the small animal imaging experiments were carried out to observe the distant metastasis of tumor cells in the living body after 30 days.Results1?GPX3 is decreased in esophageal squamous cell carcinoma and cell linesThe application of MS-PCR to detect the esophageal cancer tissues,methylation and down-regulation of GPX3 was found in esophageal cancer tissues,there is a tight relation between T-stage and N-stage and GPX3 methylation compared esophageal carcinoma tissues with normal tissues.Nevertheless,We have further demonstrated that gene methylation is a reversible state in the squamous cell carcinoma cells.2.GPX3 can inhibit the proliferation and invasion of esophageal squamous cell carcinoma cellsThe results of the CCK-8 and single cell cloning experiments showed that GPX3 could inhibit the proliferation of esophageal squamous cell carcinoma cells.The wound-healing,migration and invasion experiments and in vivo experiments have confirmed that GPX3 can inhibit the distant metastasis and invasion of cells.3.GPX3 inhibits the distant invasion and migration of esophageal cancer by activating the FAK/AKT signaling pathwayThe expressions of related proteins such as P-AKT,P-FAK,and RAS in the FAK/AKT pathway changed After over-expression and down-regulation of GPX3?while the expression of AKT and FAK remains unchanged,these findings suggested that GPX3 could activate the FAK/AKT signal pathway,whereas the inhibition of the expression of MMP-9 was found after the activation of the FAK/AKT pathway,This suggested that the expression of MMP-9 might be mediated via FAK/AKT pathway and thus regulates the invasion and migration of esophageal squamous cell carcinoma cells.To further verify that MMP-9 is regulated by the FAK/AKT signaling pathway,the positive and negative regulation of FAK/AKT pathway was performed,Subsequently,MMP-9 will also make a response to the changes.These accumulating findings hinted that GPX3 inhibits the distant invasion and migration of esophageal cancer by suppressing the expression of MMP-9 via activating the FAK/AKT signaling pathway.DiscussionWe first revealed the methylation and antitumor effect of GPX3 in esophageal squamous cell carcinoma and its under-lying mechanism through the study of GPX3 in esophageal cancer tissues and cells.We found that GPX3 methylation contributes to the down-regulation of GPX3,then inhibits the proliferation and invasion of esophageal squamous cell carcinoma cells.Further mechanism analysis also demonstrated that the FAK/AKT signaling pathway participates in the inhibitory function of GPX3 on the invasion and migration of esophageal squamous cell carcinoma.These findings reveal the mechanism of GPX3 down-regulation in esophageal squamous cell carcinoma and provide a solid theoretical basis for GXP3 to play a role in tumor suppression in esophageal squamous cell carcinoma.