Preliminary Study On The Mechanism Of EIF6 On The Esophageal Squamous Cell Carcinoma

Objective:In 2020,the incidence rate and mortality rate of Esophageal cancer（EC）ranked tenth and sixth respectively in the world.Among them,the main histological type of esophageal cancer is esophageal squamous cell carcinoma（ESCC）.At present,the treatment methods of ESCC are mainly surgical treatment,radiotherapy,and chemotherapy.Neoadjuvant radiotherapy and chemotherapy plus surgical resection are recommended for advanced patients.Although these treatments have greatly saved the lives of patients,some patients have not benefited.The research of ESCC targeted therapy mainly focuses on EGFR and PD-1.At present,these two targeted drugs cannot significantly improve the 3-year and 5-year survival rates of patients,so it is necessary to explore more effective and accurate treatment schemes.Eukaryotic translation initiation factor 6（e IF6）is mainly located in the cytoplasm of mammalian cells and can affect the maturation of 60S ribosomal subunit.e IF6,as a speed limiting factor in the last step of translation initiation,can effectively prevent the premature binding of 60s subunit and 40s subunit.In recent years,it has been found that eif6 is overexpressed in a variety of tumors and is closely related to the occurrence and development of tumors.However,whether e IF6 can promote tumorigenesis and development in ESCC and its potential molecular mechanism are unclear.The purpose of this experiment is to preliminarily explore the role and mechanism of e IF6 in ESCC from the tissue and cell levels,to provide basis for relevant research.Methods:1.The expression difference of e IF6 protein in esophageal squamous cell carcinoma was analyzed by bioinformatics,and then the expression of e IF6 in human esophageal squamous cell carcinoma tissue microarray（HEso S180Su08）was detected by Envision immunohistochemistry.Combined with the corresponding medical history data,the relationship between the expression and clinicopathological parameters was analyzed.2.RT-q PCR and Western Blot were used to measure the expression of e IF6 in human esophageal squamous cell carcinoma cell line Kyse-150,TE-1 and normal esophageal squamous epithelial cell line HECC.3.Lentivirus mediated sh RNA stable transfection:observed the fluorescence signal under the inverted fluorescence microscope to confirm the successful transfection.Determined the knockdown efficiency by RT-q PCR and Western blot,and then conduct scratch test,CCK8 test,Transwell test and plate cloning test to detect the biological behavior difference between the stable knockdown group and the control group of cell line kyse-150.4.Detection of EMT protein:E-cadherin,N-cadherin and vimentin proteins were detected by Western blot in stable transfected cell lines,and the changes of mean fluorescence signal intensity of E-cadherin,N-cadherin and vimentin proteins were observed by immunofluorescence method.5.Detection of PI3K/AKT/mTOR pathway:p-AKT^ser473,AKT,p-mTOR and mTOR proteins were detected by Western blot in stably transfected cell lines.Results:1.Immunohistochemical results:the expression of e IF6 protein was up-regulated in esophageal squamous cell carcinoma,and the difference was statistically significant（?²=7.234,P=0.007）.This result was consistent with the results of biological information.The expression level of e IF6 was statistically correlated with the degree of differentiation.The worse the degree of differentiation,the higher the expression level of e IF6,and there was no statistical correlation with age and gender.2.CCK8 experiment:after stable knockdown of e IF6,the OD values of knockdown group and control group were detected at four time points of 24h,48h,72h and 96h.The OD values of knockdown group was significantly lower than those of control group,suggesting that knockdown of e IF6 can reduce the proliferation activity of esophageal squamous cell carcinoma cells in vitro.3.Scratch test:after stable knockdown of e IF6,the migration of the knockdown group in 24 hours was less than that of the control group,suggesting that knockdown of e IF6 can reduce the migration ability of esophageal squamous cell carcinoma cells in vitro.4.Transwell experiment:after stable knockdown of e IF6,the number of cells passing through the chamber in the knockdown group was lower than that in the control group,suggesting that knockdown of e IF6 can reduce the invasion ability of esophageal squamous cell carcinoma cells in vitro.5.Plate cloning experiment:after stable knockdown of e IF6,the number of colony formation in the knockdown group was lower than in the control group,suggesting that knockdown of e IF6 can reduce the colony formation ability of esophageal squamous cell carcinoma cells.6.Detection of EMT protein:in the stably transfected cell line,compared with the control group,E-cadherin protein increased while N-cadherin and vimentin protein decreased.Immunofluorescence showed that the mean fluorescence signal intensity of E-cadherin increased while the signal of N-cadherin and vimentin decreased.7.Detection of PI3K/AKT/mTOR pathway:in the stably transfected cell line,the expression of p-AKT^ser473 and p-mTOR protein decreased compared with the control group.Conclusion:1.The expression of e IF6 in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues,which was consistent with the results of biological information.Clinicopathological analysis showed that the expression level of e IF6 was related to the degree of differentiation.The worse the degree of differentiation,the higher the proportion of high expression.It was speculated that e IF6played a role in promoting the occurrence and development of esophageal squamous cell carcinoma.2.After lentivirus stable transfection knockdown the expression of e IF6,the proliferation activity,migration,invasion and colony forming ability of kyse-150 cell line in vitro decreased,suggesting that e IF6 may play a role in promoting esophageal squamous cell carcinoma.3.e IF6 can promote the progression of EMT in esophageal squamous cell carcinoma,which may be related to PI3K/AKT/mTOR pathway.