Expression Of ISG15 In Esophageal Squamous Cell Carcinoma And Its Correlation With USP18 And PKM2

Background and ObjectiveEsophageal cancer is one of the most common gastrointestinal malignancies in the world,with high morbidity and mortality,which is a big threat to human life and health.China is one of the countries with the highest incidence of esophageal cancer,especially in Henan,and the histological type is mainly of squamous cell carcinoma.A large number of literatures have confirmed that there are many factors related to the pathogenesis of esophageal cancer.However,the mechanism of tumorigenesis and development of esophageal cancer is still unclear.Thus most patients have a poor therapeutic effect and poor prognosis.Therefore,further study of the occurrence and development mechanism of esophageal cancer has become the focus and challenge of current researchs.Energy metabolism is a key factor in tumorigenesis.Recent studies have found that aerobic glycolysis is the main metabolic form of most tumor cells and is closely related to the occurrence and development of tumors.M2 Pyruvate Kinase M2(PKM2)is an important rate-limiting enzyme in the glycolysis pathway,which plays a major role in the energy metabolism of tumors.In our previous study,we confirmed by immunohistochemistry that PKM2 was highly expressed in esophageal squamous cell carcinoma and may be related to its occurrence and development.In vivo,the function and activity of PKM2 is regulated by many factors,and the ubiquitin-like modification by interferon-stimulated gene 15(ISG15)is one of the major regulatory pathways.Ubiquitin specific protease 18(USP18)is a specific depolymerase of ISG15.It participates in the regulation of protein functions mainly by deubiquitination.In recent years,studies have found that USP18 can also influence tumorigenesis by regulating the production of interferons and cell proliferation.Our previous study showed that the expression of USP18 in esophageal squamous cell carcinoma was increased and related to the heterogeneity of esophageal squamous epithelium,which led to the follow-up studies.ISG15 is the first Ubiquitin-like protein(UBL)that had been identified.Similar to ubiquitin,ISG15 can covalently modify various proteins and participate in the regulation of multiple signaling pathways,which is very important for innate immune response.In recent years,studies have found that dysfunction of the ubiquitin-modifying enzyme system can lead to tumors.The expression of ISG15 is significantly increased in many tumor cells,and it can regulate the proliferation,invasion,and metastasis of these cells.However,there is no relevant literature has been reported about the expression and role of ISG15 protein in the occurrence and development of esophageal squamous cell carcinoma or its correlation with PKM2 and USP18.In this study,we detected the expression of ISG15 in esophageal squamous cell carcinoma tissue by immunohistochemistry(IHC),and Western Blot was used to detect the expression of ISG15 in esophageal cancer cell lines.Exploring the role of ISG15 in the tumorigenesis and development of esophageal squamous cell carcinoma,and analysis the correlation between ISG15,USP18 and PKM2.This in turn provides a theoretical basis for the further exploration about the role of ISG15 in tumors and the pathogenesis of esophageal cancer.Methods1.The specimen of 70 cases of esophageal squamous cell carcinoma,60 cases of esophageal intraepithelial neoplasia and 30 cases of normal esophageal mucosa tissues were collected and made into histopathological section.Cultured esophageal squamous cell carcinoma cell line KYSE70 and normal esophageal mucosal epithelial cell line HET-1A,then extracted the protein.2.The immunohistochemical SP-9000 three-step method was applied to detect the localization and expression of ISG15 protein in esophageal squamous cell carcinoma,esophageal intraepithelial neoplasia and normal esophageal mucosa tissues.Using Western Blot to detect the expression of ISG15 protein in KYSE70 and HET-1A cell lines.3.Statistical analysis: SPSS21 software was used for data analysis and process.Using t test to analysis counting data and the results were showed as mean±standard deviation.Qualitative data was analyzed by ?2 test.The correlation analysis was analyzed by Spearman rank correlation analysis.Inspection level was set as ?=0.05.Results1.Immunohistochemical results showed that the positive expression rate of ISG15 in esophageal squamous cell carcinoma,esophageal squamous intraepithelial neoplasia and normal esophageal mucosa tissues were 74.3%,33.3% and 6.7% espectively,and the difference was statistically significant between the three groups(?2=45.1,P<0.05).Then performed a pairwise comparison and the differences between the groups all had statistically significant.The positive rate of ISG15 in high-grade squamous intraepithelial neoplasia tissues(46.7%)was higher than that in low-grade(20%),and the difference showed statistical significance(?2 =4.8,P <0.05).The analysis of related clinical characteristics demonstrated that the expression of ISG15 protein had no correlation with age,sex,size,differentiation,invasion or metastasis(P>0.05).Correlation analysis showed that the expression of ISG15 and PKM2 in esophageal squamous cell carcinoma was positively correlated(r =0.672,P <0.05),while there was no correlation between the expression of ISG15 and USP18 or PKM2 and USP18(P>0.05).2.The result of Western Blot showed that the expression of ISG15 was(0.23±0.07)and(1.20±0.34)respectively in HET-1A cell line and KYSE70 cell line,it was significantly higher in esophageal cancer cells than that in normal esophageal mucosal epithelial cells,the difference was statistically significant(t = 8.04,P< 0.05).Conclusion1.The expression of ISG15 was increased in esophageal squamous cell carcinoma,suggesting that ISG15 may be involved in the tumorigenesis of esophageal cancer.2.PKM2 protein and ISG15 protein may have synergistic effect in the occurrence of esophageal squamous cell carcinoma,and jointly promote tumorigenesis.