PKM2 Mediates The Promoting Effect Of ISG15 On The Proliferation And Migration Of Esophageal Squamous Cell Carcinoma

Background and objectiveEsophageal cancer is one of the most invasive malignant tumors in gastrointestinal tumors with extremely low rate of 5-year survival,and about 90% of the cases are esophageal squamous cell carcinoma(ESCC).Although the current diagnosis and treatment technology is constantly improving,the overall five-year survival rate is often not satisfactory.Therefore,in-depth study of the mechanism of ESCC and finding its potential biomarker is particularly necessary.Interferon-stimulated gene 15(Interferonstimulatinggene15,ISG15)is a ubiquitin-like protein molecule whose abnormal expression involves many types of cancer.ISG15 can not only play a role as a free molecule,but also as a protein posttranslational modification molecule to modify the target protein(ISGylation),which is closely related to the occurrence and development of tumor.The purpose of this study is to clarify the role of free ISG15 in tumorigenesis.Previous studies have showed that ISG15 was highly expressed and promoted the proliferation and migration of ESCC cells.The disorder of energy metabolism is the characteristic of cancer cells,and many studies have shown that aerobic glycolysis is the main mode of metabolism of cancer cells.Pyruvate Kinase M2(PKM2)is a key rate-limiting enzyme in glycolysis pathway and plays an important role in cancer cell metabolism.Our results show that the high expression of PKM2 in ESCC is related to the malignant progression of ESCC cells,which indicates that PKM2 may be abnormally activated in ESCC.PKM2 can aggregate in the nucleus and regulate gene expression through post-translational modification.Signal transducer and activator of transcription 3(STAT3)plays an important role in the regulation of aerobic glycolysis.It has been reported that PKM2 can translocate into the nucleus through post-translational modification to stimulate the activity of transcription factors including β-catenin and STAT3,thus increasing the expression of gene products needed for tumor growth.In this study,molecular cloning,immunohistochemistry,Western blotting,q RTPCR,immunofluorescence,cell proliferation(CCK-8)and migration(scratch)experiments were used to explore the expression level and role of ISG15 and PKM2 in ESCC,as well as the interaction between them and the downstream molecules that mediate the occurrence and development of ESCC,which will provide new ideas for targeted therapy of ESCC.Materials and Methods1.Collection of ESCC tissues and cell lines30 cases of ESCC and matched normal tissues were from Anyang Cancer Hospital,ESCC cell EC9706,KYSE140,KYSE150,KYSE180,KYSE270,KYSE410,KYSE450,KYSE510,KYSE520 and normal esophageal epithelial cell Het-1A were from the cell bank of Shanghai Chinese Academy of Sciences.2.Expression of ISG15 in ESCC tissue and cell line and its effect on biological behavior of ESCCThe m RNA expression of ISG15 in esophageal carcinoma and normal esophageal tissues was analyzed by GEPIA database,and the expression of ISG15 protein was detected by immunohistochemistry and Western Blot technique.Overexpression of ISG15 in KYSE140 and KYSE180,Western Blot was used to detect the efficiency of overexpression.CCK-8 and scratch test were used to detect the effect of overexpression of ISG15 on the biological behavior of ESCC.3.Expression of PKM2 in ESCC tissue and cell line and its effect on biological behavior of ESCCThe expression of PKM2 protein was detected by immunohistochemistry and Western Blot.Overexpression of PKM2 in KYSE150 and KYSE510,Western Blot was used to detect the efficiency of overexpression.CCK-8 and scratch test were used to detect the effect of overexpression of PKM2 on the biological behavior of ESCC.4.Analysis of the relationship between ISG15 and PKM2 in ESCC(1)The expression of ISG15 and PKM2 in ESCC tissues and cells were analyzed by Pearson.(2)After overexpression of ISG15 in KYSE140 and KYSE180,Western Blot and cellular immunofluorescence assay was used to detect the expression and localization of PKM2,respectively.(3)Construct low expression PKM2 plasmid p LKO.1-sh RNA1/2-PKM2,KYSE140 and KYSE180 were selected to verify the down-regulation efficiency,and p LKO.1-sh RNA1-PKM2 was selected for following experiment.In order to further verify the interaction between the two molecules,overexpression plasmid of ISG15 and the low expression plasmid of PKM2 were simultaneously transfected into the above two kinds of cells.Firstly,the overexpression and down-regulation efficiency of the two were verified by Western Blot,and then the subsequent cell function experiments were carried out,CCK-8,clone formation and scratch assay were used to detect the effects of the two plasmids on the biological behavior of ESCC.5.Detection of related molecules in downstream signal pathwayOverexpression of ISG15 in KYSE140 and KYSE180,Western Blot was used to detect the expression of β-catenin,p-STAT3 and STAT3.The m RNA expression levels of c-Myc,c-Jun,survivin and FRA1 were detected by q RT-PCR.6.Statistical analysisSPSS23.0 statistical software was used to analyze the experimental results,and the data were expressed by mean ±standard deviation,and the differences between groups were compared by t-test or analysis of variance,the test level was α = 0.05,and in this paper,*P<0.05,**P<0.01 and ***P<0.001.Results1.ISG15 was highly expressed in ESCC tissues and KYSE450,KYSE520,but low in KYSE140 and KYSE180;Overexpression of ISG15 in those two low expression cells can promote the proliferation and migration of ESCC cells.2.PKM2 was highly expressed in ESCC tissues and KYSE140,KYSE180,but low in KYSE150 and KYSE510;Overexpression of PKM2 in those two low expression cells can promote the proliferation and migration of ESCC cells.3.There was a positive correlation between the expression of ISG15 and PKM2 in ESCC tissues and cells.In KYSE140 and KYSE180,overexpression of ISG15 could up-regulate the expression of PKM2 and promote its nuclear translocation.The overexpression plasmid of ISG15 and the low expression plasmid of PKM2 were simultaneously transfected into the above two cells,CCK-8,clone formation and scratch assay results indicated that the down-regulation of PKM2 could weaken the promotion effect of ISG15 on the proliferation and migration of ESCC.4.Overexpression of ISG15 can induce phosphorylation of STAT3 and upregulate the expression of apoptosis inhibitor gene survivin in KYSE140.Conclusion1.Down-regulation of PKM2 can weaken the promoting effect of ISG15 on the proliferation and migration of ESCC cells.2.Overexpression of ISG15 can up-regulate the expression of PKM2 and promote its nuclear translocation,which induce nuclear STAT3 phosphorylation,and upregulate the expression of apoptosis inhibitor gene survivin,and then mediate the occurrence and development of ESCC.