HSP27 Promotes The Progression Of Esophageal Squamous Cell Carcinoma By Regulating PKM2

Objective: To investigate the mechanism of heat shock protein 27(HSP27)regulating the malignant phenotype of esophageal squamous cell carcinoma;to explore the regulation of HSP27 on PKM2 in esophageal squamous cell carcinoma;to reveal HSP27 in esophageal squamous cell carcinoma Functions in invasion,proliferation,and migration.Methods:(1)Clinical phenotype: immunohistochemistry(IHC)was used to detect the expression of HSP27 in neoplastic tissues of esophageal carcinoma and corresponding normal tissue of adjacent domains.Statistical analysis of HSP27 and clinicopathological parameters(patient age,gender,tumor)Correlation between size,pathological type,and lymph node metastasis and prognosis,and relationship with PKM2.(2)Cell function: the background expression of HSP27 in esophageal cancer cell lines was detected by Western blot.Short hairpin RNA(shRNA),which interferes with HSP27 expression,was transfected into HSP27-expressing esophageal squamous carcinoma cell KYSE450 and KYSE150 using lentiviral transfection.Flow cytometry(FCM)Cell lines stably transfected and knocked down HSP27 were sorted,and the knockdown effect was verified by Western blot and the expression levels of PKM2 and other related biomarkers were detected when HSP27 expression levels were different.Immunoprecipitation(IP)experiments and immunofluorescence experiments were used to explore the interaction between protein molecules.Cell scratch test was used to verify the effect of HSP27 expression on the migratory aptitude of esophageal squamous carcinoma cells,thiazolyl blue colorimetric assay(MTT)to verify the changes of esophageal squamous carcinoma cells in proliferation and Transwell assay to verified the effect of HSP27 expression on the invasion ability of esophageal squamous carcinoma cells.Results:(1)Clinical phenotype: The relative expression level of HSP27 in esophageal squamous cell carcinoma(85/100,85%)was more massive than that in adjacent normal tissues(19/80,23.8%).The difference was statistically significant(P<0.05).And HSP27 expression was associated with pathological grade and T stage of esophageal squamous cell carcinoma(P<0.05).Kaplan-Meier survival analysis demonstrated shorter suvival in patients with high HSP27 expression(Log-rank test,P<0.05),compared with low expression of HSP27.The prognosis is poor.(2)Cell function: Western blot results displayed that the level of HSP27 was highest in KYSE450 cells,followed by KYSE150,KYSE30,EC9706,TE-1,Eca109 cells.The expression of HSP27 was successfully knocked down in KYSE450 and KYSE150 cell lines(P<0.05),and the expression level of PKM2 and EMT-related protein Vimentin was significantly decreased(P<0.05),and the expression of EMT-related protein E-cadherin was increased(P< 0.05).The results of IP experiments showed that there was no direct interaction between HSP27 and PKM2,but HSP27 and SUMO2/3 could directly bind to play a regulatory role,and immunofluorescence results showed that HSP27 and SUMO2/3 co-localized to the cytoplasm and morphologically fuse each other.In addition,after knocking down SUMO2/3,the expression levels of HSP27,PKM2 and EMT-related protein Vimentin decreased,and the expression of EMT-related protein E-cadherin increased.The statistics associated to cell function trials implied that the proliferation ability(P<0.05),migration ability(P<0.05)and invasion ability of esophageal squamous carcinoma cells were curbed(P<0.05),since the expression of HSP27 was down-regulated.Conclusion: HSP27 can promote the progression of esophageal squamous cell carcinoma by combining SUMO2/3 with self-supplementation and stabilize PKM2 expression,suggesting that HSP27 may be a therapeutic target for esophageal squamous cell carcinoma.