Preliminary Study Of The Effect Of EF2 On STAT Signaling Pathway And Apoptosis In Tumor Cells

Objective: Eukaryotic translational elongation factor 2(EF2)was selected from our laboratory by fluorescence differential two-dimensional gel electrophoresis(2D-DIGE)and matrix-assisted desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)for lung squamous cell carcinoma.The differential protein expressed between the tumor tissue and the adjacent tissues of the patient is higher in the lung squamous cell carcinoma than in the adjacent tissues.At present,domestic and foreign studies on the relationship between EF2 and tumors have mainly focused on the detection of differential protein expression levels,and the clear mechanism for EF2 to promote tumor development is unclear.In the early work of the laboratory,We found that EF2 may activate the STAT signal transduction pathway in 293 T cells using the dual luciferase reporter gene.Therefore,we propose a scientific hypothesis: whether EF2 promotes the development of cells to a more malignant phenotype through the STAT pathway.In this study,exogenous EF2 gene was transfected into Hela cells of human cervical cancer and H520 cells of lung squamous cell carcinoma to explore the effect of EF2 on the STAT signaling pathway and its mechanism,In order to understand the possible functions of EF2 in the development of tumors,explore whether blocking STAT pathway can inhibit the tumor-promoting effect of EF2,and provide new ideas and methods for tumor treatment.Methods:(1)Selection of cell lines and construction of EF2 high expression and knock down cell lines: Hela and H520 were selected as EF2 high expression and knock downcell lines to verify the high expression and knock down efficiency respectively.(2)Effect of EF2 on phosphorylation of STAT1 and STAT3 key proteins in STAT pathway:cells were collected 48 hours after the transient transfection of EF2,and the total protein was extracted.Western blot was used to detect the changes of phosphorylated proteins in STAT1 and STAT3 in the STAT pathway.(3)Effect of EF2 on the expression and subcellular localization of p-STAT1(Tyr701)and p-STAT3(Y705)proteins:immunofluorescence and nucleolith separation were used to detect whether EF2 promoted the entry of p-STAT1(Tyr701)and p-STAT3(Y705)into the nucleus to play a transcription factor role.(4)Whether EF2 activates STAT3 upstream kinase JAK2.(5)Blocking the effect of JAK2/STAT3 pathway on EF2 localization: JAK2/STAT3 pathway inhibitor was added to Hela cells,and Western blot was used to detect the expression of EF2 in nucleus and cytoplasm of cytoplasmic fractionated protein.(6)Detection of apoptotic phenotype: DAPI staining and flow cytometry were used to detect the effect of EF2 on apoptosis.(7)Effect of EF2 on the downstream apoptosis target gene Bcl-x and Bcl2 of JAK2/STAT3 pathway.Results:(1)Hela and H520 cells were selected as the study objects,and EF2 high expression and knockout cell lines were established successfully.(2)EF2 activates the STAT pathway:Western blot results showed that EF2 affected the expression of p-STAT3(Y705)and p-STAT1(Tyr701)proteins.In other words,the expression of p-STAT3(Y705)and p-STAT1(Tyr701)were increased in cell lines with high EF2 expression,and the difference was statistically significant(P<0.05).In EF2 knockdown cell lines,the expression of p-STAT3(Y705)and p-STAT1(Tyr701)were decreased,and the difference was statistically significant(P<0.05).(3)The results of nuclear cytoplasm extraction and immunofluorescence assay showed that EF2 promoted the nucleation of p-STAT1(Tyr701)protein and inhibited the nucleation of p-STAT3(Y705)protein.(4)EF2 activates STAT3 upstream kinase JAK2: a.Western blot results showed that JAK2 and p-JAK2 expression were increased after EF2 was highly expressed,and the difference was statistically significant(P<0.05).The expression of JAK2 and p-JAK2 were decreased after EF2 knockout,and the difference was statistically significant(P<0.05).(5)Blocking the JAK2/STAT3 pathway reduced EF2 expression in the cytoplasm and nucleus:When the JAK2/STAT3 pathway inhibitor AG490 was added to Hela cells,Western blot results showed decreased expression of EF2 in cytoplasm and nucleus.(6)DAPI staining and flow cytometry showed that EF2 inhibited the apoptosis of Hela cells.(7)EF2 increased the expression of downstream apoptosis target gene Bcl-xl in JAK2/STAT3 pathway: a.q PCR results showed that after EF2 was highly expressed,the expression of Bcl-xl m RNA was increased,the difference was statistically significant,and there was no significant change in the expression of Bcl2 m RNA.b.Western blot results showed that the expression of Bcl-xl was decreased in EF2 knockdown cell lines.Conclusions: 1.EF2 activates the STAT signaling pathway.2.Blocking JAK2/STAT3 pathway inhibits EF2 expression in cytoplasm and nucleus.3.EF2 inhibits tumor cell apoptosis.4.EF2 promotes the expression of Bcl-xl.