Effects Of PI3K/AKT/GSK-3β Signaling Pathway Mediated By Ebp1 On The Migration And Invasion Of Cervical Cancer HELA Cells

Objective:Cervical cancer is one of the common malignant tumors and the gynecological malignant tumor with the highest mortality rate.In recent years,gene-targeted therapy has become a research hotspot in the treatment of cervical cancer,but the treatment of advanced cervical cancer is not ideal.The main reason is that cervical cancer cells have strong migration and invasion capabilities.Therefore,it is very important to find safer and more effective gene targets to treat cervical cancer.It has been reported in the literature that the Erb B3 binding protein Ebp1 encoded by the human PA2G4 gene is highly expressed in a variety of cancers including cervical cancer.Previous studies have found that Ebp1 can induce the proliferation of cervical cancer cells,but Ebp1 has an effect on the migration and invasion of cervical cancer cells.still not clear.Therefore,this study mainly explores the influence of Ebp1 on the malignant behavior of cervical cancer and the possible molecular mechanism,and provides a new route for the development of cervical cancer targeted drugs.Methods:In this study,the Ualcan software was used to analyze the expression of PA2G4 in various cancers and the difference in expression between cervical cancer tissues and normal cervical tissue.Then the recombinant lentiviral transfection method was used to knock down the expression of Ebp1 in cervical cancer HELA cells.After successful transfection,the scratch experiment was used to detect the migration ability of the cells in the sh-NC group and sh-Ebp1 group,and the Transwell experiment was used to detect the invasion ability of the cells in the sh-NC group and the sh-Ebp1 group.The STRING software constructs the Ebp1 and EMT-related protein network,and then uses Western blot to detect the effect of Ebp1 on the expression level of EMT-related proteins.The STRING software constructs the protein network between Ebp1 and PI3K/AKT/GSK3β,and then uses Western blot to detect the effect of Ebp1 on the phosphorylation level of key proteins in the PI3K/AKT/GSK3β pathway.Finally,scratch experiment,Transwell experiment and Western blot were used to detect the migration,invasion ability and EMT-related protein expression levels of Control group,LY294002 group,Perifosine group,TWS119 group and sh-Ebp1 group,respectively.Results:1.Analysis of Ualcan database shows that Ebp1 protein is positively expressed in a variety of cancer tissues.The positive expression level of Ebp1 in cervical cancer tissue was significantly higher than that in normal cervical tissue(P<0.05).Detection of cell proliferation ability: The CCK-8 assay showed that the KD group had slower growth and weaker proliferative ability than the NC group.2.Western blot detection of lentiviral transfection efficiency showed that the expression of Ebp1 protein in the sh-Ebp1 group was significantly lower than that in the sh-NC group(P<0.001),indicating that the HELA cell line knocked down the Ebp1 gene was successfully constructed.3.The results of the scratch experiment showed that compared with the sh-NC group,the scratch healing rate of the sh-Ebp1 group was reduced(24h: P<0.01,48h:P<0.001),indicating that the migration ability of HELA cells after knocking down the Ebp1 gene Weaken.Apoptosis detection: Flow cytometry and TUNEL method were used to detect the apoptosis of HELA cells.The results showed that the proportion of apoptotic cells in KD group was higher than that in NC group(P<0.05).This indicates that Ebp1 gene silencing increases the apoptosis of HELA cells.4.The results of the Transwell experiment showed that compared with the sh-NC group,the number of cells passing through the lower chamber of the Transwell in the sh-Ebp1 group was reduced(P<0.001),indicating that the invasive ability of HELA cells was weakened after the Ebp1 gene was silenced.5.The STRING software constructs a protein network between Ebp1 and EMT,which shows that Ebp1 is related to EMT-related proteins.Western blot results showed that knocking down the Ebp1 gene can up-regulate the expression of E-cadherin protein(P<0.001),and decrease the expression of E-cadherin,Vimentin,Snail,Slug and MMP2 protein(P<0.001).6.The construction of the protein network related to Ebp1 and PI3K/AKT/GSK3β pathway by STRING software shows that Ebp1 is related to the related proteins of PI3K/AKT/GSK3β pathway.Western blot detection results showed that compared with the Control group,the expression of p-PI3 K,p-AKT,and p-GSK3β in the LY294002 group was significantly decreased(P<0.001),and there was no significant change in the expression of Ebp1 protein;in the Perifosine group,the expression of p-AKT and p-AKT and The expression of p-GSK3β protein decreased significantly(P<0.001),and the expression of Ebp1 and p-PI3 K protein did not change significantly;the expression of p-GSK3β protein decreased in TWS119(P<0.01),and the expression of Ebp1,p-PI3 K and p-AKT protein did not Significant changes;the expression of Ebp1,p-PI3 K,p-AKT and p-GSK3β protein decreased in the Sh-Ebp1 group.7.The results of the scratch experiment and the Transwell experiment showed that the migration and invasion ability of HELA cells in the LY294002 group,Perifosine group,TWS119 group and sh-Ebp1 group were all weakened compared with the Control group(scratch: P<0.01,Transwell: P<0.001).Western blot results showed that: Compared with the Control group,the E-cadherin protein levels in the LY294002 group,Perifosine group,TWS119 group and sh-Ebp1 group were up-regulated(P<0.001),while N-cadherin,Vimentin,Snail,Slug,MMP2 The protein level was down-regulated(P<0.001).Conclusion:1.Knockdown of Ebp1 gene can reduce the migration and invasion ability of cervical cancer HELA cells.2.Knockdown of Ebp1 gene can inhibit the EMT process of cervical cancer.3.Ebp1 regulates the EMT process through the PI3K/AKT/GSK3β signaling pathway,and promotes the migration and invasion of cervical cancer HELA cells.