Effects Of Ebp1 Silencing On Cervical Cancer Cells Proliferation,Invasion And Metastasis

Objective:Ebpl,encoded by the human PA2G4 gene,is one of the major binding proteins for the ErbB3 receptor,a member of the epidermal growth factor receptor family.It has been reported that Ebpl plays an important role in many cancers such as breast cancer,prostate cancer,oral squamous cell carcinoma and liver cancer,Previous research found that Ebpl expressed highly in cervical cancer tissues and lowly in adjacent tissues,but its role in cervical cancer is still unclear.Therefore,the purpose of this study was to investigate the effect of Ebpl on the proliferation,invasion and metastasis of cervical cancer cell line HELA cells.Methods:In this study,recombinant lentivirus infection assay was used to knock down the expression of Ebpl protein in cervical cancer HELA cells.The experiment was divided into two groups:PA2G4-RNAi virus group(KD group)and negative control virus group(NC group).The expression of Ebpl protein in HELA cells was detected by cell immunofluorescence and Western Blotting after 72 h of infection.A significant decrease in Ebpl protein expression of KD group indicated successful transfection.Then CCK-8 was used to detect the cell viability of the two groups and the proliferation curve was drawn.Plate colony formation and the soft agar colony formation assays were performed to detect the proliferative capacity and colony forming ability of the two groups.Flow cytometry was used to analyze the cell cycle of the two groups.The apoptosis of the two groups was detected by TUNEL and flow cytometry.The invasion and migration ability of the two groups was detected by transwell assay and scratch test.Results:1.Cellular immunofluorescence assay results showed that Ebpl protein highly expressed in the NC group of HELA cells and mainly in the cytoplasm.The green fluorescence of the KD group was significantly weaker than that of the NC group,indicating that Ebpl expressed in the KD group significantly lower than that in the NC group.Similar to immunofluorescence results,Western Blotting assay showed that the expression of Ebp1 protein in KD group was prominently lower than that in NC group(P<0.001).The above results indicated that HELA cells with Ebp1 low expression was successfully constructed.2.The CCK-8 assay showed that KD group had slower growth and weaker proliferative ability than the NC group.3.The results of plate and soft agar colony formation assay showed that colony forming and cell proliferation ability of KD group cells were weaker than the NC group(P<0.001).4.Flow cytometry was used to detect cell cycle differences between NC and KD cells.The results showed that the G0/G1 phase proportion in KD cells was significantly higher than that in NC cells(P<0.01),while the S phase and G2 phase proportion in KD cells was lower.These results showed that Ebpl gene silencing blocked HELA cells in the G0/G1 phase.5.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 Ebpl gene silencing increases the apoptosis of HELA cells.6.Scratch test results showed that the healing ability of KD group cells was lower than that of NC group(P<0.05),indicating that the migration ability of HELA cells was weakened after Ebpl gene silencing.The results of Transwell assay showed that the invasion ability of KD group was significantly weaker than that of NC group(P<0.01),indicating that Ebpl gene silencing weakened the invasion ability of HELA cells.Conclusion:1.Ebpl gene silencing weakens the proliferation of HELA cells.2.Ebpl gene silencing reduces the invasion and metastasis ability of cervical cancer HELA cells.3.Ebp1 gene silencing induces apoptosis of cervical cancer HELA cells.