Ebp1 Promotes Liver Cancer Cell Proliferation And Migration Through PI3K/Akt/mTOR Signaling Pathway

Objective:Hepatocellular carcinoma is a common malignant tumor.Currently,the treatment effect of liver cancer is not good.Finding more effective treatments has become a current research hotspot.Ebpl is one of the major intracellular binding proteins of ErbB3,and its coding gene is human PA2G4.Ebpl is widely present in various tissues including human liver.A large amount of literature shows that abnormal expression of Ebpl can be seen in tumors such as prostate cancer,oral squamous cell carcinoma and liver cancer.We experimentally explore the effect of Ebp1 on the malignant behavior of hepatocellular carcinoma and its molecular mechanism,providing new ideas for the development of targeted drugs for liver cancer.Methods:1.The expression level of PA2G4(Ebp1)in various cancers was analyzed by CCLE database,and the Ualan and Oncomine databases were used to compare PA2G4(Ebpl)expression differences in normal tissues and hepatocellular carcinoma.And Kaplan-Meier survival curve was used to analyze the relationship between PA2G4(Ebpl)expression and prognosis of hepatocellular carcinoma.Use STRING to build a PPI network diagram of PA2G4(Ebpl).2.Recombinant lentiviral transfection method was used to knock down the expression of Ebpl in sk-hepl and HepG2 cellsand the knockdown level was tested by western blot.3.CCK-8 method was used to detect the survival and growth of cells in sh-NC group and sh-Ebp1 group;plate cell clone formation test and soft agar clone formation test were used to detect cell proliferation ability;flow cytometry was used to detect cells cycle in both groups;scratch test was used to compare the migration ability of the two groups;Nude mouse tumor formation experiments were used to study the effect of Ebpl on tumor proliferation in vivo.4.Western blot was used to explore the protein expression levels of PI3K/Akt/mTOR signaling pathway related genes in sh-NC group and sh-Ebpl group.After using the Akt inhibitor,observe the protein expression levels of the two groups of cells.Results:1.Analysis of CCLE database shows that Ebpl expression is higher in hepatocellular carcinoma;analysis of Ualcan and Oncomine databases shows that the expression level of Ebp1 in hepatocellular carcinoma is significantly higher than that of normal liver tissue;Kaplan-Meier survival curve analysis Ebpl is closely related to the prognosis of patients with hepatocellular carcinoma.The PPI network diagram using STRING shows that Ebpl is closely related to the PI3K/AKT/mTOR signaling pathway.2.Detection of lentivirus transfection efficiency showed that the expression of Ebpl in sh-Ebpl group was significantly lower than that in sh-NC group,indicating that Ebpl gene silencing of sk-hepl and HepG2 cell lines was successfully constructed(P<0.0001).3.The results of the CCK-8 experiment showed that the cell growth viability of cells in the sh-NC group was significantly higher than that in the sh-Ebpl group in both sk-hep1 and HepG2 cell lines.4.Both the plate cell clone formation experiment and the soft agar clone formation experiment showed that compared with the sh-NC group,the proliferation ability and colony forming ability of the sh-Ebp1 group of sk-hep1 and HepG2 cell lines were significantly reduced(P<0.0001).5.The results of cell cycle detection by flow cytometry showed that Ebpl gene silencing caused sk-hep1 cells to block in G2 phase(P<0.01)and HepG2 cells to block in G1 phase(P<0.01).6.The results of the scratch test showed that after knocking down Ebp1,the migration capacity of sk-hepl(P<0.0001)and HepG2(P<0.001)cells decreased significantly.7.Tumor formation experiments in nude mice showed that the subcutaneous tumor-bearing volume of the sh-NC group was significantly larger than that of the sh-Ebpl group.According to the tumor growth curve,the tumor growth rate in nude mice became slower after knocking down Ebp1.8.After Ebpl was silenced,Western blot results showed that the expression of p-Akt and p-mTOR decreased(P<0.001).Conclusion:1.Ebpl gene silencing reduced the proliferation of sk-hepl cells and HepG2 cells,its caused cell arrest.2.Ebpl gene silencing reduced the migration ability of sk-hepl cells and HepG2 cells.3.Ebpl gene silencing can significantly inhibit tumor growth in nude mice bearing tumorsand provide basic theoretical support for clinical transformation.4.Ebpl promotes the proliferation and migration of liver cancer cells through the PI3K/Akt/mTOR signaling pathway,providing a theoretical basis for the development of clinical targeted drugs.