Translation Initiation Factor EIF3d Promotes The Progression Of Gallbladder Carcinoma By Regulating The Stability Of GRK2 Protein

Object Gallbladder cancer is most common malignancy in the biliary tract system,high mortality,poor prognosis and five-year survival rate less than 5%.EIF3d as a member of the e IF3 subunit can selectively regulate the translation of specific protein which involved in cell proliferation and thus promote the progression and development of tumor.Investigating the function and mechanism of eIF3d in gallbladder cancer provides us theoretical basis for gallbladder cancer targeted therapyMethod Immunohistochemistry analysis was performed to detect eIF3d protein expression in gallbladder cancer tissue.The association between the expression of eIF3d and the clinicopathological characteristics and prognosis of gallbladder cancer were analyzed statistically.EIF3d was knocked down by lentivirus-mediated gene knockdown method or over-expressed by plasmid overexpression means in gallbladder cancer cells,respectively.CCK-8 assay,clone formation assay,wound heal assay and transwell invasion assay were used to evaluate the proliferation and migration of gallbladder cancer cells.The cell cycle progression and cell apoptosis of gallbladder cancer cells were analyzed by flow cytometry.Nude mouse xenograft model and mouse metastasis model were used to assess the tumorigenic and metastatic ability of the stable cells in vivo.Immunoblotting analysis was performed to analyze cell cycle and apoptosis related proteins expression.Using the yeast two-hybrid to screen the eIF3d interacting protein.Co-immunoprecipitation assay was carried out to validate the specific interaction.Cycloheximide pulse-chase experiments detect the protein degradation speeds.The GRK2 kinase inhibitor was obtained by screening the natural product library.The inhibitory effect of ginsenoside Rg-3 on GRK2 kinase was detected by GRK2 kinase activity assay kit.CCK-8,colony formation,cell cycle and apoptosis assay were used to evaluate the effect of ginsenoside Rg-3 on gallbladder cancer cells.Nude mouse xenograft assay were used to assess the effect of ginsenoside Rg-3 on gallbladder cancer cell tumorigenic ability in vivoResult EIF3d is highly expressed in gallbladder cancer patients,which is related to the clinical stages,metastasis and prognosis of gallbladder cancer patients.The cell biological function study shows that eIF3d promotes the proliferation of gallbladder cancer cells via promoting cell cycle progression and inhibiting cell apoptosis.Moreover,EIF3d promotes the invasion and metastasis of gallbladder cancer cells by regulating the EMT procession.The yeast two-hybrid system and immunoprecipitation results showed that eIF3d could directly interact with GRK2.EIF3d enhances GRK2 protein stability by inhibiting the ubiquitin-proteasome degradation of GRK2.Upregulation of GRK2 activated PI3K/AKT signaling pathway to promote proliferation and metastasis of gallbladder cancer cells.Finally,ginsenoside Rg-3 as a GRK2 kinase inhibitor was obtained through screening the natural product sample library.Ginsenoside Rg-3 could significantly suppress the proliferation of gallbladder cancer cells both in vitro and in vivo.Conclusion Overexpression of eIF3d promotes the gallbladder cancer progression through the GRK2-mediated activation of PI3K/AKT signaling pathway.Ginsenoside Rg-3 inhibits GRK2 kinase activity and blocks e IF3d-GRK2 axis in gallbladder cancer to suppress gallbladder cancer progression,which provides us a new treatment for gallbladder cancer.