Study Of The Mechanism On The Stabilization Of CCND1 By EIF3H Promotes The Progression Of Intrahepatic Cholangiocarcinoma

Background:Cholangiocarcinoma is a heterogeneous group of tumors that grow along the bile duct tree and is the second most common liver tumor,after hepatocellular carcinoma(about 15%of cases)and about 3%of gastrointestinal malignancies.Global morbidity and mortality from cholangiocarcinoma have increased over the past few decades.Intrahepatic cholangiocarcinoma accounts for the majority(approximately 85%)of patients with primary liver cancer in East Asia.A wider range of epidemiological and clinical practice suggests that HBV and HCV-related liver disease are absolute risk factors for cholangiocarcinoma and are more associated with intrahepatic cholangiocarcinoma.Occult HBV infection is a new risk factor for intrahepatic cholangiocarcinoma.The incidence of intrahepatic cholangiocarcinoma increased at the end of the last century,which is also related to the increased burden of HCV infection in the population.For intrahepatic cholangiocarcinoma,the global age-standardized mortality rate is also increasing year by year,with the highest in Hong Kong.There is growing evidence that eukaryotic translational initiation factor 3H(EIF3H)is associated with a variety of physiological and pathological mechanisms.Studies have found that the expression level of EIF3H is closely related to a variety of tumors,and there is amplification and overexpression of EIF3H in many malignant tumors.Current studies have confirmed that EIF3H can promote the progression of intrahepatic cholangiocarcinoma,but its underlying molecular mechanism is not well understood and further research is needed.Materials and Methods:Analysis of public databases found that EIF3H is highly expressed in cholangiocarcinoma.Western blot,qPCR,and immunohistochemical staining were used to detect EIF3H expression in intrahepatic cholangiocarcinoma tissues and cell lines.The effect of EIF3H on the proliferation,migration and invasion of intrahepatic cholangiocarcinoma cells was evaluated by colony formationassay,MTT assay,CCK8 assay,scratch assay,transwell assay and apoptosis detection by cell flow cytometry.After knocking out EIF3H,the whole genome was sequenced,and the sequencing results were submitted to IPA software to analyze the enrichment of pathways,and the results showed that the Wnt/β-catenin signaling pathway was related to EIF3H.Western blot detects changes in the expression of key proteins of the Wnt/β-catenin signaling pathway and downstream targets.Rescue experiments in vivo and in vitro a proved the cancer-promoting effect of EIF3H/CCND1 axis in intrahepatic cholangiocarcinoma and the upstream and downstream regulatory relationship between EIF3H and CCND1.Ki67 immunohistochemical staining of tumor sections after subcutaneous tumorigenesis in nude mice demonstrated the effect of EIF3H on tumor growth in vivo.Cycloheximide chase assay was used to assess changes in the stability of CCND1 proteins.MG132 treats cells to demonstrate whether CCND1 is degraded by the ubiquitin-proteasome system.Co-immunoprecipitation of ubiquitin molecules and CCND1 proteins detected changes in the level of CCND1 ubiquitination modification after knocking down EIF3H.Results:Bioinformatics analysis showed high expression of EIF3H in cholangiocarcinoma.At the same time,we found that the level of EIF3H mRNA transcription and protein translation were higher in intrahepatic cholangiocarcinoma cell lines.The results of EIF3H immunohistochemical staining in intrahepatic cholangiocarcinoma tissues showed that EIF3H was highly expressed in intrahepatic cholangiocarcinoma tissues and was mainly localized to the cytoplasm,and the plasmonuclear separation experiment also proved this result.EIF3H expression was significantly up-regulated in intrahepatic cholangiocarcinoma tissues as well as cell lins.Moreover,the expression level of EIF3H was positive correlated with tumor grade.Patients with high level of EIF3H expression showed a poor overall survival.In contrast,experimental downregulation of EIF3H caused the suppression of proliferation and migration and augment of proportion of apoptosis in cancer cells.Microarray analysis and following experiments indicated the Wnt/β-catenin signaling was suppressed after silencing EIF3H,suggesting EIF3H mediating intrahepatic cholangiocarcinoma via Wnt/β-catenin signaling.Inhibition of Wnt/β-catenin signaling pathway can alleviate the effect of overexpression of EIF3H on the enhancement of viability and apoptosis reduction of intrahepatic cholangiocarcinoma cells.Rescue experiments in vitro and in vivo demonstrated that the axis of EIF3H/CCND1 plays a tumor-promoting role in intrahepatic cholangiocarcinoma,suggesting EIF3H was an upstream regulator of CCND1 in intrahepatic cholangiocarcinoma.The score of Ki67 immunohistochemical staining in tumor sections after subcutaneous tumorigenesis in nude mice showed the role of EIF3H/CCND1 axis in promoting tumor growth in vivo.Cycloheximide chase indicated that the half-life of CCND1 protein becomes shorter After treatment with MG132,increased levels of CCND1 protein were observed.Co-immunoprecipitation experiments of ubiquitin molecule and CCND1 protein showed that the level of ubiquitination modification of CCND1 protein became higher after knocking down EIF3H.Conclusion:EIF3H stabilizes CCND1 to mediate proliferation,migration and apoptosis of intrahepatic cholangiocarcinoma cells via Wnt/β-catenin signaling.Overall,our findings imply that EIF3H may be a potential therapeutic target for intrahepatic cholangiocarcinoma treatment.