The Mechanism Of ENT3Promoting The Progression Of Hepatocellular Carcinoma By Regulating AKT/mTOR Signaling Pathway

Objective: Hepatocellular carcinoma(HCC)is one of the most dangerous types of cancer with high mortality and early metastasis.The radical treatment of HCC includes hepatectomy and orthotopic liver transplantation.However,most HCC patients are associated with liver cirrhosis and the possibility of hepatectomy is low.At present,most patients with HCC are mainly treated with sorafenib,but the overall survival rate is low,and the prognosis is poor.Equilibrative nucleoside transporter 3(ENT3)is an intracellular nucleoside and nucleobase transporter located in lysosomes and mitochondria,which is important for intracellular nucleoside and nucleobase transport.To study the mechanism of ENT3 promoting the proliferation,metastasis and invasion of HCC cells by regulating AKT/mTOR signal pathway,to explore the prognostic markers of HCC,to lay a theoretical foundation for possible targeted therapy.Methods: GEPIA,UALCAN and TIMER databases were used to analyze the differential expression of ENT3 in pan-cancer and its correlation with clinicopathological parameters and disease prognosis.The Escherichia coli with knockdown ENT3 expression was constructed by sh RNA technology,and then separated and purified by plate marking,and then cultured to extract plasmid DNA.The knockdown plasmid DNA was transfected into the hepatoma cell line by in vitro transfection technique,and the knockdown efficiency was verified by Western blotting.The effects of knockdown ENT3 on the proliferation,migration and invasion of HCC cells were verified by cell biological function experiments in vitro,such as cell proliferation,cell cloning,wound healing,Transwell migration and invasion.Flow cytometry was used to detect the cell cycle and apoptosis after knockdown of ENT3.The data of hepatoma gene expression group in TCGA database were analyzed by R language,and the ENT3 gene set enrichment analysis(GSEA).STRING database was used to explore the interaction between ENT3-encoded proteins and key proteins in the enrichment pathway.Through the Linked Omics database,the transcriptome data of ENT3 in HCC were analyzed to explore the enrichment of ENT3-related gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG).Western blotting was used to detect the correlation between knockdown ENT3 and AKT/mTOR signal pathway.SC79,an AKT agonist,was used to activate AKT/mTOR signal pathway to observe whether SC79 could restore the inhibitory effect of knockdown ENT3 on AKT/mTOR signal pathway.Results: The expression of ENT3 was up-regulated in Pheochromocytoma and Paraganglioma(PCPG),Rectum Adenocarcinoma(READ),Liver Hepatocellular Carcinoma(LIHC)and other malignant tumors.The abnormal activation of ENT3 in HCC was closely related to the poor prognosis and clinicopathological indexes of patients.In the in vitro cell model,knockdown of ENT3 can inhibit HCC cell viability,cell proliferation,cell migration and invasion,induce S-phase arrest of cell cycle,and promote cell apoptosis.GO/KEGG results showed that ENT3-related genes were related to cell infection,nucleotide metabolism,DNA binding and transcriptional regulation.GSEA pathway enrichment analysis showed that ENT3-related genes could activate AKT/mTOR signal pathway and star oncogene MYC,DNA repair process.Knockdown of ENT3 can reduce the expression of p-AKT and p-mTOR protein,inhibit the expression of p-p70S6K1 and promote the expression of p-4EBP1protein-the downstream effect factor of AKT/mTOR pathway.SC79 can partially restore the inhibitory effect of knockdown ENT3 on p-AKT,p-mTOR and p-p70S6K1 protein expression,and reduce the promoting effect of knockdown ENT3 on p-4EBP1 protein expression.Conclusions: ENT3 was highly expressed in HCC and was related to poor prognosis.ENT3 promoted the proliferation,metastasis and invasion of HCC cells by regulating AKT/mTOR signal pathway.