USP3 Regulates Proliferation,invasion And Glycolysis Of Gastric Cancer By Activating The Wnt/MYC Pathway

Objective:To explore the role of USP3,a member of the USPs family,in cholangiocarcinoma.USP3 can activate the Wnt/MYC pathway by promoting the glycolysis of cholangiocarcinoma cells,and inhibit its ubiquitination level by binding with MYC,and ultimately promote the proliferation and invasive metastasis of cholangiocarcinoma.To confirm that USP3 may be a potential prognostic marker of cancer and can regulate the survival and invasion of cancer.It is expected to become a new molecular target for cancer treatment.Methods:Clinical samples were collected from * * * hospitals from July 2015 to May2022,and a total of * * pairs of cholangiocarcinoma tissues and corresponding normal tissues were collected.All surgical specimens were frozen and stored at-80 ℃ for further RNA and protein analysis,and embedded in the wax block for subsequent IHC experiments.Statistical analysis: Use Graph Pad Prism 8.0(Graph Pad Software,La Jolla,CA,USA)to analyze and present data.Use SPSS software to build Kaplan-Meier survival curve.The statistical difference between the two groups was analyzed by t-test,and the difference was statistically significant with P<0.05.Results:1)The prognosis of cholangiocarcinoma patients with high expression of USP3 is poor.The expression of USP3 in two kinds of gastric adenocarcinoma cells is higher than that in normal gastric mucosa epithelial cells.2)Knock down the expression of USP3 can significantly inhibit the proliferation of tumor cells,and knock down the expression of USP3 can significantly inhibit the invasion and migration of cholangiocarcinoma cells.3)The glycolysis pathway in cancer metabolism has changed significantly.After USP3 knockout,the glucose uptake of cholangiocarcinoma cells significantly decreased.In addition,the concentration of lactic acid and ATP in cholangiocarcinoma cells decreased significantly after the expression of USP3 was knocked out.Decreasing the expression level of USP3 can significantly reduce the glycolytic ability of tumor cells.After the USP3 gene is knocked out,the expression of GLUT1,HK2,PDM2 and LDHA target genes is down-regulated.4)Compared with the control group,the proliferation ability of tumor cells in nude mice after USP3 knockout was significantly reduced.Compared with the control group,the tumor volume and weight were significantly reduced after the USP3 expression was knocked out.Compared with the knockout group(sh-USP3),the proliferation-related indexes Ki67,PCNA,GLUT1,HK2,PKM2 and LDHA in the control group were significantly higher.5)After overexpression of USP3,the level of ubiquitin in MYC increased significantly,and there was no significant change in MYC m RNA after knockdown or overexpression of USP3.USP3 significantly activated the activity of Wnt/MYC signal pathway,and USP3 directly promoted the m RNA expression of Cyclin D1,c-Myc,VEGF and Survivin.6)Knocking down MYC or adding MYC inhibitor reversed the promotion of proliferation and invasive metastasis of cholangiocarcinoma cells induced by USP3 overexpression.The overexpression of USP3 can promote intracellular glucose uptake and lactate production,and promote intracellular glycolysis.The promotion of intracellular glucose uptake and lactate production induced by USP3 overexpression can be reversed by knocking out MYC or adding inhibitor.Similarly,the same trend was observed in the intracellular production of ATP.The m RNA level of its main metabolism-related target genes GLUT1,HK2,PKM2 and LDHA was up-regulated after the overexpression of USP3,while the m RNA level was almost down-regulated after MYC knockdown or adding inhibitors in the control group.Conclusion:The prognosis of patients with cholangiocarcinoma with high expression of USP3 is poor.The expression of USP3 in the two kinds of cholangiocarcinoma cells is higher than that in the normal bile duct mucosa epithelial cells.Reducing the expression of USP3 can significantly inhibit the proliferation of tumor cells,and reducing the expression of USP3 can significantly inhibit the invasion and migration of cholangiocarcinoma cells.After knocking out the USP3 gene,the glucose uptake of cholangiocarcinoma cells decreased significantly,indicating that USP3 can promote the glycolysis of tumor cells,improve energy production,and promote the expression of target genes of GLUT1,HK2,PDM2 and LDHA metabolism in tumor,thus promoting tumor growth.