PI3K/AKT/FOXO1 Promotes The Proliferation Of Bladder Cancer Cell By Regulating HnRNP-F Expression

BACKGROUD&OBJECTIVE:Bladder cancer is the tenth highest incidence of urinary system malignant epithelial tumors in the world.There is a gender difference in its incidence,ranking seventh among male cancer patients and seventeenth among female cancer patients.The development of bladder cancer is inseparable from the activation of proto-oncogenes and the inactivation of tumor suppressor genes,which in turn leads to abnormal proliferation of bladder cancer cells and invasion to other parts of the body.In recent years,with the development of bladder cancer research,people’s understanding of bladder cancer is constantly improved,and the treatment methods are also developing.However,the problem of easy recurrence after bladder cancer treatment leads to a poor prognosis.Therefore,studying the proliferation mechanism of bladder cancer has important basic and clinical significance,which is helpful for us to find new treatment methods.Studies have shown that Heterogeneous nuclear ribonucleoprotein F(hnRNP-F)is elevated in multiple malignancies and plays a role in promoting tumor progression.In previous studies,we confirmed that in human bladder cancer tissues,hnRNP-F mRNA levels and protein levels are both increased and related to poor patient prognosis;in vitro and in vivo experiments have demonstrated that hnRNP-F can promote the invasion ability of bladder cancer cells,Migration ability and EMT process.Based on previous research,this research project intends to explore the effect of hnRNP-F protein on the proliferative capacity of human bladder cancer cells and explore the molecular mechanism that regulates hnRNP-F expression.Part 1:HnRNP-F promotes bladder cancer cell proliferationMethods:Establishment of stable knock-down HnRNP F cell lines.The effects of hnRNP-F on the proliferation of bladder cancer cells were measured by CCK8 proliferation assay and plate cloning experiment.The effect of hnRNP-F on the cycle distribution of bladder cancer cells was detected by flow cytometry.Results:HnRNP-F can promote the proliferation of bladder cancer cells;hnRNP-F can promote the G1/S phase transition of bladder cancer cells and accelerate the cell cycle process.Part 2.Bioinformatics predicts the relationship between HnRNP-F and PI3K/AKT signaling pathwayMethods:Download the data of 403 bladder cancer patients from TCGA database,and use bioinformatics method to enrich and analyze the genes and signal pathways related to hnRNP-F.The optimal dose of PI3K/AKT pathway inhibitor LY294002 in bladder cancer cells was determined by using the median lethal dose.Western blot was used to detect the relationship between hnRNP-F and PI3K/AKT pathway proteins in bladder cancer cells.The effect of LY294002 on bladder cancer cells was determined by Western blotting.Results:HnRNP-F is highly related to the PI3K/AKT pathway.hnRNP-F cannot affect the expression of PI3K/AKT pathway protein,and PI3K/AKT pathway protein can promote the expression of hnRNP-F.Part 3.PI3K/AKT regulates the expression of hnRNP-F in bladder cancer cellsMethods:Bioinformatics was used to predict the transcription factors that could act on hnRNP-F.The promoter region of hnRNP-F binding FOXOl was detected by chromatin immunoprecipitation assay(CHIP).The full-length plasmid of hnRNP-F promoter region was constructed,and the effect of FOXO1 on hnRNP-F was determined by dual luciferase report experiment.Construct and transfect plasmids that interfere with hnRNP-F and FOXO1,CCK8 proliferation experiment and plate cloning experiment were used to detect the effect of FOXO1 on the proliferation of bladder cancer cells after using LY294002,and flow cytometry was used to detect effect of FOXO1 on bladder cancer cell cycle after using LY294002.RESULTS:In bladder cancer cells,FOXO1 binds to the promoter of hnRNP-F and inhibits its transcription.In bladder cancer cells,PI3K/AKT regulates hnRNP-F protein expression through FOXO1 to promote cell cycle progression and cell proliferation;interfering with FOXO1 protein expression can restore PI3K/AKT inhibitors regulating hnRNP-F to inhibit the bladder cancer cell cycle progression and proliferation.CONCLUSION:PI3K/AKT promotes bladder cancer cell proliferation by regulating hnRNP-F expression through FOXO1.