MiRNA-96 Targeting FOXO1 Regulates Carcinogenesis Of Akt/GSK-3?/?-catenin Pathway In Hepatoma Cells

BackgroundHepatocarcinoma is a malignant tumor that threatens the safety of human life in the world.The incidence rate of liver cancer is increasing year by year,but its prognosis was not improved significantly.Therefore,effective therapeutic targets is the breakthrough in the treatment of hepatocellular carcinoma.The role of miRNA in cancer has been recognized by many studies,and it is closely related to migration,invasion,immunity,proliferation,apoptosis,angiogenesis and other aspects of cancer.Some studies have shown that serum levels of miR-96 are elevated in patients with hepatocellular carcinoma,but the carcinogenic mechanism is not yet clear.FOXO1 is a key tumor-associated negative regulatory transcription factor that slows cancer cell growth and reduces cancer metastasis.Activation of AKT/GSK-3?/?-catenin signaling pathway is one of the most important tumor-related signaling pathways and plays a key role in the occurrence and development of tumors.Thus,blocking the activation of AKT/GSK-3?/?-catenin signaling pathway and the entry of ?-catenin into the nucleus can inhibit tumor development.Phosphorylation of AKT at Ser9 regulates GSK-3? activity.Phosphorylation of AKT promotes phosphorylation of GSK-3?,thereby impairs the ability of GSK-3? to degrade ?-catenin.?-catenin is a key molecule in this signaling pathway.After the degradation of ?-catenin is affected,it accumulates in the cytoplasm and eventually translocates to the nucleus.In the nucleus,?-catenin further activates its downstream genes,exacerbating tumor progression.Part I Abnormal expression of miR-96 and FOXO1 in hepatocarcinoma ObjectiveExplore the expression of miR-96 and FOXO1 in hepatocarcinoma tissues and cell lines,and explore their possible relationship.MethodsqRT-PCR and Western blot were used to detect the expression levels of miR-96 and FOXO1 in hepatocarcinoma tissues and liver cancer cells.ResultsThe expression level of miR-96 in hepatocarcinoma tissue and hepatocarcinoma cells was abnormally increased,and the expression level of FOXO1 in hepatocarcinoma tissue and hepatocarcinoma cells was abnormally decreased.Part? miR-96 targets FOXO1ObjectiveTo explore the regulatory role of miR-96 and FOXO1 in the process of hepatocarcinoma,establish a hepatocarcinoma cell model that inhibited miR-96 expression and overexpressed FOXO1,and then explored its effects on the proliferation,migration and invasion of hepatocarcinoma cells.Methods1.CCK-8 method and Transwell chamber method were used to detect the effects of inhibiting miR-96 expression and FOXO1 overexpression on the proliferation,migration and invasion of hepatocarcinoma cells.2.Bioinformatics prediction,dual luciferase reporter gene detection experiment,Western blot were used to detect the targeting relationship between miR-96 and FOXO1.Results1.Inhibition of miR-96 expression and overexpression of FOXO1 could inhibit the proliferation,migration and invasion of hepatocarcinoma cells.2.miR-96 targetd and negatively regulated FOXO1.Part III: Regulation of miR-96/FOXO1/AKT/GSK-3?/?-catenin signaling pathway on hepatocarcinoma cell and tumor growth ObjectiveTo explore the mechanism of miR-96 / FOXO1 / Akt / GSK-3? / ?-catenin signaling pathway on the proliferation,migration and invasion of hepatocarcinoma cells and the regulation of hepatocarcinoma growth.Methods1.Experimental groups: anti-miR-NC group,anti-miR-96 group,pcDNA 3.1group,pcDNA 3.1-FOXO1 group,miR-NC+pcDNA 3.1 group,miR-96+pcDNA 3.1group,miR-96+pcDNA 3.1-FOXO1 group.2.CCK-8 method and Transwell chamber method were used to detect the proliferation,migration and invasion of hepatocarcinoma cells respectively.3.Western blot was used to detect protein expression of key genes in Akt /GSK-3? / ?-catenin signaling pathway in hepatocarcinoma cells.4.Immunofluorescence experiment was used to detect the nuclear displacement of ?-catenin in hepatocarcinoma cells.5.Tumorigenesis test in nude mice was used to detect tumor growth.6.Immunohistochemical method was used to detect the positive amount of FOXO1 in tumorigenesis tissues of nude mice.Results1.Compared with the anti-miR-NC group,the protein expression of p-AKT and p-GSK-3? was significantly reduced and the protein expression of p-?-catenin was significantly increased in the anti-miR-96 group.Compared with the pcDNA 3.1group,the protein expression of p-AKT and p-GSK-3? in the pcDNA 3.1-FOXO1 group were significantly reduced,and the the protein expression of p-?-catenin was significantly increased.2.Compared with the miR-NC+pcDNA 3.1 group,the expression of p-AKT and p-GSK-3? protein in the liver cancer cells of the miR-96+pcDNA 3.1 group were increased significantly,and the protein expression of p-?-catenin was decreased significantly.Compared with the miR-96+pcDNA 3.1 group,the protein expression of p-AKT and p-GSK-3? in hepatocarcinoma cells of the miR-96+pcDNA3.1-FOXO1 group were significantly reduced,and the protein expression of p-?-catenin was significantly increased.3.Compared with the miR-NC+pcDNA 3.1 group,the miR-96+pcDNA 3.1group had a significantly higher proliferation ability of hepatocarcinoma cells,and the number of migration and invasion cells was significantly increased.Compared with the miR-96+pcDNA 3.1 group,cell proliferation ability of the miR-96+pcDNA3.1-FOXO1 group was significantly reduced,and the number of migration and invasion cells was significantly reduced.4.Compared with the miR-NC+pcDNA 3.1 group,the expression of ?-catenin in the nucleus in the miR-96+pcDNA 3.1 group was up-regulated.Compared with the miR-96+pcDNA 3.1 group,the expression of ?-catenin in the nucleus in the miR-96+pcDNA 3.1-FOXO1 group was decreased.5.Compared with the miR-NC+pcDNA 3.1 group,the tumor volume of nude mice in the miR-96+pcDNA 3.1 group was significantly increased,and the positive rate of FOXO1 in the tumor tissue was significantly reduced.Compared with the miR-96+pcDNA 3.1 group,the tumor volume of nude mice in the miR-96+pcDNA3.1-FOXO1 group was significantly reduced,and the positive rate of FOXO1 in the tumor tissue was significantly increased.Conclusion:miR-96 can promote the proliferation,migration and invasion of hepatocarcinoma cells and the growth of liver cancer tumors.The mechanism is related to the targeting of negative regulation of FOXO1,which in turn activates the Akt/GSK-3?/?-catenin signaling pathway.