Effect And Mechanism Of MiR-6838-5p Targeting Polycomb Protein CBX4 On Liver Cancer Stem Cells Through ERK Signaling Pathway

ObjectiveTo clarify the expression of polycomb protein CBX4 in liver cancer stem cells and its impact on the biological behavior of liver cancer stem cells such as stemness and self-renewal.To predict the mi RNAs targeting the polycomb protein CBX4 and study their effects on liver cancer stem cells,and further elaborate the specific mechanism of mi RNA targeting polycomb protein CBX4 on liver cancer stem cells.Methods1.Bioinformatics methods were used to analyze the expression differences of polycomb protein CBXs in pancarcinoma and paracancer tissues,liver cancer and parahepatic tissues,and also liver cancer at different stages of differentiation,as well as its correlation with the prognosis of patients with liver cancer by GEPIA2,UALCAN and TCGA database platforms.2.Flow cytometry was employed to sort liver cancer stem cells with CD44+and CD133+,and their stemness was evaluated by sphere formation capability and colony formation tests.The expression differences of stem-related genes Oct4,Sox2,Nanog and polycomb protein CBX4 were detected by QT-PCR and WB.3.Lentivirus was used to construct a cell line with low expression of polycomb protein CBX4,and the effects of knocking down polycomb protein CBX4 on sphere formation,colony formation,biological behavior,and expression of stemness related genes Oct4,Sox2 and Nanog of LCSCs were analyzed.4.Mircro RNAs that may target polycomb protein CBX4 were predicted by Target Scan,mi RWalk and mi RDB databases,and their expression in LCSCs were analyzed by QT-PCR and WB,and mi RNAs with low expression in LCSCs were selected as research objects.5.The expression of the predicted mi RNA in liver cancer tissues was clarified,and the effects of overexpression of mi RNAs on polycomb protein CBX4 and the biological behavior of LCSCs were studied.6.The interaction between predicted mi RNA and polycomb protein CBX4 was verified by dual luciferase reporting assay.7.QT-PCR and WB were used to detect the expression of ERK in LCSCs,as well as the effects of mi RNA and polycomb protein CBX4 on p ERK1/2 and ERK1/2,so as to further study the effects of mi RNA targeting polycomb CBX4 on the biological behavior of LCSCs through the ERK signaling pathway.8.In vivo experiments were conducted to further verify the effects of mi RNA targeting polycomb protein CBX4 on the tumorigenicity,cell cycle,angiogenesis,and epithelial mesenchymal transformation of LCSCs.Results1.The expression of polycomb protein CBXs are different in different tumor tissues,and are also significantly different in the same cancer and adjacent tissues.The expression of polycomb protein CBXs in liver cancer and parahepatic tissues are different,and there are also differences at different stages of liver cancer,which are closely related to the prognosis of patients with liver cancer.2.The LCSCs sorted by CD44+CD133+ had the strongest sphere formation and colony formation ability,and the expressions of stemness related genes Oct4,Sox2 and Nanog were significantly increased.3.The expression of polycomb protein CBX4 was increased in LCSCs.After knocking down the polycomb protein CBX4,the expressions of stemness related genes Oct4,Sox2 and Nanog were significantly decreased,and the sphere formation and colony formation rate of LCSCs were decreased.The proliferation rate of LCSCs was significantly slowed down,and the apoptosis rate was significantly increased,and the migration and invasion ability were also affected.4.Bioinformatics analysis results showed that mi R-200c-3P,mi R-485-3p,and mi R-6838-5P could target the polycomb protein CBX4.QT-PCR showed that mi R-6838-5P was low expressed in LCSCs and liver cancer,which was used as the object of follow-up research.5.Overexpression of mi R-6838-5P can inhibit the expression of polycomb protein CBX4,and the targeting effect of mi R-6838-5P on polycomb protein CBX4 was verified by dural luciferase reporting experiment.6.Overexpression of mi R-6838-5P can significantly inhibit the ability of the sphere formation and colony formation rate of LCSCs,and inhibit their proliferation,migration,and invasion,and promote the apoptosis.7.The expressions of p ERK1/2 and ERK1/2 were increased in LCSCs,and mi R-6838-5p targeting polycomb protein CBX4 plays a regulatory role in the sphere formation,colony formation,proliferation,and apoptosis of LCSCs through ERK signaling pathways.ERK inhibitor LY3214996 can block the regulatory effect of mi R-6838-5p targeting polycomb protein CBX4.8.In vivo experiments showed that mi R-6838-5p could significantly reduce the tumorigenicity of LCSCs,which was enhanced by the intervention of polycomb protein CBX4.Meanwhile,mi R-6838-5p can inhibit tumor cell proliferation,angiogenesis and epithelial mesenchymal transformation,while polycomb protein CBX4 can promote tumor cell proliferation,angiogenesis and epithelial mesenchymal transformation.9.In vivo experiments showed that mi R-6838-5p could inhibit the expression of p ERK1/2 and ERK1/2,while polycomb protein CBX4 could promote the expression of p ERK1/2 and ERK1/2.ConclusionPolycomb protein CBX4 is highly expressed in liver cancer stem cells,and can regulate the stemness and self-renewal ability of liver cancer stem cells.Polycomb protein CBX4 is the direct target of mi R-6838-5p.The mi R-6838-5p targeting polycomb protein CBX4 regulates the biological characteristics and tumorigenicity of liver cancer stem cells in vivo and in vitro by regulating ERK signals.The mi R-6838-5p targeting polycomb protein CBX4 can provide a new idea for effective treatment of liver cancer.