The Study On The Mechaisms Of MicroRNA-1305 Inhibits The Stemness Of Liver Cancer Stem Cells

Objective: to study the interaction between MicroRNA-1305 and UBE2 T and its effect on hepatoma stem cells,and to explore its mechanism.Methods: the significant differential genes and common genes between hepatocellular carcinoma cells and normal cells were compared and analyzed by in situ hybridization and GEO microarray database,and the interaction network map was drawn.The expression of UBE2 T in CD133+ CD13+ hepatoma stem cells was analyzed by flow cytometry,q RT-PCR and Western blot.The effects of silencing UBE2 T gene on the ability of spherical cell formation,colony formation and proliferation of LCSCs and the volume and weight of tumor after inoculation in nude mice were observed.The interaction between miR-1305 and UBE2 T was further verified by luciferase reporter gene system.In order to further verify the interaction between miR-1305 and UBE2 T in LCSCs,miR-1305 and UBE2 T were cotransfected into LCSCs.The effects of miR-1305 and UBE2 T on the ability of spherical cell and colony formation,proliferation and tumorigenicity of LCSCs were observed.In order to further study the effect of the interaction between miR-1305 and UBE2 T on LCSCs signal pathway.QRT-PCR and Western blot were used to analyze the effects of miR-1305 and UBE2 T on the expression of phosphorylated p-Akt and p-GK3 βin Akt signal pathway,respectively.LYC294002 Akt signal pathway inhibitor was used to inhibit Akt signal pathway,and the effects of UBE2 T and miR-1305 on the expression of p-Akt and p-GSK3 β protein in LCSCs were observed.The effects of UBE2 T on the formation,proliferation and migration of spherical and colony cells of LCSCs and the volume and weight of tumor in nude mice were observed by LY294002 treatment.Results: significant differential genes and common genes between hepatocellular carcinoma cells and normal cells were compared and analyzed by in situ hybridization and GEO microarray database,and the interaction network map was drawn.UBE2 T is located in the center of the interaction network map.Flow cytometry,q RT-PCR and Western blot analysis showed that UBE2 T was highly expressed in CD133+ CD13+ liver cancer stem cells,and the expression level of Oct4,Sox2,Nanog related to self-renewal ability of stem cells was significantly higher than that of Huh7 cells.By silencing UBE2 T gene,the ability of spherical cell formation,colony formation and proliferation of LCSCs were significantly inhibited,and the volume and weight of tumor after LCSCs inoculation in nude mice were significantly reduced.Furthermore,the interaction between miR-1305 and UBE2 T was verified by luciferase reporter gene system.Mi R-1305 could significantly inhibit the expression of relative luciferase in UBE2 T group,but could not inhibit the expression level of relative luciferase in UBE2 Tmut group.In order to further verify the interaction between miR-1305 and UBE2 T in LCSCs,miR-1305 and UBE2 T were cotransfected into LCSCs.The results showed that miR-1305 could significantly inhibit the ability of spherical cell and colony formation,proliferation and tumorigenicity of LCSCs,while the overexpression of UBE2 T could significantly improve the self-renewal ability and tumorigenicity of LCSCs.In order to further study the effect of the interaction between miR-1305 and UBE2 T on LCSCs signal pathway.The results of q RT-PCR and Western blot analysis showed that miR-1305 and UBE2 T could significantly reduce and increase the expression of phosphorylated p-Akt and p-GK3 β in Akt signal pathway,respectively.In this paper,LYC294002 Akt signal pathway inhibitors are used to inhibit Akt signal pathway.The results showed that UBE2 T could significantly up-regulate the expression of p-Akt and p-GSK3 β protein in LCSCs,miR-1305 could significantly down-regulate the expression of p-Akt and p-GSK3 β protein in LCSCs,and LY294002 could also significantly down-regulate the expression of p-Akt and p-GSK3 β protein in LCSCs by UBE2 T gene.LY294002 treatment could significantly reduce the formation,proliferation and migration of spherical cells and colony cells overexpressing LCSCs in UBE2 T,and also significantly reduce the volume and weight of tumors in nude mice inoculated with UBE2 T overexpressed LCSCs.Conclusion: miR-1305 targets UBE2 T to inhibit Akt signaling pathway,thus inhibiting the self-renewal and tumorigenicity of LCSCs.These findings may enhance the understanding of miR-1305 as a therapeutic target to limit the progress of LCSCs.This study will provide a new type of small molecules for inhibiting the progress of liver cancer stem cells and provide a scientific basis for the analysis of the molecular mechanism of the progression of liver cancer cells.