The Mechanism Research Of MiR-504 Targeted Negative Regulation Of CDK6 On Proliferation Of Oral Cancer Cells

Objective:To clarify the effects of miR-504 on the biological behavior of oral cancer cells Tca-8113,including proliferation,migration,invasion,plate cloning and apoptosis,and screening and validation of downstream target genes.We explore the role of miR-504 in the biological behavior of Tca-8113 and its regulatory mechanism.And our research lays the theoretical foundation for an in-depth understanding of the molecular mechanisms of oral cancer and the search for new molecular targeted therapeutic targets.Methods:(1)To study the function of miR-504,we transfected Tca-8113 cells with chemically synthesized double-stranded oligonucleotides(miR-504 and NC)to regulate the expression level of miR-504.The expression of miR-504 was detected by real-time quantitative PCR analysis.The NC was used as control.(2)The effects of miR-504 were assessed by cell viability,cell migration,invasion assay,colony formation and apoptosis assay through up-regulating the expression of miR-504.(3)The target genes of miR-504 was predicted by the online microRNA target gene prediction tools,we also repeated the luciferase assay to validate CDK6 as a target of miR-504 in Tca-8113 cells and western blotting was used to protein expression.(4)We study the mechanism of miR-504-5p inhibiting Tca-8113 proliferation of oral cancer cells by western blotting and RT-qPCR.Results:(1)The results demonstrated that cells transfected with miR-504 were significantly increased the expression level of miR-504 compared with the NC group.(2)Up regulation of miR-504 inhibited the viability of Tca8113 cells,cell migration,invasion assay,colony formation,but there was no significant change in apoptosis assay of Tca8113 cells.(3)The target gene of miR-504 was predicted by bioinformatics,and the target gene was screened by RT-qPCR.Finally,CDK6 was selected as the follow-up experiment.(4)Luciferase assay demonstrated that the luciferase activity of the Wt CDK6-3'UTR construct was significantly inhibited after the introduction of miR-504.(5)The results of western blotting showed that after transfection of miR-504,the expression of CDK6 protein was down-regulated than NC group.(6)The expression of E2F1,Cyclin D1,and Beclin 1 in miR-504 overexpression group was significantly lower than that in negative control group,but p21 protein expression was up-regulated in miR-504 overexpression group compared with negative control group.Conclusions:This study is based on the abnormal expression of miR-504 in oral cancer.We explored the role and mechanism of miR-504,and discovered that miR-504 is an actor as tumor suppressor gene.The up-regulation of miR-504 inhibits proliferation,migration,invasion and plate cloning ability of oral cancer cells in vitro.Meanwhile,the targeted regulatory relationship between miR-504 and CDK6 was clarified.Our study may provide ideas and theoretical basis for clinical treatment of oral cancer.