The Mechanism And Effect Of MiR-424 In Endometria Cancer

Objective: mi RNA,a small noncoding RNA that regulates target gene or protein expression by combining with m RNA on transcription level,involved in progresses of so many diseases including tumors.Study will explore the role of mi R-424 in endometrial cancer,so that the study shall lay the groundwork for target treatment and mi R-424-target network in EC.Methods: The model of uregulated expression miR-424 and knockdown miR-424 was established through instantaneously transfected mi R-424 mimics and mi R-424-ASO into two EC cell lines HEC-1-A and Ishikawa,and the transfected system is 50 n M and transfected vector is Lipofectamine3000.The experiment was divided into 4 groups: Control group,mi R-424 mimics group,mi R-424-ASO group and ASO-NC group.1.Transwell invasive assay detected the changes of ability of invasion when EC cells were ransfected by mi R-424 mimics and mi R-424-ASO and ASO-NC after 48 hours.And Wound healing assay detected the ability of migration in24 h and 48 hours then evaluted the rate of wound healing of EC cells were upregulated and downregulated by mimics and ASO-mi RNA.2.Western Blot assay detected the total cell protein expression of E-cadherin and Vimentin that was marker of epithelial-to-mesenchymal transition(EMT).And Immunofluorescence assay detected cell protein expression and verified the location of E-cdherin through staining.3.Dual luciferase reporter assay verified the fact that 3? UTR of E2F6 could be complementary paired by mi R-424.The previously designed mut E2F6 3?UTR or E2F6 3?UTR plasmid and mi R-424 mimics or ASO-mi R-424 or ASO-NC and helper transfection reagent were transfected into EC cells.The experiment groups were:Control,mi R-424 + wild E2F6 3?UTR group/mut E2F6 3?UTR group,ASO-424 +wild E2F6 3?UTR group/mut E2F6 3?UTR group,ASO-NC + wild E2F6 3?UTR group/mut E2F6 3?UTR group.The fluorescence detection kit and Promega Dual-Luciferase system measured the fluorescence of each group.4.Then the RT-q PCR assay detected the expression of m RNA of E2F6 and Western Blot assay detected the expression of E2F6 protein,that verified the mutual interaction between mi R-424 and E2F6.5.To reversely verify the E2F6 made influences for mi R-424,EC cells transfected by mi R-424 mimics were transfected by E2F6 overexpressed plasmid or without E2F6 upregulated plasmid.The experiment were individed into two groups:mi R-424 mimics + pc DNA3.1-E2F6 group,mi R-424 mimics + pc DNA3.1 group.6.Then invasion ability of EC cells were detected by Transwell invasive assay,and migration ability was detected through Wound healing assay.Results: Transwell assay showed that invasion cell counts of mi R-424 mimics significant decreased vs control group(P<0.05),and invasion cell counts of ASO-424 group significant increased vs ASO-NC group(P<0.05).And Wound healing assay showed that wound healing ratio of mi R-424 mimics significantly decrease vs Control group(P<0.05),and wound healing ratio of ASO-424 was markedly higher than ASO-NC group(P<0.05).So knockdown of mi R-424 in cell lines of EC facilitated migration and invasion ability of EC cell and invasion and migration ability was disrupted by overexpressed mi R-424.Western Blot assay showed that the expressed level of protein E-cadherin in mi R-424 mimics that was marker of EMT was dramatically increased compared with Control group(P<0.05).The expression of Vimentin protein was evidently decreased in mi R-424 mimics compared with Control group(P<0.05).And the E-cadherin protein expression in ASO-424 group was lower than ASO-NC group(P<0.05).Expressed level of Vimentin protein in ASO-424 group was higher than ASO-NC group(P<0.05).Immunofluorescence assay discovered that EMT-related E-cadherin protein located at around of EC cell membrance.To verify the E2F6 was target of mi R-424,Dual luciferase reporter assay was designed.The assay displayed that luciferase intensity of mi R-424 + wild E2F6 3?UTR group was incaresed vs Control group(P<0.05),and compared with ASO-NC + E2F6 3?UTR group,the luciferase intensity of ASO-424 + wild E2F6 3?UTR was higher then ASO-NA E2F6 3?UTR group(P<0.05).And the luciferase intensity had no significance of mi R-424 + mut E2F6 3?UTR and ASO-NC + mut E2F6 3?UTR respectively vs Control and ASO-NC.Then the RT-q PCR study showed that expression of m RNA of E2F6 was decreased in mi R-424 mimics group compared with Control group(P<0.05),and expression of m RNA of E2F6 in ASO-424 group was increased compared with ASO-NC group(P<0.05).The luciferase reporter assay indcated mi R-424 could directly combined with 3?UTR of E2F6 in EC cells.Western Blot study showed that expression of E2F6 protein was decreased in mi R-424 mimics group compared with Control group(P<0.05),and expression of E2F6 protein in ASO-424 group was increased compared with ASO-NC group(P<0.05).To reversely verify the E2F6 made influences to mi R-424,designing the assay that EC cells were transfected by mi R-424 mimics with E2F6 upregulated plasmid or without E2F6 upregulated plasmid.The transwell assay and wound healing experiment showed that the invaded cell numbers and wound healing ration were markedly increased in mi R-424 mimics + pc DNA3.1-E2F6 group vs mi R-424 mimics + pc DNA3.1group(P<0.05).Conclusion: This work demonstrates a new meschanism that mi R-424 actes as a tumor suppressor to inhibite migration,invasion and EMT progression by regulating target E2F6 in EC.And E2F6 weaken the anti-carcinoma influence of mi R-424 in EC cells.