MiR-96 Targets EPB41L3 To Promote The Proliferation And Migration Of T24 In Bladder Urothelial Carcinoma

Objective: To screen the target gene EPB41L3 of mir-96,study the expression difference of EPB41L3 in bladder cancer tissue and adjacent tissues,and explore the effect of mir-96 targeting tumor suppressor gene EPB41L3 on the proliferation and migration of bladder cancer T24 cells.Methods: The target gene prediction website predicted the target gene of mir-96,constructed the wild-type vector Wt-EPB41L3-3?UTR and the mutant vector Mut-EPB41L3-3?UTR,and co-transfected HKE293 cells.Experimental grouping: Wt-EPB41L3-3?UTR-pmir GLO group,Mut-EPB41L3-3?UTR-pmir GLO group,Wt-EPB41L3-3?UTR-pmir GLO + NC mimics group,Mut-EPB41L3-3?UTR-pmir GLO + NC mimics group,Wt-EPB41L3-3?UTR-pmir GLO + mi R-96-5p mimics group,Mut-EPB41L3-3?UTR-pmir GLO + mi R-96-5p mimics group,Lucifersae detection and analysis of the target relationship between Mir-96 and EPB41L3;Western blot and q RT-PCR detection of EPB41L3 protein and mRNA expression,experimental groups: blank group,Mir-96 mimic group,Mir-96 mimic NC group,Mir-96 inhibitor group,Mir-96 inhibitor NC group;Oncomine database online analysis of EPB41L3 in the bladder The expression of cancer tissue and normal tissue was different.The human bladder cancer tissue chip was used as the experimental research object.The expression of EPB41L3 gene in bladder cancer tissue chip was detected by immunohistochemistry.The EPB41L3-SAP-pc DNA3.0 overexpression plasmid was constructed.Bladder cancer T24 cells were used as experimental research objects,co-transfected with mir-96 mimic and NC,experimental groups: mir-96 mimic + EPB41L3 overexpression vector group,mir-96 mimic + empty vector group,NC + EPB41L3 overexpression vector group,NC + empty vector group;scratch test to detect cell migration,CCK8 to detect cell Proliferation,further confirmed that EPB41L3 is a direct functional target gene of mir-96.Result: 1.Bioinformatics predicts that the target gene of mi R-96 is EPB41L3.2.The wild-type vector EPB-mir-96-wt and the mutant vector EPB-mir-96-mut were successfully constructed,and the double luciferase report experiment proved that mir-96 targets and regulates the EPB41L3 gene.3.Western blot experiments and q RT-PCR detection of mir-96 mimic and mir-96 inhibitor transfection,the expression levels of EPB41L3 protein and mRNA in mir-96 mimic group were significantly lower than those in NC group,compared with mir-96 inhibitor group EPB41L3 protein and mRNA expression levels in mir-96 mimic group were significantly increased.4.Oncomine and UALCAN database online analysis results show that the expression of EPB41L3 in normal bladder tissue is higher than that in bladder cancer tissue(p <0.05).5.Human bladder cancer tissue chip immunohistochemistry experiments showed that the expression of EPB41L3 gene in tissues adjacent to bladder cancer was significantly higher than that in bladder cancer tissues(P = 0.001).6.The results of the scratch experiment and CCK8 experiment showed that the mir-96 mimic + EPB41L3 overexpression vector group compared with the NC + EPB41L3 overexpression vector group,mir-96 counteracted the effect of EPB41L3 on inhibiting cell proliferation and migration,further proving that EPB41L3 is a mir-96 direct functional target gene.Conclusion: EPB41L3 is a target gene directly regulated by mi R-96 in bladder cancer T24 cells.The expression of EPB41L3 in bladder cancer tissues is lower than that in adjacent tissues,and it shows the characteristics of tumor suppressor genes.Proliferation and migration of cancer T24 cells.