MiR-9-5p Negatively Regulates Choroidal Melanoma Cells By Targeting BRAF

Introduction: To investigate the mechanism of miR-9-5p regulating the function of choroidal melanoma cells by targeting BRAF.Methods: 1.The regulation role of miR-9-5p on choroidal melanoma cells.Human invasive choroidal melanoma cells MUM-2B and MUM-2C were cultured in vitro and were instantaneously transfected with miR-9-5p mimics,miR-9-5p inhibitor,and their respective negative control(NC and inhibitor NC)with cationic liposomes as the medium.RT-qPCR was conducted to evaluate the expression of miR-9-5p.EDU assay was applied to measure the state of cell proliferation.Transwell assay was utilized to measure the state of cell migration and invasion.2.miR-9-5p binds to BRAF and inhibits the expression of BRAF.Through bioinformatics website TargetscanHuman7.2 and microRNA.org,the binding sequence of miR-9-5p to the 3'UTR region of BRAF mRNA was predicted.Double luciferase reporter gene assay was conducted to confirm the binding sequence between miR-9-5p and the 3'UTR region of BRAF mRNA.RT-qPCR was conducted to evaluate the expression of BRAF mRNA.Western Blotting was conducted to detect the expression of BRAF protein.3.The influence of BRAF on wild-type CM cells.With gene interference technique,siRNAs were transfected into choroidal melanoma cells MUM-2B and MUM-2C.Western Blotting was conducted to detect the expression of BRAF protein.EDU was used to measure choroidal melanoma cells' proliferation.Transwell assay was used to detect cells' migration and invasion.4.The regulation role of miR-9-5p on choroidal melanoma cells by directly targeting BRAF.Western Blotting was conducted to detect the expression of BRAF protein when stable cell lines overexpressing BRAF were constructed,meanwhile Western Blotting was conducted to detect the expression of BRAF protein when stable cell lines overexpressing BRAF being transfected with miR-9-5p mimics.EDU was used to detect the proliferation of choroidal melanoma cells.Transwell was used to examine the migration of choroidal melanoma cells.Results:1.The evidence of RT-qPCR indicated that the expression of miR-9-5p is significantly up-regulated in choroidal melanoma cells after being transfected with miR-9-5p mimics,while the expression of miR-9-5p is significantly down-regulated in choroidal melanoma cells after being transfected with miR-9-5p inhibitor.EDU assay results showed that the ability of cell proliferation is reduced in miR-9-5p mimics group,while the ability of cell proliferation is enhanced in miR-9-5p inhibitor group.Transwell assay results indicated that cell migration and invasion is significantly reduced in miR-9-5p mimics group,while cell migration and invasion is significantly enhanced in miR-9-5p inhibitor group.2.Analysis of Targetscan Human7.2 and microRNA.org predicted the binding sequence between miR-9-5p and the 3'UTR region of BRAF mRNA.The results of double luciferase assay verified the binding sequence between miR-9-5p and the 3'UTR region of BRAF mRNA.The evidence of RT-qPCR indicated that the expression of BRAF is significantly down-regulated in choroidal melanoma cells after being transfected with miR-9-5p mimics,while the expression of BRAF is significantly up-regulated in choroidal melanoma cells after being transfected with miR-9-5p inhibitor.Western Blotting results indicated that the expression of BRAF protein is significantly down-regulated in miR-9-5p mimcs group,while the expression of BRAF protein is significantly up-regulated in miR-9-5p inhibitor group.3.The results of Western Blotting showed that siRNAs significantly down-regulate the expression of BRAF protein of choroidal melanoma cells.EDU assay results showed that the ability of cell proliferation is reduced when silencing BRAF.Transwell assay results indicated that cell migration and invasion is significantly reduced when silencing BRAF.4.BRAF protein expression was sharply up-regulated in stable cell lines overexpressing BRAF,meanwhile Western Blotting results showed that the expression of BRAF is down-regulated in stable cell lines overexpressing BRAF when being transfected with miR-9-5p mimics.Experimental results of cell function in vitro manifested that the ability of cell proliferation and migration is notably enhanced in stable cell lines overexpressing BRAF,meanwhile cell proliferation and migration is reduced when stable cell lines overexpressing BRAF being transfected with miR-9-5p mimics.Conclusions: 1.miR-9-5p inhibits proliferation,migration and invasion of choroidal melanoma cells.2.BRAF promotes the proliferation,migration and invasion of wild-type choroidal melanoma cells.3.miR-9-5p negatively regulates the expression of BRAF and the proliferation,migration and invasion of choroidal melanoma cells by directly targeting BRAF.