MiR-15b-5p Mediates Choroid Melanoma Cells Proliferation By Targeting CDK4

Introduction : Choroidal melanoma is the most common primary intraocular malignant tumor in adults.Choroidal melanoma can be induced by many mechanisms,such as gene mutation,chromosome deletion or translocation and abnormal activation of signaling pathways.Among them,the P16-cyclin D-CDK4/6-RB pathway is related to the cell cycle,and many studies have shown that it is closely related to the occurrence and development of choroidal melanoma.CDK4,as a core molecule,plays an important role in the pathway.With the increasing researches on micro RNAs in recent years,a variety of micro RNAs have been confirmed to be involved in the occurrence and development of choroidal melanoma.According to the micro RNA classical mechanism,RNA sequence complementation is predicted by bioinformatics website between mi R-15b-5p and CDK4 3’UTR region.Therefore,this study will explore the inhibition effects of mi R-15b-5p on proliferation of choroid melanoma cell line through targeting CDK4.Methods:1.CCK8 was used to detect the effect of Fascaplysiny,a CDK4 specific inhibitor,on cell proliferation in MUM-2B and MUM-2C cell lines of human eye invasive choroidal melanoma.2.Dual-luciferase assay was used to verify the direct binding site between mi R-15b-5p and the 3’UTR region of CDK4 m RNA.3.Liposomes were used to transfect negative control RNA,mi R15b-5p mimics,inhibitor N.C RNA and mi R-15b-5p inhibitor into MUM-2B cells,respectively.Real-time quantitative PCR was used to detect the expression level of mi R-15b-5p in4 groups after transfection to verify the transfection efficiency.4.Western Blot was used to detect the protein expression of CDK4 in 4 groups.5.CCK8 was used to detect the proliferation ability of 4 groups.6.Flow cytometry was used to detect the cell cycle of 4 groups.Results : 1.CCK8 test results showed that the proliferation ability of cells in the dosing group(100n M)was significantly decreased compared with the control group(0n M).In MUM-2B cells,the relative OD value in the dosing group was 58.1% of that in the control group at 72h(t = 10.57,P < 0.001);in MUM-2C cells,the relative OD value in the dosing group was 49.8% of that in the control group at 72h(t = 10.09,P < 0.001).2.The results of double luciferase reporter analysis showed that compared with the control group(WT+nc group),the luciferase activity of the group co-transfected with CDK4 3’ UTR wild-type plasmid and mi R-15b-5p mimics(WT+mi group)was significantly decreased(t = 7.81,P < 0.01);there was no significant difference in luciferase activity between the two groups co-transfected with CDK4 3’UTR mutant plasmids(MT+nc group and MT+mi group).3.Real-time quantitative PCR results showed that mi R-15b-5p expression in the mimics group was significantly increased compared with that in the nc group(t = 25.01,P < 0.0001);mi R-15b-5p expression in inhibitor group was significantly reduced compared with inhibitor nc group(t = 25.01,P < 0.0001).4.Western Blot results showed that the protein expression level of CDK4 decreased after overexpression of mi R-15b-5p(t =4.92,P < 0.01);the protein expression level of CDK4 increased after under-expression of mi R-15b-5p(t = 3.33,P < 0.05).5.The results of CCK8 detection showed that the cell proliferation ability was weakened after overexpression of mi R-15b-5p.At 72 h,the relative OD value in the mimics group was 67.22% of that in the nc group(t = 9.32,P < 0.001).The cell proliferation ability was enhanced after mi R-15b-5p under-expression of mi R-15b-5p.After 72 hours,OD value of inhibitor group was 1.37 times higher than that of inhibitor nc group(t = 5.72,P < 0.01).6.The results of flow cytometry showed that the percentage of G1 cells increased after overexpression of mi R-15b-5p.The percentage of G1 cells in nc group and mimics group was [(75.98 ± 0.50)% and(81.79 ± 0.75)%],the difference was statistically significant(P < 0.01).The percentage of G1 cells decreased after under-expression of mi R-15b-5p.The percentage of G1 cells in inhibitor nc group and and inhibitor group was [(76.36 ± 0.98)% and(70.82 ± 1.01)%],the difference was statistically significant(P < 0.05).Conclusions:1.CDK4 promotes the malignant proliferation of choroidal melanoma cell lines.2.mi R-15b-5p can target CDK4 and inhibit the expression of CDK4.3.mi R-15b-5p can cause G1/S phase block and inhibit the malignant proliferation of choroidal melanoma cell lines.