MiR-147a Inhibits Choroidal Melanoma Cells Proliferation,Migration And Invasion By Targeting MCM3

Objective: Choroidal melanoma(CM)is the most common uveal malignant tumor in adults.Due to its complex condition,high degree of malignancy,easy invasion and metastasis,the prognosis is extremely poor.Therefore,it is of great significance to study the pathogenesis of choroidal melanoma in order to reduce its incidence and mortality.Researches in recent years have confirmed that micro RNA(mi RNA)is related to the occurrence and development of various tumors including choroidal melanoma,and can affect the biological behavior of tumor cells,such as growth,differentiation,metastasis,apoptosis and so on.In addition,minichromosome maintenance protein 3(MCM3)is one of the components of DNA prereplication complex,which participates in the initiation and extension of DNA replication and the repair of gene damage.And MCM3 plays an important role in the growth,proliferation and invasion of tumor cells.Above all,it is speculated that mi RNA and MCM3 may be involved in regulating the biological process of choroidal melanoma cells.In this project,we will study the effects of mi R-147 a and MCM3 on the proliferation,migration and invasion of choroidal melanoma cells,and explore whether mi R-147 a plays its biological role through targeting regulation of MCM3.Methods: 1.To explore the effects of mi R-147 a on biological function of choroidal melanoma cells.Human choroidal melanoma MUM-2B cells were transfected with liposome,and four groups were set up: mi R-NC,mi R-147 a mimics,inhibitor-NC,mi R-147 a inhibitor.EDU was used to detect cell proliferation in each group;Transwell assay was used to detect cell migration and invasion.2.To explore mi R-147 a is targeted to bind to MCM3 and regulate its expression.The downstream target genes of mi R-147 a were predicted and analyzed by Targetscan7.2 bioinformatics website.Dual luciferase reporter gene assay was used to verify whether mi R-147 a is targeted to bind to the 3’UTR region of MCM3 m RNA.Western Blot was conducted to detect the target gene MCM3 protein expression content.3.To explore the effects of MCM3 on the biological function of choroidal melanoma cellsSmall RNA interference technique was used to knock down the MCM3 gene in choroidal melanoma MUM-2B cells.In NC RNA and MCM3-si RNA groups,Western Blot was used to verify the efficiency of MCM3 knockdown.EDU test was used to detect cell proliferation,and Transwell test to detect cell migration and invasion.4.To explore the effects of MCM3 on proteins related to PI3K/AKT signal pathway.Western Blot was used to detect the protein expressions of p110α,phosphorylated AKT and total AKT in control group and MCM3 knockdown group.Results:1.The results of EDU test showed that the number of proliferation of MUM-2B cells with up-regulated mi R-147 a was significantly lower than that of the control group(t=5.11,P<0.01),on the contrary,the number of down-regulated mi R-147 a was higher than that of the control group(t=3.43,P<0.05).Transwell assay indicated that the numbers of migration and invasion of MUM-2B cells with up-regulated mi R-147 a were lower compared with the control group(t=5.90,P<0.01;t=3.05,P<0.05),the numbers of down-regulated mi R-147 a were higher compared with the control group(t=8.12,P<0.01;t=4.52,P<0.05).2.There is a complementary sequence between mi R-147 a and the 3’UTR region of MCM3 m RNA.Dual luciferase assay showed that compared with the control group(transfected with mi R-NC and MCM3 3’UTR wild-type plasmids),the relative luciferase activity of the cells transfected with mi R-147 a and MCM3 3’UTR wild type plasmids was decreased significantly(t=15.10,P<0.001),but there was no significant difference in luciferase activity between the two groups transfected with mi R-NC/mi R-147 a and MCM3 3’UTR mutant plasmids(t=0.25,P=0.82).Western Blotting results indicated that up-regulated mi R-147 a can decrease the level of MCM3 protein in MUM-2B cells(t=4.06,P<0.05),down-regulated mi R-147 a can increase the level of MCM3 protein(t=8.24,P<0.01).3.Compared with the control group,the result of Western Blot test showed that MCM3-si RNA significantly down-regulated the expression of MCM3 protein of choroidal melanoma cells(t=5.12,P<0.01).EDU assay results showed that the ability of cell proliferation was reduced after knockdown of MCM3(t=7.30,P<0.01).Transwell assay results indicated that the ability of cell migration and invasion is significantly reduced after knockdown of MCM3(t=11.57,P<0.001;t=5.98,P<0.01).4.Compared with the control group,the contents of p110α and phosphorylated AKT protein in the knockdown MCM3 group were confirmed to be decreased(t=6.48,P<0.01;t=5.05,P<0.01),but there was no significant change in total AKT content between the two groups(t=0.27,P=0.80).Conclusion: 1.mi R-147 a can inhibit the malignant proliferation,migration and invasion of choroidal melanoma cells.2.mi R-147 a binds to MCM3 m RNA 3 ’UTR and inhibits the expression of MCM3 protein.3.The knockdowned MCM3 can also inhibit the proliferation,migration and invasion of choroidal melanoma cells,which may be related to the inhibition of PI3K/AKT signal pathway.