MiRNA-451 On Biological Characteristics Of RF/6A Retinal Vascular Endothelial Cells

Objective We investigated the effect and possible mechanism of miRNA-451 on proliferation, migration and lumen formation ability of RF/6A retinal vascular endothelial cells to provide theoretical basis for treatmenting proliferative diabetic retinopathy.Methods 1. Transfection efficacy was measured after transfecting RF/6A cells with miRNA-451 mimic and inhibitor through lipofectamine. 2. RF/6A cells were divided into four groups: miRNA-451 mimic group?miRNA-451 mimic control group?miRNA-451 inhibitor group and miRNA-451 inhibitor control group. Cellular proliferation were detected by cell counting kit-8(CCK-8). Cell scratch and Transwell test were employed to show its migration ability. Lumen formation capacity were measured by Matrigel test. Besides that, we employed RT-PCR to detect the m RNA expression of certain genes associated with biological behavior. 3. Targeting miRNA-451, we performed bioinformatic analysis on its target with databases such as mi RBase?mi Randa?Target Scan and Pic Tar. 4. SPSS 19.0 software was employed to make the statistical analysis of experimental results.We used One-way ANOVA to compare data between two groups and P<0.05 was considered to be statistically significant.Results 1. Transfection efficacy results after six hours demonstrated miRNA-451 mimic group with high transfection efficacy in 20 um and miRNA-451 inhibitor group with high transfection efficacy in 80 um. 2. According to CCK-8 assay, miRNA-451 mimic group showed significantly lower results compared to that in mimic control group in 24 h and 48h(P<0.05) and inhibitor group showed higher results than that in its control group in 48h(P<0.05). No statistical differences existed between groups of 72 h(P>0.05). 3. Cell scratch test showed the results were lower in miRNA-451 inhibitor group than that in inhibitor control group in 6h and 12h(P<0.05). There were no significant differences between mimic and mimic control group(P > 0.05). 4. Transwell test which represents cell migration ability as well revealed that in 24 h results in miRNA-451 inhibitor group was lower than that in control group(P < 0.05) while no significant differences could be seen in miRNA-451 mimic and mimic control group(P > 0.05). 5. Lumen formation research demonstrated that miRNA-451 mimic group was higher and inhibitor group was lower than that in their respective control group with significant results( P < 0.05). 6. RT-PCR showed that m RNA expression of Cyclin A1?Cyclin D1?PDGFR? and MMP2 were all changed with miRNA-451 expression. Cyclin A1 m RNA was lower in mimic group and inhibitor group in 24 h. It was the same as MMP2. Expression of Cyclin D1 in mimic group was lower and PDGFR? was higher in inhibitor group in 24h(P < 0.05). 7. Bioinformatic analysis showed that miRNA-451 is mainly involved in regulating cell development, negative regulation of cell proliferation and a variety of enzymes and other biological process(P<0.05). It achieved its effect possibly through various signaling pathways such as transforming growth factor-betamitogen-activated protein kinase-, epidermal growth factor-,Wnt-,chemokine-, fatty acid metabolism- and mammalian target of rapamycin signaling pathway(P < 0.05).Conclusions 1. Through transfection method of lipofectamine, miRNA-451 mimic and inhibitor can effectively up-regulate or down-regulate the expression of miRNA-451 in RF/6A cells. It is more significant in down-regulating miRNA-451.When expression of miRNA-451 was down-regulated, proliferation was not changed significantly while ability of migration and lumen formation was restrained with significant results showing promising prospects in treating PDR with miRNA-451. 2. When miRNA-451 was down-regulated, expression of Cyclins?PDGFR? and MMP2 was in accordance with behavioral changes, demonstrating that Cyclins?PDGFR? and MMP2 might be involved with this process. Cyclins and PDGFR? showed the ability of proliferation and MMP2 reflected the migration ability.3. The influence of miRNA-451 on RF/6A cells might be mediated by a series of signaling pathways and possible side effects should be paid attention to.