| Background: Diabetic Retinopathy(DR) is one of the most common and serious complications of diabetes mellitus. It is also an important cause of blindness in adults. With the development of economics and improvement of our living standards, the incidence rate of diabetes mellitus is increasing dramatically. According to the alteration of the fundus oculi, diabetic retinopathy can be divided into two types: proliferative diabetic retinopathy and nonproliferative diabetic retinopathy. The proliferative diabetic retinopathy is pathologically characteristic of angiogenesis, easy to lead to retinal detachment and blindness, and is the main cause of serious visual function impairment. The process of angiogenesis is mainly regulated by vascular endothelial growth factor(VEGF), and a critical element in this process is the proliferation and invation of endothelial cells. VEGF is a strong vascular endothelial cell-specific mitogen. It can induce vascular endothelial cell proliferation, and promote angiogenesis. It was reported that the expression of VEGF is significantly upregulated in diabetic retinopathy model, and this upregulation is associated with hemorrhages and effusion of the retinal blood vessels. Overexpression of VEGF is an important pathogenesis of the development of diabetic retinopathy. Inhibition of VEGF expression has been an important therapeutic strategy for diabetic retinopathy.MicroRNAs(miRNAs) are a group of newly discovered endogenous non-coding RNA with regulatory function in eukaryotes in recent years. miRNAs can interact with the 3’UTR of a certain target gene in a sequence-specific way, thus regulate the protein expression of the target gene. The regulatory functions of miRNAs is widespread, including cellular proliferation, apoptosis, differentiation, growth and development, insulin secretion, tumorigenesis, organogenesis, haematopoiesis, and infection prevention to microbes including virus. These functions are highly conservative and organ-specific. Previous studies indicated that some miRNAs have the effect of regulating angiogenesis. miR-126 is one of the miRNAs that are closed associated with vascular endothelial cells and the functions of blood vessels. It is a kind of angiogenesis signal-regulating factor, which is important for the maintenance of vascular endothelial cells and blood vessels integrity in the body. It is reported that miR-126 can downregulate the angiogenic growth factors such as VEGF, and promote new blood vessels formation. It is also reported that in many kinds of tumor cells, the expression of miR-126 is decreased compared with normal controls. miR-126 is considered as a factor that inhibit the growth of cells, and it acts on the insulin receptor substrate-1(IRS-1) gene. The IRS-1 gene plays a critical role in the insulin signal transduction and the development of diabetes mellitus. Previous reports indicated that the retina expresses large amount of IRS-1 and PI3 K, and insulin can induce the transcription of VEGF mRNA and expression of VEGF in murine aortic smooth muscle cells via the IRS-1/PI3K/AKT pathway and the RAS/MAPK pathway. The upregulation of VEGF expression in vascular endothelia cells by insulin may be via activation of the PI3K/AKT pathway, and VEGF was predicted to lie downstream the IRS-1/PI3K/AKT pathway by the KEGG pathway database. Based on these evidences, we speculate that miR-126 may participate the regulation of retinal new blood vessels formation by acting on the IRS-1 gene via the IRS-1/PI3K/AKT pathway.Objectives: In this study, we aimed to detect the expression level of miR-126 in the endothelial cells(EC)and retinal pericytes(RP)from a mouse model of diabetic retinopathy, explore the target gene of miR-126. We also aimed to detect the mRNA and protein expression levels of the IRS-1 gene in the EC cells and RP cells from the mouse model of diabetic retinopathy. In order to confirm that the IRS-1 gene is the genuine target gene of miR-126, we detect the expression level of IRS-1 after the overexpression or inhibition of the miR-126 expression by transfecting miR-126 mimics or miR-126 inhibitor into the EC cells and RP cells from the mouse model of diabetic retinopathy. In order to elucidate whether miR-126 has regulatory effect on the expression of VEGF, and to elucidate whether the regulatory effects of miR-126 on IRS-1 and VEGF was via the IRS-1/PI3K/AKT pathway, we detected the expression levels of VEGF, PI3 K and AKT after the overexpression or inhibition of the miR-126 expression by transfecting miR-126 mimics or miR-126 inhibitor into the EC cells and RP cells from the mouse model of diabetic retinopathy. Furthermore, we aimed to explore the effects of miR-126 on the proliferative ability and invasive ability of the EC cells and RP cells from the mouse model of diabetic retinopathy. Finally, we aimed to confirm that the inhibitory effects of miR-126 on VEGF, PI3 K and AKT expression, and on EC and RP cell proliferation and invasion were via regulating IRS-1 expression, by interfering IRS-1 with siRNA. With the above experiments, we aim to explore the potentiality of miR-126 as a novel therapeutic target for diabetic retinopathy.Methods: We detected the mRNA expression levels of miR-126 and IRS-1 in the EC cells and RP cells from the mouse model of diabetic retinopathy by real-time PCR, and detected the protein expression level of IRS-1 in the EC cells and RP cells from the mouse model of diabetic retinopathy by Western blot. We overexpressed and inhibited the endogenous miR-126 expression the EC cells and RP cells from the mouse model of diabetic retinopathy by transfecting synthetic miR-126 mimics and miR-126 inhibitor into the cells, and then confirm the effects by detecting miR-126 expression levels after trasfection by real-time PCR.We predicted the target gene of miR-126 with the bioinformatic analytic software TargetScan. We used the dual luciferase reporter gene detection system to validate that the IRS-1 is the genuine target of miR-126. We first cloned the IRS-1 gene 3’UTR DNA fragment containing the predicted binding sites into the pGL-3-promotor plasmid to construct reporter gene plasmid. Then we cotransfected the EC cells with the luciferase reporter vector plasmid containing the wild-type or mutant IRS-1 3’UTR(pMIR-REPORT- IRS-1 wt and pMIR-REPORT- IRS-1 mut)and miR-126 mimics or its control. After 48 hours, we detected the luciferase activity of each group. At the same time, we detected the protein expression level of IRS-1 in the EC cells and RP cells from the mouse model of diabetic retinopathy by Western blot after transfection with miR-126 mimics or miR-126 inhibitor or their controls. Then we used the MTT assay to detect the proliferation rate of EC cells and RP cells after transfection with miR-126 mimics or miR-126 inhibitor or their controls, and detected the invasive ability of EC cells and RP cells with the transwell invasion assay after transfection with miR-126 mimics or miR-126 inhibitor or their controls. Next, we used Western blot to detect the protein expression levels of the PI3K/AKT pathway-related molecules including PI3 K, AKT and VEGF in the EC cells from diabetic retinopathy mouse model when miR-126 was overexpressed or inhibited by transfection with miR-126 mimics or miR-126 inhibitor. Finally, we interfered the expression of IRS-1 in the EC cells from diabetic retinopathy mouse model with siRNA, and detected the protein expression levels of PI3 K, AKT and VEGF by Western blot when the cells were cotransfected with IRS-1-siRNA and miR-126 mimics or miR-126 inhibitor. Again, we detected the proliferation rate and invasive ability of EC cells and RP cells with the MTT assay and the transwell invasive assay when the cells were cotransfected with IRS-1-siRNA and miR-126 mimics or miR-126 inhibitor.Results: In this study, we found that the expression of miR-126 in both EC cells and RP cells of the diabetic retinopathy mouse model were significantly decreased compared with normal controls. We transfected miR-126 mimics or miR-126 inhibitor and their controls into the EC cells and RP cells of the diabetic retinopathy mouse model, and detected the expression levels of miR-126 in the cells after transfection. As a result, the miR-126 expression levels in EC and RP cells transfected with miR-126 mimics were significantly higher than those transfected with miR-126 mimics control, and the miR-126 expression levels in the EC and RP cells transfected with miR-126 inhibitor were significantly higher than those transfected with miR-126 inhibitor control, suggesting that transfection with miR-126 mimics or miR-126 inhibitor can effectively overexpress miR-126 or inhibit the endogenous miR-126 expression level in the EC and RP cells of the diabetic retinopathy mouse model. IRS-1 was predicted to be the target gene of miR-126 in the EC and RP cells of the diabetic retinopathy mouse model using the bioinformatic analytic software TargetScan. Real-time PCR and Western blot analysis revealed that the mRNA and protein expression level of IRS-1 in the EC and RP cells of the diabetic retinopathy mouse model was increased compared with normal control. Then we used the dual luciferase reporter gene assay to validate that IRS-1 is the genuine target of miR-126 in the EC and RP cells of the diabetic retinopathy mouse model. We cotransfected the EC cells with the luciferase reporter vector plasmid containing the wild-type or mutant IRS-1 3’UTR(pMIR-REPORT- IRS-1 wt and pMIR-REPORT- IRS-1 mut)and miR-126 mimics or its control. We detected the luciferase activity of each group 48 hours after transfection. As a result, the luciferase activity in the IRS-1-wt with miR-126 mimics group was significantly reduced compared with the other three groups. The results suggested that miR-126 inhibitor reversed the reduction in the expression level of luciferase with wild-type IRS-1 3’UTR in EC cells. Next, we detected the protein expression level of IRS-1 in the EC and RP cells from diabetic retinopathy mouse model by Western blot after transfection with miR-126 mimics or miR-126 inhibitor into the cells, and found that the expression level of IRS-1 was significantly downregulated by overexpression of miR-126 in EC and RP cells from diabetic retinopathy mouse model, whereas the expression level of IRS-1 was significantly upregulated by inhibition of endogenous miR-126 expression in the EC and RP cells from diabetic retinopathy mouse model. The MTT assay revealed that the proliferation rates of EC and RP cells from diabetic retinopathy mouse model were repressed when transfected with miR-126 mimics compared with the miR-126 mimics control, and the proliferation rates of EC and RP cells from diabetic retinopathy mouse model were increased when transfected with miR-126 inhibitor compared with the miR-126 inhibitor control, suggesting that miR-126 could inhibit the proliferative ability of the EC and RP cells from diabetic retinopathy mouse model. Meanwhile, the results of transwell invasive assay revealed that the number of invading EC and RP cells was decreased when transfected with miR-126 mimics compared with the miR-126 mimics control, whereas the number of invading EC and RP cells was increased when transfected with miR-126 inhibitor compared with the miR-126 inhibitor control, suggesting that miR-126 could inbihit the invasive ability of EC and RP cells from diabetic retinopathy mouse model. Furthermore, we detected the protein expression levels of PI3 K, AKT and VEGF in the EC and RP cells from diabetic retinopathy mouse model by Western blot after transfection with miR-126 mimics or miR-126 inhibitor and their controls. Our results showed that overexpression of miR-126 could significantly downregulate the protein expression levels of PI3 K, AKT and VEGF in the EC and RP cells from diabetic retinopathy mouse model, while inhibition of miR-126 expression could upregulate the protein expression levels of PI3 K, AKT and VEGF in the EC and RP cells from diabetic retinopathy mouse model. Finally, we interfered the expression of IRS-1 with siRNA, and detected the protein expression levels of PI3 K, AKT and VEGF, and the proliferation rate and invasive ability in the EC and RP cells cotransfected with IRS-1-siRNA and miR-126 inhibitor. The results showed that under the condition of IRS-1 silence, the effects of miR-126 inhibitor on PI3 K, AKT, VEGF expression and on the proliferative and invasive ability of EC and RP cells from diabetic retinopathy mouse model were nullified.Conclusions: In our study, we found that the expression of miR-126 was downregulated in the EC and RP cells from diabetic retinopathy mouse model, and that IRS-1 was the target gene of miR-126 in the EC and RP cells from diabetic retinopathy mouse model. Overexpression of miR-126 could downregulate the expression of the PI3K/AKT pathway proteins, such as VEGF, PI3 K and AKT by downregulating its target gene IRS-1. In addition, overexpression of miR-126 could inhibit the proliferative ability and invasive ability of the EC and RP cells from diabetic retinopathy mouse model. Silencing of IRS-1 by siRNA nullified the effects of miR-126 inhibitor on PI3 K, AKT, VEGF expression and on the proliferative and invasive ability of EC and RP cells from diabetic retinopathy mouse model. The results of this study provided a potential target for diabetic retinopathy therapeutic strategy. |
PDF Full Text Request