The Protective Mechanisms And Effects Of MiRNAs Targeting HIF-1α/SP1/Robo4 In The Development Of Diabetic Retinopathy

BackgroundDiabetic retinopathy(DR)is the leading cause of visual disability and blindness in the working-age adults(20–65 years).DR is one of the most serious microvascular complications of diabetes mellitus(DM).The pathogenesis of DR is multifactorial and the molecular mechanisms are still not fully understood.DR is primarily caused by the long-term detrimental effects of high glucose,following by oxidative stress,inflammation response,accumulation of glycation end-products and neural dysfunction,which lead to retinal microvascular defects and the breakdown of blood-retinal barrier.These pathophysiological changes will cause the macular edema and retinal neovascularization.Therefore,it is fundamental to explore the pathophysiological and molecular mechanisms involved in protecting the integrity of blood-retinal barrier and inhibiting the neovascularization to prevent and treat DR.Roundabout 4(Robo4)is specifically expressed in vascular endothelial cells and is involved in angiogenesis and the maintenance of blood vessel stability.Moreover,Robo4 promotes pathological angiogenesis through various signaling pathways.Recently,Robo4 was found to be related to the progression of DR.Robo4 expression and distribution have been studied in the fibrovascular membranes(FVMs)of patients with proliferative DR.It was increased not only in FVMs,but also in the endothelial cells of new vessels.It has been reported that the expression level of Robo4 is increased with the time prolonging in human retinal endothelial cells(HREC)under hypoxia,and the knockdown of Robo4 could enhance the cell viability and inhibit the cell migration.Furthermore,Robo4 was detected in human retinal pigment epithelium(RPE)cells,downregulation of Robo4 could enhance the ability of cell viability,migration and tube formation under hypoxic conditions.Thus,Robo4 may have a role in the formation of FVMs and could exert physiologic effects on retinal cells.These implied that Robo4 may play important roles in the development of DR,however,the molecular and pathophysiological mechanisms are not fully understood.We previously detected that hypoxia induced factor-1α(HIF-1α)was colocalised with Robo4 in the vessels of FVMs,and HIF-1α could positively regulate Robo4 in HREC under hypoxia or normoxia.HIF-1α is an oxygen-sensitive transcription factor that is associated with angiogenesis during the progression of DR and FVM development.Under conditions of low oxygen,hundreds of proteins related to cell proliferation,survival and angiogenesis can be activated by HIF-1α signalling pathways.However,through the prediction by PROMOTER 2.0,there is no binding site of HIF-1α on the promoter region of Robo4.There may be some modulatory mechanisms mediated HIF-1α regulating Robo4.Further analyzing the promoter region of Robo4,there are multiple specificity protein 1(SP1)binding sites.Thus,SP1 may be the transcriptional factor that can activate Robo4.Specificity protein 1(SP1)and HIF-1α cooperate to promote tumor progression and activate genes related to cell adaption for hypoxia.Additionally,SP1 is necessary for full basal expression of Robo4 in macrovascular endothelial cells.DNA methylation of the proximal promoter of Robo4 inhibits SP1 binding,inducing low Robo4 expression in nonendothelial cells.Thus,aberrant levels of Robo4 induced by HIF-1α may be mediated via SP1 in DR.MicroRNAs(miRNAs)are a group of short(approximately 21–23 nts long)and highly conserved sequences of endogenous RNAs that do not code for any protein.miRNAs modulate gene expression through transcriptional and post-transcriptional regulation,inducing mRNA degradation or inhibiting protein translation by binding to the seed region on the 3′-UTR of target genes.Recent studies showed that miRNAs play important roles in the progression of DR and are involved in multiple signalings and pathogenic changes related to DR.By the prediction of TargetScan,miR-125b-5p may target SP1 and Robo4,while miR-146a-5p may target HIF-1α and Robo4.Thus,HIF-1α may modulate Robo4 by the mediation of SP1 or miR-146a-5p regulation,while SP1 may binding to the promoter region of Robo4 directly or through miR-125b-5p mediation.The modulation of Robo4 by transcriptional or miRNA regulation could exert potential effects on the development of DR.ObjectiveThe study aimed to explore the expression levels of HIF-1α,SP1 and Robo4 in the rat retinas and RPE cells under diabetic conditions,and the variations of related miRNAs.The transcriptional regulation and miRNA-mediated modulation of Robo4 expression was evaluated in RPE cells under hyperglycemic or hypoxic conditions in vitro.Furthermore,we explored the effects of downregulating Robo4 expression on the related retinal cell functions during the progression of DR.Methods1.Exploring the expression levels of HIF-1α,SP1,Robo4 and miRNAs in the diabetic retinasForty 8-week male SD rats were randomised into negative control group(NC,20 rats)and diabetes group(DM,20 rats).Diabetes was induced by a single intraperitoneal injection of streptozotocin(65 mg/kg,in citrate buffer,pH 4.5),and control rats received an identical volume of citrate buffer.Rats were considered diabetic when their blood glucose exceeded 16.7 mmol/L at 72 h and 1 week after STZ administration.Control or diabetic rats were maintained for 4,6,and 8 weeks.Six eyeballs from each group were excised for preparation of retinal tissue sections(6 μm)to perform HE and immunofluorescent staining.Retinal tissue for protein analysis and mRNA extraction was conducted in six eyes from each group.2.Detecting the expression levels of HIF-1α,SP1,Robo4 and miRNAs in ARPE-19 under hyperglycemic conditionsRPE cells were cultured with high glucose medium(25 mmol/L D-glucose)for 1,3,5,and 7 days.Protein levels of HIF-1α,SP1,Robo4 were analyzed by western blot,while transcript levels of HIF-1α,SP1,Robo4 and miRNAs were analyzed by RT-qPCR.Furthermore,double immunofluorescence was performed to localize the expression of HIF-1α,SP1 and Robo4 in ARPE-19 under high glucose.3.Determining the levels of HIF-1α,SP1,Robo4 and miRNAs in ARPE-19 under hypoxic conditionsRPE cells were treated with hypoxia for 4,8,16 and 24 hours.Protein and RNA were extracted for determining the changes of HIF-1α,SP1,Robo4 and miRNA by western blot and RT-qPCR.4.Transfecting RPE cells with HIF-1α siRNA,SP1 siRNA or Robo4 siRNA under different conditions to detecting the changes of their levelsRPE cells cultured under hyperglycemia or hypoxia were transfected with X HIF-1α siRNA,SP1 siRNA or Robo4 siRNA.Western blot and RT-qPCR were performed to determine the effects of silencing HIF-1α,SP1 and Robo4 respectively.Furthermore,the mRNA and protein levels of SP1 and Robo4 were detected with knockdown of HIF-1α,while Robo4 was also measured with SP1 silencing.5.Confirming the modulatory roles of miRNAs on their targetsThrough the prediction of TargetScan,miR-125b-5p may target the 3’-UTR of SP1 and Robo4,while miR-146a-5p may target the 3’-UTR of HIF-1α and Robo4.Firstly,RPE cells were transfected with miR-125b-5p mimic under hyperglycemia,then the levels of SP1 and Robo4 were detected to confirm whether miR-125b-5p could inhibit the expression of SP1 and Robo4.Similarly,miR-146a-5p mimic was transfected to RPE cells under hypoxia,and then the levels of HIF-1α and Robo4 were measured by Western blot and RT-qPCR.Finally,luciferase assays were conducted to confirm the miRNA direct targeting sites on the 3’-UTR of target genes.6.Evaluating the effects of regulatory mechanisms mediating Robo4 expression on RPE cells functions under hyperglycemic or hypoxic conditionsRPE cells were cultured under hyperglycemic or hypoxic conditions,transfecting with specific siRNA or miRNA mimic to inhibit the expression of HIF-1α,SP1 and Robo4.MTS,monolayer permeability,tight-junction proteins and transwell assay were performed to evaluate the changes of RPE cell viability,permeability and migratory ability in different conditions.Results1.By HE staining of diabetic retinas from 4 weeks of DM rats,we found that the nerve fiber layer(NFL)and ganglion cell layer(GNL)arranged slightly disorganized,and then deteriorated in DM 6 weeks and 8 weeks.Pathological changes including edema,vacuolar degeneration and inner plexiform layer(IPL)thinner were also observed in long-term DM retina.Western blotting showed high levels of HIF-1α,SP1,and ROBO4 after 4 weeks of uncontrolled diabetes,with levels sustained at 6 and 8 weeks.HIF-1α and ROBO4 were upregulated beginning at 4 weeks,whereas SP1 tended to increase at week 4 and was significantly overexpressed at week 6.Immunofluorescence analysis showed that HIF-1α and SP1 were weakly expressed in all layers of the normal retina,particularly the NFL,GCL,IPL,and RPE.In the retinas of 8-week DM rats,HIF-1α and SP1 were upregulated not only in the NFL,GCL,IPL,and RPE but also in the outer plexiform layer(OPL)and inner section(IS).Robo4 was weakly expressed in the NFL,GCL,IPL,IS,and RPE in normal retina,but upregulated in the same layers as well as the OPL in DM rats.Thus,HIF-1α,SP1,and Robo4 were co-expressed and with stronger expression in diabetic retinas than in the normal retina.These results implied that HIF-1α,SP1,and Robo4 were involved in the development of DR and may deteriorate its progression.In addition,we found the aberrantly expressed miRNAs including miR-124-3p,miR-125b-5p,miR-135b-5p,miR-145-5p,miR-146a-5p,miR-199a-5p,which may play vital roles in the progression of DR.2.Notably,high glucose enhanced HIF-1α,SP1,and ROBO4 mRNA and protein levels in RPE cells in a time-dependent manner(1,3,5 and 7 days),whereas mannitol control did not induce any obvious changes.While RPE cells cultured under the same condition,miR-124-3p,miR-125b-5p,miR-135b-5p,miR-145-5p,miR-146a-5p,miR-199a-5p were changed with hyperglycemic time prolonging.Notably,miR-125b-5p was decreased while the levels of SP1 and Robo4 were increased.Thus,upregulation of SP1 and Robo4 may be mediated by downregulation of miR-125b-5p.Double immunofluorescence staining of high-glucose-treated ARPE-19 cells showed that HIF-1α was weakly detected in the nucleus and cytoplasm of RPE cells under normal glucose(NG)or manitol(MN)and was strongly enhanced under high glucose(HG).Robo4 showed weak immunofluorescence in the cytoplasm and cytomembrane of RPE cells under NG or MN,and upregulation was observed after treatment with HG.SP1 was expressed in the nucleus of RPE cells,with upregulation observed after culture in HG.Co-staining with Robo4 showed strong fluorescence in the cytoplasm and cytomembrane of RPE cells in HG compared with that in NG or MN.These results confirmed the localization and upregulation of HIF-1α,SP1,and Robo4 in RPE cells exposed to high glucose.3.RPE cells treated with hypoxia for 4,8,16 and 24 hours induced the increased transcriptional and protein levels of HIF-1α,SP1,and Robo4.Accordingly,changes in HIF-1α,SP1,and Robo4 in ARPE-19 cells under hypoxia were similar to those under high-glucose conditions.We then analyzed the expression of another specific miRNA,miR-146a-5p.Our results showed that miR-146a-5p was downregulated in RPE cells exposed to hypoxia for 16 h,and HIF-1α and Robo4 were elevated,suggesting that miR-146a-5p and HIF-1α/ROBO4 may interact in ARPE-19 cells.XII 4.Transfecting RPE cells under HG or hypoxia with HIF-1α siRNA,SP1 siRNA,or Robo4 siRNA,the mRNA and protein levels of these three genes were significantly inhibited by the specific siRNA.Furthermore,SP1 was repressed with HIF-1α downregulation,and Robo4 was inhibited following suppression of HIF-1α or SP1.These data implied that HIF-1α could positively modulate Robo4 expression by mediating SP1 in DR models.5.After transfection of ARPE-19 cells with miR-125b-5p mimic under HG conditions,miR-125b-5p was increased by nearly 25-fold compared with miRNA scramble transfection.This transfection reduced hyperglycemia-induced SP1 and Robo4 upregulation at both mRNA and protein levels.Luciferase assays revealed that cotransfection of HEK293 with the corresponding 3′-UTR WT plasmid and miR-125b-5p mimic led to a significant decrease in luciferase activity compared with the scrambled mimic.These results confirmed the direct downregulation of SP1 and Robo4 by miR-125b-5p in RPE cells under HG conditions.Under hypoxia,miR-146a-5p overexpression blocked hypoxia-induced HIF-1α and Robo4 upregulation in RPE cells.HIF-1α 3′-UTR or Robo4 3′-UTR luciferase activity was significantly suppressed with miR-146a-5p overexpression compared with the scramble mimic.Thus,miR-146a-5p could directly inhibit the overexpression of HIF-1α and Robo4 in ARPE-19 cells under hypoxic conditions.6.The effects of HIF-1α,SP1,and Robo4 expression on RPE cells under DR conditions were then evaluated.RPE cell viability was significantly decreased,cell monolayer permeability was increased,the tight-junction proteins were broken and cell migration was promoted compared with that in normal glucose or normoxic conditions.Transfection with SP1 or Robo4 siRNA or miR-125b-5p mimic under HG,and knockdown of HIF-1α/ROBO4 by siRNA or miR-146a-5p under hypoxic condition,could elevate the viability of RPE cells,reduce the cell permeability,increase the levels of tight-junction proteins and decrease cell motility significantly.Accordingly,knockdown of Robo4 by upstream inhibition of HIF-1α/SP1 or miRNA improved the RPE morphology in the progression of DR.ConclusionWe demonstrated the regulatory effects of HIF-1α mediated SP1 regulation and miRNAs on Robo4 expression in the development of DR.The high level of Robo4 deteriorated the progression of DR.Downregulation of Robo4 significantly improved cell functions under different conditions in DR.Therefore,targeting specific miRNAs in combinational therapy may have the potential to prevent the expression of multiple genes in multifaceted diseases,such as DR.