Investigation Into The Expression And Mechanism Of ING4 In Diabetic Retinopathy

BackgroundDiabetic retinopathy(DR)is the most common microvascular complication of diabetes and has been noted as a major public health problem that seriously threatens the vision health.The pathological changes at the early stages of DR include the thickening of the vascular basement membrane,microvascular occlusion,loss of microvascular pericytes,microaneurysm formation and blood-retinal barrier(BRB)breakdown.At later stages,proliferative diabetic retinopathy leads to retinal neovascularization,epiretinal membrane formation and subsequent traction retinal detachment.The pathogenesis of DR are extremely complicated and still not fully understood,and most studies attribute the pathological changes to accumulation of advanced glycosylation end products(AGEs),enhanced polyol pathway,activation of protein kinase C,nonenzymatic glycation and oxidative stress.The imbalance between pro-angiogenic and anti-angiogenic factors and the breakdown of BRB induced by severe hypoxia have become the important part in the development of DR.Therefore,exploring the mechanism of BRB breakdown and retinal neovascularization from the perspective of molecular biology is of great significance to find targets for prevention and treatment of DR.Inhibitor of growth 4(ING4)belongs to the inhibitor of growth(ING)family,whichs function as the type II tumor suppressor,and is responsible for the regulation of multiple cellular processes such as cell cycle,apoptosis,contact inhibition,hypoxic adaptation,tumor angiogenesis,invasion,and metastasis.Human ING4 protein is highly expressed in normal tissues,but its expression is dramatically decreased in some types of cancer and non-neoplastic disorders,suggesting that its aberrant expression may contribute to the pathogenesis of these diseases.Compelling evidence suggests that ING4 physically interacts with several key genes that regulate cellular functions,such as NF-?B,VEGF,HIF-1? and MMPs.Restoring ING4 may improved the pathological state and has been a potential treatment target.Specificity protein 1(Sp1)is widely distributed in human cells and involved in metabolism,cell proliferation,growth,cell death,as well as many pathogenic signaling pathways in various disease states.There are 12,000 Sp1 binding sites in the human genome and Sp1 regulates downstream gene transcription by binding to the GC-box or GT-box elements to affect cellular function.The abilities of Sp1 to bind to the transcriptional sites can also be regulated by upstream factors,including HIF-1?,e NOS and ERK.A high level of Sp1 can be detected in the eye tissues of DM patients,DR animals and cell models,and Sp1 exerts an angiogenic effect by regulating factors such as VEGF in most studied.Since the regulatory relationship between ING4 and a variety of important genes involved in the pathogenesis of DR has been shown,it suggests that ING4 may play a certain role in DR by interacting with these key factors.ObjectiveThe study aimed to detect the expression of ING4 in the retina of normal and diabetic rats.ARPE-19 was cultured under hypoxia conditions to simulate the hypoxic microenvironment during the pathogenesis of DR,exploring its migration and angiogenesis regulation in hypoxic environment.Furthermore,we investigated the possible regulatory relationship between ING4,Sp1 and several factors involved in cell migration and angiogenesis.Methods1.Exploring the expression levels of ING4 in the normal and diabetic retinas Forty healthy male Sprague-Dawley rats were randomly divided into normal control group(Control,20 rats)and diabetic group(Diabetic,20 rats).A type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin(STZ)and control rats received an identical volume of citrate buffer.Rats were sacrificed 4,8 or 12 weeks later,and eyeballs from each group were removed.The pathological changes of retina were observed by hematoxylin-eosin staining using the paraffin section,and the expression and localization of ING4 protein in retinal tissues were detected by immunohistochemical staining.The total RNA and protein of the retinas were extracted to detect the expression of ING4.2.Detecting the effects of ING4 on the cellular function of ARPE-19 in hypoxic environmentAfter transfection with an ING4 overexpression lentiviral vector,ARPE-19 migration under hypoxia was tested by wound healing and transwell assays.The angiogenic effect of conditioned medium(CM)from ARPE-19 cells was examined by assessing human retinal endothelial cell(HREC)capillary tube formation.3.Detecting the effect of ING4 on the Sp1 expression in ARPE-19 under hypoxic conditionsAfter culturing ARPE-19 under hypoxia for 24,48 and 72 hours,ING4 m RNA and protein levels were detected by RT-q PCR and Western blot.Normal ARPE-19,as well as the cells transfected with negative control and ING4-overexpressing lentivirus was cultured in hypoxia for 24 hours,then RT-q PCR and Western blot were conducted to detect the regulation of ING4 on the Sp1 expression.4.Determining the effect of ING4 on the expression of VEGF-A,MMP-2 and MMP-9 in ARPE-19 under hypoxic conditionsING4 overexpressing and control cells were treated under hypoxia for 24 hours,and RT-q PCR and Western blot were performed respectively to detect the effect of hypoxia and/or overexpression of ING4 on the m RNA and protein expression levels of VEGF-A,MMP-2 and MMP-9.5.Transfecting ARPE-19 cells with ING4 or Sp1 si RNA under hypoxia conditions to detect the changes of VEGF-A,MMP-2 and MMP-9ARPE-19 was transfected with Sp1 si RNA and then cultured under hypoxia for 24 hours.RT-q PCR and Western blot were performed to determine the expression of ING4,Sp1,VEGF-A,MMP-2 and MMP-9,respectively.Furthermore,the m RNA and protein levels of ING4,Sp1,VEGF-A,MMP-2 and MMP-9 were also detected with ING4 silencing.Results1.Blood glucose levels of rats met the diagnostic criteria for diabetes after the single intraperitoneal injection of STZ,demonstrating that diabetic model was established successfully.By hematoxylin-eosin staining of diabetic retinas,we found that the structure of nerve fiber layer(NFL)and ganglion cell layer(GCL)were slightly disordered in 4 weeks of diabetic rats.In the diabetic retinas from 8 weeks and 12 weeks,histopathological damage included edema and vacuolar degeneration in NFL,disordered and loose arrangement in the GCL,inner and outer nuclear layer.Besides,the boundaries between inner and outer segments of the photoreceptor were not clear.Immunohistochemical staining showed that diffuse ING4 expression seemed to decrease throughout the diabetic rat retinas compared with normal rats.Quantitative analysis showed reduced ING4 m RNA and protein levels in the retina of diabetic rats.2.After ARPE-19 was infected with ING4 overexpression lentiviral vector,the expression of green fluorescent protein was detected.ING4 m RNA and protein expression were significantly higher than the basal expression level,suggesting that ARPE-19 cell line stably overexpressing ING4 had been successfully obtained.Hypoxia exposure significantly increased the migratory capacity of ARPE-19 cells,and hypoxic CM efficiently promoted HREC tube assembly in vitro.These effects were reversed by ING4 overexpression.3.We examined ING4 expression levels in ARPE-19 under hypoxia conditions and detected no significant changes at any time point,demonstrating that ING4 was not affected by hypoxic,which is consistent with previous results in another cell line.Hypoxia exposure upregulated Sp1 expression and this phenomenon was prevented by the ectopic overexpression of ING4 using lentivirus transfection.4.Hypoxia induced increased expression levels of VEGF-A,MMP-2 and MMP-9,and ING4 overexpression partly reduced their expression.These data indicated that ING4 exerted potential inhibitory effects on these pro-angiogenic factors under hypoxia.5.Si RNA-mediated Sp1 depletion in ARPE-19 under hypoxia was shown to restore the expression of VEGF-A,MMP-2 and MMP-9 that was induced by hypoxia,but failed to alter ING4 expression.However,with the downregulation of ING4 expression,the expression levels of Sp1,VEGF-A,MMP-2 and MMP-9 were higher than that simply under hypoxia.ConclusionING4 is expressed in the normal retinas and decreases in the diabetic ones,and could inhibit the hypoxia-induced migration and angiogenesis regulation of ARPE-19 cells in vitro.ING4 exerts an inhibitory effect on cell migration and proangiogenesis-related genes such as VEGF-A,MMP-2 and MMP-9,and this regulatory pathway is dependent on the transcription factor Sp1,demonstrating that ING4 may have protective effects in the development of DR.