Circular RNA-ZNF532 Regulates Diabetes-induced Retinal Pericyte Degeneration And Vascular Dysfunction

Objective:Diabetic retinopathy(DR)is one of the main causes of blindness in adults,which seriously threatens the quality of diabetic patients' life and causes serious social economic load.Pathological neovascularization is the main manifestation of DR.Previous studies have shown that endothelial cell dysfunction is closely related to pathological neovascularization,yet the role of pericyte degeneration in DR is poorly understood.CircularRNAs(circRNAs)represent a class of widespread and diverse endogenousRNAs which could regulate cell proliferation,differentiation,apoptosis and migration.Increasing studies demonstrate that circRNAs are key regulators of various physiological and pathological processes and are expected to become new biomarkers of various human diseases.In this study,we investigated the role of circularRNA-ZNF532 in pericyte biology and diabetes mellitus-related microvascular dysfunction.Methods:1.Part ?: We used CircularRNA microarray to identify high glucose-regulated circRNAs in pericytes and retina.Hierarchical clustering was used to analysis the high glucose-regulated circRNAs.Real-time reverse transcription PCR(q RT-PCR)was conducted to detect the expression pattern of 5 upregulated and 5 downregulated circRNAs identified by circRNA microarray.In vivo,cZNF532 expression was detected by q RT-PCRs in retinal vessels isolated from nondiabetic retinas and diabetic retinas.In vitro,the expression of cZNF532 in pericytes and HRVECs were also detected.Bioinformatics analysis and chromatin immunoprecipitation were used to detect the upstream target molecules which may regulate cZNF532 under diabetic conditions.2.Part ?: In vitro,siRNAs or plasmid were transfected into cells to intervene the expression of cZNF532.MTT,Ki67 immunofluorescence staining,PI staining,and caspase-3/7 activity assay were used to detect the role of cZNF532 on pericyte viability,proliferation and apoptosis.The expression of pericyte markers was detected by q RT-PCRs.Matrigel assays combined with immunofluorescence staining were conducted to detect the role of cZNF532 on pericyte recruitment.Measurement of barrier permeability was used to detect the role of cZNF532 on endothelial barrier permeability.In vivo,we used intravitreous injection of cZNF532 shRNA to reduce the expression of cZNF532 or conditional knockdown cZNF532 in pericytes.Evans blue and PAS staining assays were used to detect the role of cZNF532 on pericyte coverage and diabetic vascular pathology.3.Part ?: Bioinformatics analysis and luciferase activity assays were conducted to demonstrate the interaction between cZNF532 and mi R-29a-3p.q RT-PCR was used to detect the relative expression abundance of cZNF532 and mi R-29a-3p.Target Scan was used to predict the target genes of mi R-29a-3p.Luciferase activity was used to detect the interaction between mi R-29a-3p and its target genes.4.Part ?: q RT-PCR assay was conducted to detect the expression of cZNF532/mi R-29a-3p/CSPG4(NG2),LOXL2 and CDK2 in human vitreous specimens.To reveal the potential clinical significance of cZNF532 /mi R-29a-3p/target gene on pericytes and diabetic retinal microvascular dysfunction,pericytes were incubated with the vitreous from PDR patients and intervened the expression of CZNF532 /mi R-29a-3p,the apoptosis of pericytes was detected.Human diabetic vitreous and cZNF532 or mi R-29a-3p antagonist was injected into the vitreous cavity in C57BL/6 mice,retinal vasopermeability and pericyte degeneration were then detected.Results:1.Part ?: Using comprehensive circularRNA expression profiling,we identified identified 1523 differentially expressed circRNAs in retinal vascular pericytes under hyperglycemic stress.Venn diagrams were generated to identify high glucose-regulated circRNAs and exclude osmotic pressure-regulated circRNAs.Eighty-six upregulated and 97 downregulated circRNAs were specifically regulated by high glucose.Hierarchical clustering analysis of high glucose – regulated circRNAs.real-time reverse transcription PCR(q RT-PCR)and Sanger sequencing showed that cZNF532 is constitutively expressed in pericytes and significantly up-regulated upon diabetes mellitus-related stresses.2.Part ?: In vitro,MTT assay,Ki67 staining,transwell assay,and q PCR assays showed that cZNF532 silencing significantly reduced the pericyte viability,proliferation,and recruitment,and affected the pericyte differentiation.In vivo,Evans blue,PAS staining,and ELISA assays shown that cZNF532 silencing could aggravate retinal vascular dysfunction as shown by decreased pericyte coverage and increased vascular leakage and retinal acellular capillaries.3.Part ?: Bioinformatics analysis,luciferase activity assays,andRNA pull-down assays showed that cZNF532 could act as an endogenous mi R-29a-3p sponge to inhibit mi R-29a-3p activity,which led to increased CSPG4(NG2),LOXL2,and CDK2 expression.4.Part ?: q RT-PCRs showed that cZNF532 expression was upregulated in the vitreous of patients with DME,PDR,or NVI and its expression was correlated with disease severity.ELISAs showed that NG2,LOXL2,or CDK2 concentration was upregulated in the vitreous of patients with DME,PDR,or NVI compared with nondiabetic control samples.Pericytes were incubated with the vitreous from PDR patients.Caspase-3/7 activity assay or PI staining assays revealed that induction of cZNF532-mediated signaling by overexpression of cZNF532 or inhibition of mi R-29a-3p protected pericytes against diabetic vitreous-induced pericyte apoptosis.Coinjection of cZNF532 overexpression vector or mi R-29a-3p antagomir reduced diabetic vitreous–induced effect on retinal vasopermeability and pericyte degeneration.Conclusions:1.Hyperglycemia induce the expression of SP1 transcription factor.SP1 could bind to the promoter region of cZNF532 and activate cZNF532 transcription in pericytes.2.In vitro,cZNF532 silencing reduced pericyte viability,proliferation,and recruitment,and affected pericyte differentiation.3.In vivo,cZNF532 silencing aggravated retinal vascular dysfunction as shown by decreased pericyte coverage and increased retinal acellular capillaries,vascular leakage,and inflammation.4.Mechanistically,cZNF532 acted as an endogenous mi R-29a-3p sponge to sequester and inhibit mi R-29a-3p activity.5.The circularRNA cZNF532 plays a vital role in pericyte biology and diabetes mellitus-induced retinal vascular dysfunction,suggesting that circularRNA is a potential target to control diabetic vascular complications.