Targeting Pericyte-Endothelial Cell Crosstalk By Circular RNA CPWWP2A Inhibition Aggravates Diabetes Induced Retinal Microvascular Dysfunction

Objective: Diabetic retinopathy(DR)is a common blind diabetes-induced microvascular dysfunction.Circular RNAs(circRNAs)are a novel class of non-coding RNAs that participate in many physiological and pathological processes.We aim to use circRNAs microarray to find differential expression of circRNAs in the retinas of DR mouse,and to observe its function in diabetic induced retinal microvascular dysfunction in vitro and vivo.Methods: 1.CircRNAs microarray profiling is conducted to investigate the potential involvement of circRNAs in the retinas of DR mouse.We conduct qRT-PCR to randomly verify 20 up-regulated and 20 circRNAs down-regulated circRNAs and selecte a specific circular RNA-PWWP2 A as the target for the following study.2.Silencing and overexpressing of cPWWP2 A in the retinas of DR mouse by cPWWP2 A shRNA and mimic in vivo,we use Evans Blue leakage assay,ELISA,the whole mount of retinal immunofluorescence staining and trypsin digestion of retina with PAS staining to observe the function of cPWWP2 A on DR microvascular dysfunction.3.Knockdown of cPWWP2 A in pericytes by cPWWP2 A siRNA in vitro,we use immunofluorescence staining,Ki67 staining,PI staining and Caspase 3/7 activity assay to evaluate the effects of cPWWP2 A expression on pericytes.Tube formation combined CD31/NG2 cell immunofluorescence staining and barrier function assay to observe the function of low expressed cPWWP2 A in pericyts on endothelial cells.4.Using RNA fluorescence in situ hybridization(RNA-FISH),luciferase reporter assay and RNA-Pulldown to confirm targeted binding of cPWWP2 A with miR-579.We silence or overexpress cPWWP2 A / miR-579 to observe the effects on diabetes-induced retinal microvascuar dysfunction and pericytes by Evans Blue leakage assay,the whole mount of retinal immunofluorescence staining,trypsin digestion of retina with PAS staining,Ki67 staining,PI staining and Caspase 3/7 activity assay in vivo and vitro.A luciferase reporter is used to analyze the target of miR-579 for Angiopoietin 1(Ang 1)/ Occludin / Silent mating type information 2 homolog 1(SIRT 1).Results: 1.The results of Circular RNA microarray indicate that 884 circRNAs are differentially expressed including 433 are up-regulated and 411 are down-regulated.We use qRT-PCR to randomly verify the 14 of 20 up-regulated and 16 of 20 down-regulated circRNAs,three of them are significantly up-regulated.We select the circular RNA cPWWP2 A as the target of this study.2.We find that silencing of cPWWP2 A in retinas of DR mouse in vivo aggravate diabetes-induced retinal microvascular dysfunction.Evans blue assay shows much retinal vascular leakage;ELISA assay shows interleukin(IL)-2,IL-6,tumor necrosis factor-α(TNF-α),vascular endothelial growth factor(VEGF)and human monocyte chemotactic protein(MCP-1)are increased;Immunofluorescence staining shows that pericytes coverage is decreased;Trypsin digestion of retina shows increased abnormal microvascular morphology.However,cPWWP2 A overexpression treatment protects against above diabetes-induced retina microvascular injury.3.We find that knocking down of cPWWP2 A in pericytes damage its functions in vitro.Immunofluorescence staining shows the decreased pericytes coverage;Ki67 cell proliferation assay shows reduced proliferation of pericytes;PI staining and Caspase 3/7 activity assay shows the increased level of apoptosis;Tube formation and CD31/NG2 immunofluorescence staining shows the poor ability of pericytes recruit to endothelials and destroy endothelial barrier function.4.Firstly,it is confirmed by RNA-FISH,luciferase reporter and RNA-Pulldown that cPWWP2 A targeted with miR-579 in the cytoplasm.Secondly,we find that miRNA-579 overexpress in vitro and vivo,leading to decreased NG2 expression in immunofluorescence staining;Ki67 cell proliferation assay shows the decreased proliferative ability;PI staining and Caspase 3/7 activity assay shows the increased level of apoptosis;Tube formation and CD31/NG2 immunofluorescence staining show the poor recruitment.After co-transfection with miRNA-579 and cPWWP2 A,cell proliferation and recruitment is significantly higher than that of miRNA-579 alone,and cellular apoptosis is significantly decreased.However,after antagonizing the expression of miR-579,the above manifestations are improved.Finally,luciferase reporter confirms that Angiopoietin 1/ Occludin/ SIRT 1 are the downstream genes of cPWWP2A/miR-579.Conclusion: 1.Diabetes-induced retinal microvascular dysfunction is associated with the expression of circular RNAs.We find that circular RNA-PWWP2 A is highly expressed under high glucose in vivo and vitro.2.In vivo,silencing of cPWWP2 A aggravates the pathological process of diabetes-induced retinal microvascular dysfunction.3.In vitro,silencing of cPWWP2 A hurts the function of pericytes and pericytes-endothelial crosstalk.4.cPWWP2 A acts as an endogenous miR-579 sponge to sequester and inhibit miR-579 activity,leading to increased expression of Angiopoietin 1,Occluding and SIRT 1.Thus,cPWWP2 A involves in the regulation of pericytes function and pericytes-endothelial crosstalk.