The Mechanism Of Transthyretin Affecting Diabetic Retinopathy Through Circ＿0007411

Diabetic retinopathy(DR)is a progressive and complex complication that seriously harms the health of diabetic patients,and it is becoming the main cause of blindness in working age people nowadays.Its main clinical manifestations include vascular permeability changes,capillary microaneurysms,capillary degeneration and angiogenesis.It has been found that transthyretin(TTR)can affect the formation of neovascularization and further affect the development of DR.Circ RNA is a kind of non-coding RNA widely existing in human body.Therefore,starting from the effect of TTR on neovascularization,this paper elaborated the molecular mechanism of TTR’s influence on DR process from the perspective of circ RNA.In this study,circ RNA sequencing was used to analyze the changes of circ RNA expression levels in Human Retinal Endothelial Cells(HRECs)with exogenous TTR,and the differences of circ RNA expression were screened.The molecular mechanism of DR was analyzed by in vivo and in vitro experiments.The results show that:(1)Recombinant TTR protein was identified as tetramer TTR protein by SDS-PAGE and gel filtration chromatography.Circ RNA sequencing results showed that TTR affected the expression of circ＿0007411 in HRECs under high glucose environment.Circ＿0007411 was derived from PTPN12 gene.Compared to the high glucose condition,the expression of circ＿0007411 and PTPN12 in HRECs increased to 1.60 and 1.27 times,respectively,when TTR was added.(2)The results of Ed U,flow cytometry,transwell,Matrigel and scratch healing experiments showed that circ＿0007411 inhibited the proliferation,migration,tube formation and scratch healing of HRECs,and promoted the apoptosis of HRECs in high glucose environment.In the meantime,circ＿0007411 also promoted the expression of PTPN12 m RNA and PTPN12 protein.(3)Diabetic mice were injected with AAV virus which overexpressed PTPN12 and EB test,PAS test and H&E test were performed three months later.Diabetic mice showed symptoms of retinal hemorrhagic leakage,neovascularization,pericyte detachment and retinal detachment.In the experimental group,the DR mice overexpressed PTPN12 showed reduced retinal leakage,fewer neovascularization,and alleviated pericyte and retinal detachment symptoms,while the DR mice in sh PTPN12 group which the PTPN12 expression were knocked down showed increased retinal leakage,more neovascularization,and aggravated pericyte and retinal detachment symptoms.(4)Micro RNAs related to circ＿0007411 were screened out from the database.The downstream targets of PTPN12 m RNA were also screened out to find out the specific downstream target.Mi R-548 m was found to have a common binding site with both circ＿0007411 and PTPN12 m RNA.QRT-pcr analysis showed that circ＿0007411 reduced the miR-548 m level in HRECs under high glucose environment,and miR-548 m reduced the expression of circ＿0007411 and PTPN12 in HRECs in turn.In vitro,miR-548 m mimics accelerated cell proliferation,migration,tube formation and scratch healing,and inhibited apoptosis of HRECs under high glucose conditions,while the overexpression of circ＿0007411saved such functional phenotype.(5)Western blot results showed that the up-regulation of miR-548 m caused the decrease of PTPN12 and Skp1 protein levels and the increase of EGFR protein levels in HRECs,which could be saved by the exoverexpression of circ＿0007411.Co-IP experiment results showed that Skp1 and PTPN12 interact directly.TTR,by increasing the expression level of circ＿0007411,promotes the binding of circ＿0007411 to miR-548 m and leads to the high expression of PTPN12.Then the expression of Skp1 and EGFR downstream were changed to affect angiogenesis.That is,circ＿0007411 plays an important role in the development of DR through miR-548m/PTPN12/Skp1/EGFR signaling pathway.