Background: To investigate the mechanism of transthyretin (TTR) induced high expression of circ_0007411 and its parent gene, protein tyrosine phosphatase nonreceptor type 12 (PTPN12) in human retinal microvascular endothelial cells (hRECs) cultivated under high glucose condition. Methods:The levels of PTPN12, circ_0007411, miR-548m, S-phase kinase associated protein 1 (SKP1) and epidermal growth factor receptor (EGFR) were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The direct interaction between circ_0007411/PTPN12 and miR-548m was investigated via Dual-luciferase reporter assay. The physiological characterization of hRECs was investigated through Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) labelling, Transwell, flow cytometry (FCM), wound healing, and tube formation assays. Co-immunoprecipitation (Co-IP) was used to detect the interaction between PTPN12 and SKP1. The function of PTPN12 against diabetic retinopathy (DR) was studied in streptozotocin (STZ) induced DR C57BL/6 mice.Results: The levels of circ_0007411 was increased in hRECs in hyperglycemia with the induction of TTR.The overexpressed circ_0007411 could significantly enhance the level of PTPN12 and repress that of miR-548m, and it could enhance apoptosis and prohibit the proliferation, migration, and tube formation of hRECs. miR-548m mimics enhanced the proliferation, migration, and tube formation of hRECs by reducing the expression level of PTPN12 and promoting that of EGFR, whereas circ_0007411 rescued it. The direct binding of PTPN12 and SKP1 was confirmed by Co-IP. Additionally, the anti-neovascularization function of PTPN12 was confirmed in a STZ-induced mouse model of DR. Conclusions:In hyperglycemia, the TTR-induced increase in circ_0007411 could repress retinal neovascularization via the miR-548m/PTPN12/SKP1/EGFR pathway.

BackgroundThe interaction between transthyretin (TTR) and heterogeneous nuclear ribonucleoprotein (hnRNP)A2B1 is involved in the neovascularization of human retinal microvascular endothelial cells (hRECs) under hyperglycemic conditions. However, whether the TTR‐hnRNPA2B1 interface can be altered and how this protein–protein interaction and associated downstream pathways are regulated is unclear.MethodsWe performed homologous sequential analysis and binding energy assays using Discovery Studio and designed substitution targeting three fragments of the interface (fragment 1: aa 34–39, ‐RKAADD‐; fragment 2, aa 61–68, ‐EEEFVEGI‐; and fragment 3, aa 96–102, ‐TANDSGP‐) to disrupt or stabilize the TTR‐hnRNPA2B1 complex and were subjected to Co‐immunoprecipitation analysis. To investigate the effect of TTR‐hnRNPA2B1 interface alterations on the physiological properties of hRECs, we performed CCK‐8, EdU, migration, wound healing and tube formation assays. To study the downstream genes, we performed qRT‐PCR and western blot.ResultsNineteen TTR substitutions were recombinantly expressed in soluble form, results indicated that reducing the binding energy stabilized the TTR‐hnRNPA2B1, while increasing the binding energy had the opposite effect. The native TTR significantly prohibited the proliferation, DNA synthesis, migration and tube formation capacities of hRECs, while fragment 1 always reduced these effects. However, the I68R and D99R substitutions in fragments 2 and 3, respectively, increased the inhibitory effect of TTR. Furthermore, our qRT‐PCR and western blot results showed that the expression and protein levels of STAT‐4, miR‐223‐3p and FBXW7 were also regulated by the alteration of the TTR‐hnRNPA2B1 interface.ConclusionThis work suggests that the formation of the TTR‐hnRNPA2B1 complex plays vital role in hyperglycemia, and modification of this interface regulates the TTR‐mediated inhibition of hREC neovascularization via the STAT‐4/miR‐223‐3p/FBXW7 pathway. This mechanism could have important implications for diabetic retinopathy treatment.