Transthyretin-induced increase in circ_0007411 represses neovascularization of human retinal microvascular endothelial cells in hyperglycemia via the miR-548m/PTPN12/SKP1/EGFR pathway

Abstract

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.

The interface targeting hnRNPA2B1 regulates the repression of transthyretin against human retinal microvascular endothelial cells in high‐glucose environment

“…The hRECs were purchased through the Cell Bank of the Chinese Academy of Science and were cultured in phenol red-free Dulbecco's Modified Eagle's Medium (DMEM; Lonza). [13][14][15][16] Cultured hRECs of 5-6 passages were plated at approximately 3000 cells/mL in six-well plates (Corning)…”

“…The proliferation of hRECs was investigated using a CCK-8 kit (Dojindo Laboratories) as described in our previous work. [13][14][15][16] The cells were seeded at 3000 cells/well in 96-well plates, after 70%-80% confluence, they were incubated in LG, LM, HG and HG + TTRs (4 μM) environments for 24 h. 10 μL CCK-8 reagent was added for a further 2 h incubation at 37°C, and A450nm was measured in 5 days.…”

“…The assays were performed according to our previously published protocols. [13][14][15][16] Briefly, for the cell migration assay, hRECs were applied to a Transwell insert, incubated for 30 h, fixed with 4% paraformaldehyde and stained with crystal violet dye. For the wound healing assay, the Wound Healing Assay Kit from Abcam (ab242285) was used.…”

“…After a 3 h incubation, the average tube length was measured as previously described. [13][14][15][16] The tube formation process was monitored using an Olympus IX-73 microscope.…”

“…15 The resulting increase in TTR-induced circ_0007411 levels represses neovascularization via the miR-548 m/PTPN12/SKP1/EGFR pathway. 16 Therefore, revealing the direct target of TTR could be of considerable clinical interest.…”