CircRNA-SCAF8 promotes vascular endothelial cell pyroptosis by regulating the miR-93-5p/TXNIP axis.

OBJECTIVES: To investigate the role and mechanism of circRNA-SR-related CTD associated factor 8 (SCAF8) in regulating endothelial cell pyroptosis in high glucose environment.

METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured and divided into six groups. The normal control group and high glucose control group were cultured in cell culture medium with 5 and 33 mmol/L glucose, respectively. The RNA control group, circRNA-SCAF8 inhibition group, miR-93-5p overexpression group and miR-93-5p inhibition group were added with non-functional siRNA, circRNA-SCAF8 inhibitor, miR-93-5p overexpression molecule and miR-93-5p inhibitor in high glucose environment, respectively. Cell viability and pyroptosis were detected by cell counting kit-8 (CCK-8) assay, flow cytometry and Hoechst 33342/propidium iodide fluorescence double staining. Western blotting and enzyme-linked immunosorbent assay were used to detect the expression of pyroptosis-related factors including apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartic acid specific protease-1 (caspase-1) and Gasdermin D (GSDMD), NOD like receptor protein 3 (NLRP-3), thioredoxin interacting proteins (TXNIP), IL-18 and IL-1β. The expression of circRNA-SCAF8, miR-93-5p and TXNIP was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fluorescence in situ hybridization (FISH) was used to locate circRNA-SCAF8 and miR-93-5p. Dual luciferase assay was used to verify the targeted regulatory relationship between miR-93-5p and upstream and downstream molecules.

RESULTS: Compared with the RNA control group, the cell survival rate of circRNA-SCAF8 inhibition group and miR-93-5p overexpression group increased (both P <0.01), the pyroptosis decreased (both P <0.01), and the expressions of pyroptosis-related factors such as TXNIP, NLRP-3, caspase-1, GSDMD, ASC, IL-18 and IL-1β were significantly decreased (all P <0.05). The expression of miR-93-5p was significantly increased after inhibition of circRNA-SCAF8 ( P <0.01), and the expression of circRNA-SCAF8 tended to decrease after overexpression of miR-93-5p, but with no statistical significance ( P >0.05). Dual luciferase assay showed that miR-93-5p downre-gulated circRNA-SCAF8 expression by binding to the 3 ´ UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 ´ UTR region of TXNIP . FISH showed that circRNA-SCAF8 and miR-93-5p were both located in the cytoplasm and were highly associated in the cells. qRT-PCR showed that the relative expression of TXNIP increased or decreased after overexpression or inhibition of miR-93-5p compared with the RNA control group, respectively (both P <0.05), suggesting that miR-93-5p could regulate TXNIP gene expression.

CONCLUSIONS: CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose.