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环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡

OBJECTIVE: 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 a...

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Formato: Online Artículo Texto
Lenguaje:English
Publicado: 《浙江大学学报》编辑部 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10495250/
https://www.ncbi.nlm.nih.gov/pubmed/37643981
http://dx.doi.org/10.3724/zdxbyxb-2023-0091
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collection PubMed
description OBJECTIVE: 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 [Formula: see text] UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 [Formula: see text] 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. CONCLUSION: CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose.
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spelling pubmed-104952502023-09-13 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡 Zhejiang Da Xue Xue Bao Yi Xue Ban Monographic Reports OBJECTIVE: 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 [Formula: see text] UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 [Formula: see text] 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. CONCLUSION: CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose. 《浙江大学学报》编辑部 2023-08-25 /pmc/articles/PMC10495250/ /pubmed/37643981 http://dx.doi.org/10.3724/zdxbyxb-2023-0091 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND 4.0 License (https://creativecommons.org/licenses/by-nc-nd/4.0/)
spellingShingle Monographic Reports
环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡
title 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡
title_full 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡
title_fullStr 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡
title_full_unstemmed 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡
title_short 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡
title_sort 环状rna-scaf8通过mir-93-5p/txnip通路促进血管内皮细胞焦亡
topic Monographic Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10495250/
https://www.ncbi.nlm.nih.gov/pubmed/37643981
http://dx.doi.org/10.3724/zdxbyxb-2023-0091
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