PMID- 37643981 OWN - NLM STAT- MEDLINE DCOM- 20230831 LR - 20230914 IS - 1008-9292 (Print) IS - 1008-9292 (Linking) VI - 52 IP - 4 DP - 2023 Aug 25 TI - CircRNA-SCAF8 promotes vascular endothelial cell pyroptosis by regulating the miR-93-5p/TXNIP axis. PG - 473-484 LID - 1008-9292(2023)04-0473-12 [pii] LID - 10.3724/zdxbyxb-2023-0091 [doi] AB - 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-1beta. 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-1beta 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. FAU - Wang, Bing AU - Wang B AD - Department of Vascular Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. 3170104484@zju.edu.cn. FAU - Yu, Xinyu AU - Yu X AD - Department of Vascular Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. 3180105451@zju.edu.cn. FAU - Chen, Tianchi AU - Chen T AD - Department of Vascular Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. FAU - Qiu, Chenyang AU - Qiu C AD - Department of Vascular Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. FAU - Lu, Wei AU - Lu W AD - Department of Vascular Surgery, Quzhou Hospital Affiliated to Wenzhou Medical University, Quzhou 324000, Zhejiang Province, China. FAU - Zheng, Xiangtao AU - Zheng X AD - Department of Vascular Surgery, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325027, Zhejiang Province, China. vaszhengxiangtao@126.com. FAU - Wu, Ziheng AU - Wu Z AD - Department of Vascular Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. wuziheng@zju.edu.cn. LA - eng LA - chi PT - Journal Article TT - 环状RNA-SCAF8通过miR-93-5p/TXNIP通路促进血管内皮细胞焦亡. PL - China TA - Zhejiang Da Xue Xue Bao Yi Xue Ban JT - Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences JID - 100927946 RN - 9001-27-8 (Factor VIII) RN - 0 (RNA, Circular) RN - 0 (Interleukin-18) RN - EC 3.4.22.36 (Caspase 1) RN - 0 (MicroRNAs) RN - 0 (TXNIP protein, human) RN - 0 (Carrier Proteins) RN - 0 (SCAF8 protein, human) RN - 0 (RNA-Binding Proteins) RN - 0 (MIRN93 microRNA, human) SB - IM MH - Humans MH - *Factor VIII MH - RNA, Circular MH - Endothelial Cells MH - Interleukin-18 MH - Pyroptosis MH - In Situ Hybridization, Fluorescence MH - Caspase 1 MH - *MicroRNAs/genetics MH - Carrier Proteins/genetics MH - RNA-Binding Proteins PMC - PMC10495250 OAB - 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-1beta. 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-1beta 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. CONCLUSION: CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose. OABL- eng OTO - NOTNLM OT - Circular RNA-SR-related CTD associated factor OT - Diabetes mellitus type 2 OT - Human umbilical vein endothelial cells OT - MiR-93-5p OT - Pyroptosis OT - Thioredoxin interacting proteins COIS- 所有作者均声明不存在利益冲突 The authors declare that there is no conflict of interests EDAT- 2023/08/30 00:41 MHDA- 2023/08/31 06:42 PMCR- 2023/08/25 CRDT- 2023/08/29 22:34 PHST- 2023/08/31 06:42 [medline] PHST- 2023/08/30 00:41 [pubmed] PHST- 2023/08/29 22:34 [entrez] PHST- 2023/08/25 00:00 [pmc-release] AID - 1008-9292(2023)04-0473-12 [pii] AID - 10.3724/zdxbyxb-2023-0091 [doi] PST - ppublish SO - Zhejiang Da Xue Xue Bao Yi Xue Ban. 2023 Aug 25;52(4):473-484. doi: 10.3724/zdxbyxb-2023-0091.