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

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

目的:探究环状RNA(circRNA)-SR相关CTD相关因子8(SCAF8)在高糖环境下对血管内皮细胞焦亡发挥的作用及机制。方法:将来源于人脐静脉内皮细胞分为正常对照组、高糖对照组分别置于5、33 mmol/L葡萄糖浓度的细胞培养基培养,RNA对照组、circRNA-SCAF8抑制组、微RNA(miR)-93-5p过表达组和miR-93-5p抑制组分别在高糖环境下加入无功能siRNA、circRNA-SCAF8抑制分子、miR-93-5p过表达分子和miR-93-5p抑制剂。采用细胞计数试剂盒8(CCK-8)法、流式细胞术和Hoechst 33342/碘化丙啶双荧光染色法检测细胞存活率和焦亡率,采用蛋白质印迹法和酶联免疫吸附法检测凋亡相关斑点样蛋白(ASC)、胱天蛋白酶1(caspase-1)、Gasdermin D蛋白(GSDMD)、NOD样受体家族蛋白3(NLRP-3)、硫氧还蛋白相互作用蛋白(TXNIP)、IL-18和IL-1β等焦亡相关因子表达,采用定量逆转录聚合酶链反应(qRT-PCR)检测circRNA-SCAF8、miR-93-5p和TXNIP的基因表达,采用荧光原位杂交法定位circRNA-SCAF8和miR-93-5p,采用双荧光素酶实验验证miR-93-5p与上下游分子的靶向调控关系。结果:与RNA对照组比较,circRNASCAF8抑制组和miR-93-5p过表达组细胞存活率升高(均P<0.01),细胞焦亡率降低(均P<0.01),TXNIP、NLRP-3、caspase-1、GSDMD、ASC、IL-18和IL-1β等焦亡相关因子的表达量显著减少(均P<0.05);抑制circRNA-SCAF8后miR-93-5p的表达量显著增加(P<0.01),过表达miR-93-5p后circRNA-SCAF8的表达有降低趋势,但差异无统计学意义(P>0.05)。miR-93-5p通过与circRNA-SCAF8的3'UTR区结合下调circRNA-SCAF8表达,miR-93-5p通过与TXNIP的3'UTR区结合下调TXNIP表达。circRNA-SCAF8和miR-93-5p均位于细胞质中,在细胞中高度关联。qRT-PCR结果显示,过表达或抑制miR-93-5p后,TXNIP的相对表达量较RNA对照组分别增加或减少(均P<0.05),证明miR-93-5p可调控TXNIP基因表达。结论:circRNASCAF8/miR-93-5p/TXNIP通路可能参与调控高糖环境下血管内皮细胞焦亡。

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 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-1βwere 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.