circ_0001879调控miR-599对脂多糖诱导HUVECs氧化应激损伤的影响

Effect of circ_0001879 regulating miR-599 on lipopolysaccharide-induced oxidative stress injury of human umbilical vein endothelial cells

目的探讨circ_0001879调控微小RNA-599(miR-599)对脂多糖(LPS)诱导人脐静脉血管内皮细胞(HUVECs)氧化应激损伤的影响。方法体外传代培养HUVECs。取传3代、对数生长期、生长状态良好的HUVECs,随机分为Control组与LPS组以及Control组、LPS组、LPS+si-NC组、LPS+si-circ_0001879组,LPS+si-NC组、LPS+si-circ_0001879组经1μg/mL LPS处理24 h后分别予si-NC、si-circ_0001879转染,LPS组仅予LPS处理,Control组不予转染且未经LPS处理。收集细胞,采用RT-qPCR法检测circ_0001879、miR-599表达,采用Annexin V-FITC/PI双染法检测细胞凋亡率,采用Western blotting法检测凋亡相关蛋白Bcl-2、Bax表达,按相关试剂盒说明检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)等氧化应激损伤指标含量。通过CircInteractome网站预测circ_0001879与miR-599的靶向调控关系。WT-circ_0001879和MUT-circ_0001879分别与miR-NC和miR-599共转染HUVECs,按双荧光素酶报告基因检测试剂盒说明检测荧光素酶活性。结果LPS组circ_0001879相对表达量显著高于Control组,miR-599相对表达量显著低于Control组(t分别为15.960、14.152,P均<0.01)。与Control组比较,LPS组和LPS+si-NC组circ_0001879相对表达量、细胞凋亡率、Bax蛋白相对表达量以及LDH、MDA含量均显著升高,而Bcl-2蛋白相对表达量以及SOD、GSH-Px含量均显著降低(P均<0.05);与LPS组和LPS+si-NC组比较,LPS+si-circ_0001879组circ_0001879相对表达量、细胞凋亡率、Bax蛋白相对表达量以及LDH、MDA含量均显著降低,而Bcl-2蛋白相对表达量以及SOD、GSH-Px含量均显著升高(P均<0.05)。经CircInteractome网站预测,circ_0001879与miR-599存在互补的核苷酸序列;与转染miR-NC+WT-circ_0001879的HUVECs细胞比较,转染miR-599+WT-circ_0001879的HUVECs细胞荧光素酶活性显著降低(t=17.483,P<0.05),而转染miR-NC+MUTcirc_0001879、miR-599+MUT-circ_0001879的HUVECs细胞荧光素酶活性比较差异无统计学意义(t=0.246,P>0.05)。结论circ_0001879可能通过靶向调控miR-599缓解LPS诱导的HUVECs氧化应激损伤。

Objective To investigate the effect of circ_0001879 regulating microRNA-599(miR-599)on lipopolypac⁃charide(LPS)-induced oxidative stress injury of human umbilical vein endothelial cells(HUVECs).Methods HUVECs were cultured in vitro.HUVECs in the third generation,which were in logarithmic growth phase and had good growth sta⁃tus,were randomly divided into the Control group and LPS group,and Control group,LPS group,LPS+si-NC group,and LPS+si-circ_0001879 group.HUVECs in the LPS+si-NC group and LPS+si-circ_0001879 group were treated with 1μg/mL LPS for 24 h and then were transfected with si-NC and si-circ_0001879,respectively.HUVECs in the LPS group were treat⁃ed with LPS only,and HUVECs in the Control group were not transfected and were not treated with LPS.The expression of circ_0001879 and miR-599 could not be detected by RT-qPCR.The apoptosis rate was detected by Annexin V-FITC/PI double staining,and the expression levels of apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting.The content of lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD),glutathione perox⁃idase(GSH-Px)and other oxidative stress injury indexes was detected according to the related kit instructions.The target ed regulatory relationship between circ_0001879 and miR-599 was predicted by CircInteractome website.HUVECs were co-transfected with WT-circ_0001879 and MUT-circ_0001879 combined with miR-NC and miR-599,respectively,and luciferase activity was detected according to the instructions of the dual luciferase reporter assay kit.Results The rela⁃tive expression level of circ_0001879 in the LPS group was significantly higher than that in the Control group,and the rela⁃tive expression level of miR-599 was significantly lower than that in the Control group(t=15.960 and 14.152,both P<0.01).Compared with the Control group,the relative expression level of circ_0001879,apoptosis rate,relative expression level of Bax protein and content of LDH and MDA in the LPS and LPS+si-NC groups increased,while the relative expression level of Bcl-2 protein and content of SOD and GSH-Px decreased(all P<0.05).Compared with the LPS group and LPS+si-NC group,the relative expression level of circ_0001879,apoptosis rate,relative expression level of Bax protein and con⁃tent of LDH and MDA in the LPS+si-circ_0001879 group significantly decreased,while the relative expression level of Bcl-2 protein and the content of SOD and GSH-Px significantly increased(all P<0.05).CircInteractome predicted that circ_0001879 and miR-599 had complementary nucleotide sequences.Compared with HUVECs transfected with miR-NC+WT-circ_0001879,the luciferase activity of HUVECs transfected with miR-599+WT-circ_0001879 significantly decreased(t=17.483,P<0.05).There was no significant difference in luciferase activity between HUVECs transfected with miR-NC+MUT-circ_0001879 and transfected with miR-599+MUT-circ_0001879(t=0.246,P>0.05).Conclusion Circ_0001879 may alleviate LPS-induced oxidative stress injury in HUVECs by regulating miR-599.