环状RNA VMA21抑制脂多糖诱导的血管内皮细胞损伤和氧化应激机制研究

Circ_VMA21 suppresses vascular endothelial cell damage and oxidative stress induced by lipopolysaccharide

目的:探讨环状RNA VMA21(circ_VMA21)对脂多糖(LPS)诱导的血管内皮细胞损伤和氧化应激的影响及其机制。方法:人脐静脉血管内皮细胞(HUVEC)随机分为Control、LPS、LPS+pcDNA-circ_VMA21、LPS+pcDNA、LPS+微小RNA(miR)-122-5p inhibitor、LPS+anti-miR-NC、LPS+pcDNA-circ_VMA21+miR-122-5p mimic组;实时定量反转录聚合酶链反应(RT-qPCR)检测circ_VMA21和miR-122-5p的表达水平;细胞计数检测细胞活性;流式细胞术检测细胞凋亡;蛋白质印迹法检测蛋白表达;酶联免疫吸附法检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)活性;双荧光素酶报告实验检测circ_VMA21和miR-122-5p的靶向关系。两组间均数比较采用t检验,多组间比较采用单因素方差分析(组间两两比较采用LSD-t检验)。结果:LPS诱导的HUVEC中circ_VMA21表达水平降低(0.17±0.02比1.00±0.00),miR-122-5p表达水平升高(3.76±0.09比1.00±0.00),细胞活性降低(0.41±0.02比1.22±0.07),细胞凋亡率(24.17±0.93比7.62±0.39)及裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved-Caspase-3)表达水平升高(0.80±0.06比0.13±0.01),MDA含量[(736.11±27.21)nmol/ml比(159.37±14.93)nmol/ml]及LDH活性升高[(486.45±16.07)U/L比120.58±9.00)U/L],SOD活性降低[(46.25±4.44)U/L比238.49±11.23)U/L,t=9.721、13.122、15.321、22.472、10.817、38.123、18.237、25.522,P<0.05];与LPS组和LPS+anti-miR-NC组比较,LPS+miR-122-5p inhibitor组miR-122-5p表达水平降低,细胞活性升高,细胞凋亡率及cleaved-Caspase-3表达水平降低,MDA含量及LDH活性降低,SOD活性升高(F=2564.169、693.501、540.990、704.667、41336.059、1170.734、414.559,P<0.05)。与LPS+pcDNA-circ_VMA21组比较,LPS+pcDNA-circ_VMA21+miR-122-5p mimic组miR-122-5p表达水平升高,细胞活性降低,细胞凋亡率及cleaved-Caspase-3表达水平升高,MDA含量及LDH活性升高,SOD活性降低(F=810.792、298.432、376.370、279.806、634.476、528.520、343.420,P<0.05)。结论:circ_VMA21通过下调miR-122-5p抑制脂多糖诱导的血管内皮细胞损伤和氧化应激。

Objective To explore the effect of circ_VMA21 on lipopolysaccharide(LPS)-induced vascular endothelial cell damage and oxidative stress and its molecular mechanism.Methods Human umbilical vein endothelial cells(HUVECs)were randomly divided into control group,LPS group,LPS+pcDNA-circ_VMA21 group,LPS+pcDNA group,LPS+microRNA(miR)-122-5p inhibitor group,LPS+anti-miR-NC group,LPS+pcDNA-circ_VMA21+miR-122-5p mimic group.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression levels of circ_VMA21 and miR-122-5p.Cell counting kit-8(CCK-8)assay was used to measure cell viability.Flow cytometry was used to examine cell apoptosis.Western blotting was used to detect protein expression.Enzyme linked immunosorbent assay(ELISA)was used to determine malondialdehyde(MDA)content,and activity of superoxide dismutase(SOD)and lactate dehydrogenase(LDH).Dual luciferase reporter experiment was done to reveal the targeting relationship between circ_VMA21 and miR-122-5p.The differences between two groups were compared by t test,and the differences between multiple groups were compared by one-way analysis of variance(the LSD-t test was used for pairwise comparison).Results The expression level of circ_VMA21 in HUVECs induced by LPS was decreased(0.17±0.02 vs.1.00±0.00),the expression level of miR-122-5p was increased(3.76±0.09 vs.1.00±0.00),the cell activity was decreased(0.41±0.02 vs.1.22±0.07),the apoptosis rate(24.17%±0.93%vs.7.62%±0.39%)and the expression level of cleaved cysteinyl aspartate-specific protease-3(cleaved-Caspase-3)were increased(0.80±0.06 vs.0.13±0.01),the MDA content[(736.11±27.21)vs.(159.37±14.93)nmol/ml]and LDH activity[(486.45±16.07)vs.(120.58±9.00)U/L]were increased,and the SOD activity[(46.25±4.44)vs.(238.49±11.23)U/L]was decreased(t=9.721,13.122,15.321,22.472,10.817,38.123,18.237,25.522,P<0.05).After overexpression of circ_VMA21 or inhibition of miR-122-5p expression,the expression level of miR-122-5p was decreased,cell activity was increased,cleaved-Caspase-3 expression level was decreased,MDA content and LDH activity were decreased,and SOD activity was increased(P<0.05).Overexpression of miR-122-5p could reverse the effect of circ_VMA21 on LPS-induced HUVECs damage and oxidative stress.Conclusion The circ_VMA21 inhibits LPS-induced HUVECs damage and oxidative stress probably by down-regulating miR-122-5p.