摘要目的:探讨环状RNA HECTD1(circ-HECTD1)是否通过调控miR-98-5p/酪氨酸蛋白激酶A4(EPHA4)的表达参与氧糖剥夺(OGD)诱导的神经元损伤。方法:体外分离培养小鼠原代皮层神经元，采用双荧光素酶报告基因实验、RNA结合蛋白免疫沉淀实验检测神经元中circ-HECTD1、miR-98-5p和EPHA4之间的靶向关系，并将神经元随机分为对照组（正常条件下培养24 h）与OGD 6 h、12 h、24 h组（予OGD处理6 h、12 h、24 h）以及OGD+Vector组与OGD+circ-HECTD1组、OGD+小干扰RNA(siRNA）阴性对照（si-NC）组与OGD+siRNA circ-HECTD1(si-circ-HECTD1)组、OGD+微小RNA(miRNA）模拟物阴性对照（miR-NC）组与OGD+miR-98-5p模拟物（miR-98-5p mimic）组、OGD+miRNA抑制物阴性对照（anti-miR-NC）组与OGD+miR-98-5p抑制物（anti-miR-98-5p）组、OGD+miR-98-5p mimic+pcDNA组与OGD+miR-98-5p mimic+EPHA4组、OGD+si-circ-HECTD1+anti-miR-NC组与OGD+si-circ-HECTD1+anti-miR-98-5p组，分别转染pCD5-ciR空载体、pCD5-ciR-circ-HECTD1过表达载体、si-NC、si-circ-HECTD1、miR-NC、miR-98-5p mimic、anti-miR-NC、anti-miR-98-5p至神经元，以及共转染miR-98-5p mimic和pcDNA3.1空载体、miR-98-5p mimic和pcDNA3.1-EPHA4过表达载体、si-circ-HECTD1和anti-miR-NC、si-circ-HECTD1和anti-miR-98-5p至神经元，OGD处理24 h后，采用实时荧光定量PCR(qRT-PCR）法检测circ-HECTD1、miR-98-5p和 EPHA4 mRNA表达，采用Western blotting实验检测EPHA4蛋白表达，采用MTT法检测细胞增殖活性，采用流式细胞仪检测细胞凋亡率，采用ELISA法检测细胞培养液中白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α含量，采用试剂盒法检测超氧化物歧化酶(SOD)、丙二醛（MDA)活性。 结果:(1)circ-HECTD1能够与miR-98-5p靶向结合，miR-98-5p能够与EPHA4 3'翻译区靶向结合。(2)与对照组比较，OGD 6 h、12 h、24 h组神经元中circ-HECTD1表达均明显升高，miR-98-5p表达均明显降低，EPHA4蛋白表达均明显升高，差异均有统计学意义( P<0.05)。(3)与OGD+Vector组比较，OGD+circ-HECTD1组神经元中circ-HECTD1的表达明显升高，miR-98-5p的表达明显降低；与OGD+si-NC组比较，OGD+si-circ-HECTD1组神经元中miR-98-5p的表达明显升高，EPHA4 mRNA和蛋白的表达均明显降低；与OGD+miR-NC组比较，OGD+miR-98-5p mimic组神经元中miR-98-5p的表达明显升高，EPHA4蛋白的表达明显降低；与OGD+anti-miR-NC组比较，OGD+anti-miR-98-5p组神经元中miR-98-5p的表达明显降低，EPHA4蛋白的表达明显升高；与OGD+si-circ-HECTD1+anti-miR-NC组比较，OGD+si-circ-HECTD1+anti-miR-98-5p组神经元中EPHA4 mRNA和蛋白的表达均明显升高，差异均有统计学意义( P<0.05)。(4)与对照组比较，OGD组神经元的细胞活力明显降低，细胞凋亡率明显升高，IL-1β、TNF-α含量明显升高，SOD活性明显降低，MDA活性明显升高；与OGD+si-NC组比较，OGD+si-circ-HECTD1组神经元的细胞活力明显升高，细胞凋亡率明显降低，IL-1β、TNF-α含量明显降低，SOD活性明显升高，MDA活性明显降低；与OGD+si-circ-HECTD1+anti-miR-NC组比较，OGD+si-circ-HECTD1+anti-miR-98-5p组神经元的细胞活力明显降低，细胞凋亡率明显升高，IL-1β、TNF-α含量明显升高，SOD活性明显降低，MDA活性明显升高；与OGD+miR-NC组比较，OGD+miR-98-5p mimic组神经元的细胞活力明显升高，细胞凋亡率明显降低，IL-1β、TNF-α含量明显降低，SOD活性明显升高，MDA活性明显降低；与OGD+miR-98-5p mimic+pcDNA组比较，OGD+miR-98-5p mimic+EPHA4组神经元的细胞活力明显降低，细胞凋亡率明显升高，IL-1β、TNF-α含量明显升高，SOD活性明显降低，MDA活性明显升高，差异均有统计学意义( P<0.05)。 结论:敲低circ-HECTD1后可通过靶向调控miR-98-5p/EPHA4的表达，从而改善OGD诱导的神经元损伤。

abstractsObjective:To explore whether circular RNA HECTD1 (circ-HECTD1) is involved in oxygen-glucose deprivation (OGD)-induced neuronal cell damage by regulating the expressions of miR-98-5p/ephrin A4 (EPHA4).Methods:Mouse primary cortical neuronal cells were isolated and cultured in vitro. The targeting relations of circ-HECTD1 and miR-98-5p with EPHA4 were detected by dual luciferase reporter assay and RNA binding protein immunoprecipitation assay. These neurons were randomly divided into control group (cultured for 24 h under normal condition) and 6, 12 and 24 h OGD treatment groups (treated with OGD for 6, 12 and 24 h, respectively), OGD+Vector group and OGD+circ-HECTD1 group, OGD+small interfering RNA (siRNA) negative control (si-NC) group and OGD+siRNA circ-HECTD1 (si-circ-HECTD1) group, OGD+micro RNA (miR) negative control (miRNC) group and OGD+miR-98-5p mimic group, OGD+miRNA inhibitor negative control (anti-miRNC) group and OGD+miR-98-5p inhibitor (anti-miR-98-5p) group, OGD+miR-98-5p mimic+pcDNA group and OGD+miR-98-5p mimic+EPHA4 group, OGD+si-circ-HECTD1+anti-miR-NC group and OGD+si-circ-HECTD1+miR-98-5p inhibitor group; pCD5-ciR empty vector, pCD5-ciR-circ-HECTD1, si-NC, si-circ-HECTD1, miR-NC, miR-98-5p mimic, anti-miR-NC or anti-miR-98-5p were transfected into the neurons, and miR-98-5p mimi and pcDNA3.1 empty vector, miR-98-5p mimic and pcDNA3.1-EPHA4 overexpression vector, si-circ-HECTD1 and anti-miR-NC, or si-circ-HECTD1 and anti-miR-98-5p were co-transfected into the neurons. After 24 h of OGD treatment, the circ-HECTD1, miR-98-5p and EPHA4 mRNA expressions were detected by real-time fluorescent quantitative PCR (qRT-PCR), the EPHA4 protein expression was detected by Western blotting, the proliferation activity was detected by MTT assay, the apoptosis rate was detected by flow cytometry, the levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in cell culture medium were detected by ELISA, and the activities of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by kit assay. Results:(1) Targeting relations between circ-HECTD1 and miR-98-5p, and EPHA4 and miR-98-5p were verified. (2) As compared with the control group, the neurons in 6, 12 and 24 h OGD treatment groups had significantly increased circ-HECTD1 and EPHA4 protein expressions and significantly decreased miR-98-5p expression ( P<0.05). (3) As compared with OGD+Vector group, OGD+circ-HECTD1 group had significantly increased circ-HECTD1 expression, and significantly decreased miR-98-5p expression ( P<0.05); as compared with OGD+si-NC group, OGD+si-circ-HECTD1 group had significantly increased miR-98-5p expression, and significantly decreased EPHA4 mRNA and protein expressions ( P<0.05); as compared with OGD+miR-NC group, OGD+miR-98-5p mimic group had significantly increased miR-98-5p expression, and significantly decreased EPHA4 protein expression ( P<0.05); as compared with OGD+anti-miR-NC group, OGD+anti-miR-98-5p group had significantly decreased miR-98-5p expression, and significantly increased EPHA4 protein expression ( P<0.05); as compared with the OGD+si-circ-HECTD1+anti-miR-NC group, OGD+si-circ-HECTD1+anti-miR-98-5p group had significantly increased EPHA4 mRNA and protein expressions ( P<0.05). (4) As compared with the control group, the OGD groups had significantly decreased cell viability and SOD activity, and significantly increased IL-1β and TNF-α levels, apoptosis rate and MDA activity ( P<0.05); as compared with the OGD+si-NC group, the OGD+si-circ-HECTD1 group had significantly decreased cell apoptosis rate, IL-1β and TNF-α levels, and MDA activity, and significantly increased cell viability and SOD activity ( P<0.05); as compared with the OGD+si-circ-HECTD1+anti-miR-NC group, the OGD+si-circ-HECTD1+anti-miR-98-5p group had significantly decreased cell viability and SOD activity, and significantly increased IL-1β and TNF-α levels, apoptosis rate and MDA activity ( P<0.05); as compared with the OGD+miR-NC group, OGD+miR-98-5p mimic group had significantly decreased cell apoptosis rate, IL-1β and TNF-α levels, and MDA activity, and significantly increased cell viability and SOD activity ( P<0.05); as compared with OGD+miR-98-5p mimic+pcDNA group, OGD+miR-98-5p mimic+EPHA4 group has significantly increased cell apoptosis rate, IL-1β and TNF-α levels, and MDA activity, and significantly increased cell viability and SOD activity ( P<0.05). Conclusion:Knockdown of circ-HECTD1 could ameliorate the OGD-induced neuronal cell damage in mice by targeting the expressions of miR-98-5p/EPHA4.