Circular RNA HECTD1 participates in oxygen-glucose deprivation-induced neuronal cell damage by regulating miR-98-5p/ephrin A4 expressions / 中华神经医学杂志

ABSTRACT

Objective: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.