摘要
目的探讨LINC00261靶向调控微小RNA(miR)-219a-5p对脊髓损伤细胞模型凋亡和氧化应激的实验研究。方法体外培养PC12细胞,缺氧诱导PC12细胞模拟建立脊髓损伤模型,细胞分为NC组、模型组、pcDNA+模型组、pcDNA-LINC00261+模型组、anti-miR-NC+模型组、anti-miR-219a-5p+模型组、miR-NC+pcDNA-LINC00261+模型组、miR-219a-5p+pcDNA-LINC00261+模型组。用实时荧光定量聚合酶链反应(RT-qPCR)检测LINC00261和miR-219a-5p的表达水平;流式细胞术检测细胞凋亡;蛋白质印迹法(Western blot)检测蛋白表达;酶联免疫吸附法(ELISA)检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性。双荧光素酶实验验证LINC00261和miR-219a-5p的靶向关系。两组间比较采用t检验,多组间比较用单因素方差分析。结果与NC组比较,模型组内LINC00261表达水平(1.00±0.10比0.38±0.04)、SOD活性[(14.86±0.71) U/mg比(6.04±0.37) U/mg]、CAT活性高于NC组[(11.24±0.84) U/mg比(4.31±0.36) U/mg],miR-219a-5p表达水平(1.00±0.11比2.43±0.17)、凋亡率[(8.24±0.61)%比(25.16±1.96)%]、裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved-Caspase-3)蛋白表达(0.38±0.03比0.86±0.07)、MDA含量[(12.36±1.01) μmol/g比(32.75±2.38) μmol/g]、p-p65(0.41±0.03比0.83±0.08)、p-IκBα(0.35±0.03比0.69±0.06)蛋白表达显著增加,差异有统计学意义(t=18.958,P<0.05)。过表达LINC00261或抑制miR-219a-5p能抑制脊髓损伤细胞模型细胞凋亡和氧化应激。LINC00261靶向负调控miR-219a-5p表达,过表达miR-219a-5p可以逆转过表达LINC00261对脊髓损伤细胞模型细胞凋亡、氧化应激的作用。与pcDNA+模型组比较,pcDNA-LINC00261+模型组内p-p65(0.85±0.07比0.43±0.04)、p-IκBα(0.71±0.07比0.38±0.03)蛋白表达低于pcDNA+模型组;与miR-NC+pcDNA-LINC00261+模型组比较,miR-219a-5p+pcDNA-LINC00261+模型组内p-p65(0.45±0.04比0.91±0.08)、p-IκBα(0.37±0.04比0.75±0.07)蛋白表达高于miR-NC+pcDNA-LINC00261+模型组,差异有统计学意义(t=26.302,P<0.05)。结论 LINC00261靶向调控miR-219a-5p/核因子-κB(NF-κB)轴减轻脊髓损伤细胞模型凋亡和氧化应激。
Objective To investigate the effect of LINC00261 targeting microRNA(miR)-219a-5p on apoptosis and oxidative stress in spinal cord injury cell models through nuclear factor-κB(NF-κB)signaling pathway.Methods PC12 cells were cultured in vitro,and PC12 cells were induced by hypoxia to simulate the establishment of spinal cord injury model.The cells were divided into normal control(NC)group,model group,pcDNA+model group,pcDNA-LINC00261+model group,anti-miR-NC+model group,and anti-miR-219a-5p+model group,miR-NC+pcDNA-LINC00261+model group,miR-219a-5p+pcDNA-LINC00261+model group.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression levels of LINC00261 and miR-219a-5p.The flow cytometry was used to detect cell apoptosis.Western blotting was used to detect protein expression.Enzyme-linked immunosorbent assay(ELISA)was used to measure malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,and catalase(CAT)activity.The dual luciferase experiment verified the targeting relationship between LINC00261 and miR-219a-5p.The t test was used for comparison between the two groups,and the one-way analysis of variance was used for the comparison between multiple groups.Results Compared with the NC group,the LINC00261 expression level(1.00±0.10 vs.0.38±0.04),SOD activity[(14.86±0.71)U/mg vs.(6.04±0.37)U/mg],and CAT activity[(11.24±0.84)U/mg vs.(4.31±0.36)U/mg]in the model group were significantly reduced,miR-219a-5p expression level(1.00±0.11 vs.2.43±0.17),apoptosis rate[(8.24±0.61)%vs.(25.16±1.96)%],cleaved-Caspase-3 protein expression(0.38±0.03 vs.0.86±0.07),MDA content[(12.36±1.01)μmol/g vs.(32.75±2.38)μmol/g],p-p65(0.41±0.03 vs.0.83±0.08),p-IκBα(0.35±0.03 vs.0.69±0.06)protein expression increased significantly(t=18.958,P<0.05).Overexpression of LINC00261 or inhibition of miR-219a-5p could inhibit cell apoptosis and oxidative stress in spinal cord injury cell models.LINC00261 targeted and negatively regulated the expression of miR-219a-5p.Overexpression of miR-219a-5p could reverse the effects of overexpression of LINC00261 on apoptosis and oxidative stress in spinal cord injury cell models.As compared with the pcDNA+model group,the p-p65(0.85±0.07 vs.0.43±0.04)and p-IκBα(0.71±0.07 vs.0.38±0.03)protein expression in the pcDNA-LINC00261+model group was significantly reduced.As compared with the miR-NC+pcDNA-LINC00261+model group,the expression of p-p65(0.45±0.04 vs.0.91±0.08)and p-IκBα(0.37±0.04 vs.0.75±0.07)protein in the miR-219a-5p+pcDNA-LINC00261+model group increased significantly(t=26.302,P<0.05).Conclusion LINC00261 targets the miR-219a-5p/NF-κB axis to reduce apoptosis and oxidative stress in the spinal cord injury cell model.
作者
江辉
王祥善
张华
曹顺海
于东方
Jiang Hui;Wang Xiangshan;Zhang Hua;Cao Shunhai;Yu Dongfang(Third Department of Spine,Zhengzhou Orthopedic Hospital Affiliated to Henan University,Zhengzhou 450015,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第9期1752-1756,共5页
Chinese Journal of Experimental Surgery
