高永强,董燕,程欣,等.miR-873-5p靶向调控VDAC1保护大鼠癫痫样神经细胞的作用机制探讨[J].中华物理医学与康复杂志,2021,43(2):109-115
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| miR-873-5p靶向调控VDAC1保护大鼠癫痫样神经细胞的作用机制探讨 |
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| DOI：10.3760/cma.j.issn.0254-1424.2021.02.002 |
| 中文关键词: miR-873-5p 电压依赖性阴离子通道1 癫痫 神经细胞损伤 |
| 英文关键词: miR-873-5p micro-RNA Voltage-dependent anion channel protein 1 Epilepsy Nerve cell injury |
| 基金项目:河南省教育厅科技计划项目（20B320030；14A320004） |
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| 中文摘要: |
| 目的 研究微RNA-873-5p（miR-873-5p）靶向调控电压依赖性阴离子通道1（VDAC1）对大鼠癫痫样神经细胞的影响及作用机制。 方法 通过无镁培养基诱导构建大鼠癫痫样海马神经细胞模型，将大鼠神经细胞分为对照组、模型组、miR-con组及miR-873-5p组。对照组采用正常培养大鼠神经细胞；模型组采用低镁细胞外液培养大鼠神经细胞；miR-con组采用低镁细胞外液培养大鼠神经细胞并转染miR-con；miR-873-5p组采用低镁细胞外液培养大鼠神经细胞并转染miR-873-5p。采用荧光定量聚合酶链式反应（qRT-PCR）检测各组神经细胞miR-873-5p及VDAC1 mRNA表达；采用Western blot技术检测神经细胞中VDAC1、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的含半胱氨酸天冬氨酸蛋白水解酶3（Cleaved caspase-3）含量；采用DCFH-DA荧光探针检测神经细胞活性氧（ROS）水平；采用硫代巴比妥酸法检测神经细胞丙二醛（MDA）含量；采用酶联免疫吸附实验（ELISA）检测神经细胞谷胱甘肽（GSH）含量；采用流式细胞术检测各组神经细胞凋亡情况；采用双荧光素酶报告基因法验证miR-873-5p对VDAC1的靶向调控作用。 结果 与对照组比较，模型组神经细胞中miR-873-5p表达显著降低（P<0.05），VDAC1表达显著升高（P<0.05），Bcl-2表达下调（P<0.05），Bax及Cleaved caspase-3表达上调（P<0.05），GSH及MDA含量显著降低（P<0.05），而ROS水平显著升高（P<0.05），细胞凋亡率显著升高（P<0.05）；与miR-con组比较，miR-873-5p组神经细胞中Bcl-2表达明显上调（P<0.05），Bax及Cleaved caspase-3表达明显下调（P<0.05），GSH及MDA含量显著升高（P<0.05），ROS水平显著降低（P<0.05），细胞凋亡率明显降低（P<0.05）；双荧光素酶报告基因实验表明miR-873-5p能抑制VDAC1表达（P<0.05）。 结论 miR-873-5p能通过负向调控靶基因VDAC1表达而减少神经细胞凋亡，同时还能抑制神经细胞氧化应激反应，对受损神经细胞具有保护作用。 |
| 英文摘要: |
| Objective To explore the regulatory effect of miR-873-5p micro-RNA targeting voltage-dependent anion channel protein 1 (VDAC1) in neurons and its mechanism. Methods Murine nerve cells were randomly divided in vitro into a control group, a model group, a mimetic negative carrier (miR-con) group and an miR-873-5p group. The epileptiform hippocampal nerve cell model was induced in all of the cells except those in the control group using magnesium-free medium. The control group was normally cultured, while the miR-con and miR-873-5p groups were transfected with miR control and miR-873-5p RNA respectively. Real-time fluorescent quantitative polymerase chain reactions were used to detect the expression of miR-873-5p and VDAC1 mRNA. Western blotting was employed to detect VDAC1, B-cell lymphoma/leukemia-2 protein (Bcl-2), Bcl-2 associated X protein (Bax) and cleared caspase-3 in the neurons. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) were measured using the DCFH-DA fluorescent probe, the thiobarbituric acid method and enzyme-linked immunosorbent assay respectively. Any apoptosis was detected using flow cytometry, while the targeting of miR-873-5p on VDAC1 was verified using the double fluorescence zymase reporter gene method. Results Compared with the control group, a significant decrease in the average expression of miR-873-5p, Bcl-2 and in GSH and MDA levels was observed in the model group, but there was a significant increase in the average level of VDAC1, Bax, cleaved caspase-3 and ROS and in the rate of apoptosis. Compared with the miR-con group, a significant decrease in the average expression of Bax, cleaved caspase-3, ROS and in the apoptosis rate was observed in the miR-873-5p group, but there was a significant increase in the average level of Bcl-2, GSH and MDA. Moreover, it was verified that miR-873-5p reduced the expression of VDAC1. Conclusion miR-873-5p protects damaged neurons by inhibiting their apoptosis through negatively regulating the target gene VDAC1 and the oxidative stress response. |
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