目的 探讨长链非编码RNA(lncRNA)核富含丰富的转录本1(NEAT1)对癫痫细胞模型海马神经元凋亡的影响及作用机制,以期为NEAT1成为癫痫治疗的新靶点提供依据。方法 于2019年8月—2020年10月期间,体外培养大鼠海马神经元细胞,无镁诱导制备癫痫海马神经元模型,实验分为:对照组(正常细胞外液)、模型组(无镁细胞外液)、转染对照组(转染非特异性siRNA+无镁细胞外液)和转染组(转染NEAT1特异性siRNA+无镁细胞外液)。实时荧光定量PCR(qPCR)检测NEAT1和miR-29b-3p的表达变化,双荧光素酶报告基因实验检测NEAT1是否靶向调控miR-29b-3p,酶联免疫吸附法(ELISA)检测白细胞介素-1(IL-1)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的含量,AnnexinV-FITC/PI双染色法检测海马神经元细胞凋亡情况,蛋白免疫印迹法(Western blot)检测各组细胞中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Toll样受体4(TLR4)和核因子-κB(NF-κB)的表达水平。结果 与对照组相比,模型组海马神经元凋亡率及NEAT1、Bax、TLR4和NF-κB的表达水平升高(F＝50.980、73.668、65.635、13.203、10.292,P＜0.05),IL-1、IL-6和TNF-α的含量增多(F＝33.107、33.857、51.129,P＜0.05),miR-29b-3p和Bcl-2的表达水平降低(F＝145.023、67.655,P＜0.05);而抑制NEAT1的表达能够降低神经元凋亡,抑制Bax、TLR4和NF-κB的表达,促进miR-29b-3p和Bcl-2的表达,减少IL-1、IL-6和TNF-α的分泌。双荧光素酶报告基因实验证实NEAT1和miR-29b-3p的靶向关系。结论 lncRNA NEAT1靶向下调miR-29b-3p的表达阻断TLR4/NF-κB信号通路来抑制癫痫模型海马神经元的凋亡。
Abstract
Objective To investigate the effect and mechanism of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1(NEAT1)on hippocampal neuron apoptosis in epilepsy cell model, in order to provide evidence for NEAT1 being the new target of epilepsy treatment. Methods The rat hippocampal neuron cells were cultured in vitro and induced without magnesium to prepare epileptic hippocampal neuron models.The experiment included control group (normal extracellular fluid), model group (magnesium-free extracellular fluid), transfection control group (transfection non-specific siRNA+magnesium-free extracellular fluid) and transfection group(transfected with NEAT1 specific siRNA+ magnesium-free extracellular fluid).The expression changes of NEAT1 and miR-29b-3p were detected by real-time fluorescent quantitative PCR (qPCR).The dual luciferase reporter gene experiment detected whether NEAT1 targets miR-29b-3p, and interleukins-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) content were tested by enzyme-linked immunosorbent assay(ELISA) .AnnexinV-FITC/PI double staining method was used to detect hippocampal neuronal cell apoptosis, protein Western blot was used to detect the expression levels of B cell lymphoma/lewkmia-2 (Bcl-2), Bcl-2 Associated X Protein (Bax), toll-like receptor-4(TLR4) and nuclear factor-κB(NF-κB) in cells of each group. Results Compared with the control group, the apoptosis rate of hippocampal neurons and the expression levels of NEAT1, Bax, TLR4 and NF-κB in the model group increased (F=50.980, 73.668, 65.635, 13.203, 10.292, P<0.05), the contents of IL-1, IL-6 and TNF-α increased (F=33.107, 33.857, 51.129, P<0.05), the expression levels of miR-29b-3p and Bcl-2 decreased (F=145.023, 67.655, P<0.05).While inhibiting the expression of NEAT1 can reduce neuronal apoptosis, inhibit the expression of Bax, TLR4 and NF-κB, promote the expression of miR-29b-3p and Bcl-2, and reduce the secretion of IL-1, IL-6 and TNF-α.The dual luciferase reporter gene experiment confirmed the targeting relationship between NEAT1 and miR-29b-3p. Conclusion lncRNA NEAT1 targets to down-regulate the expression of miR-29b-3p and block the TLR4/NF-κB signaling pathway to inhibit the apoptosis of hippocampal neurons in epilepsy models.
关键词
长链非编码RNA核富含丰富的转录本1 /
miR-29b-3p /
癫痫 /
海马神经元凋亡
Key words
lncRNA NEAT1 /
miR-29b-3p /
epilepsy /
hippocampal neuron apoptosis
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基金
河南省医学科技公关计划省部共建项目和软科学项目(SBGJ202002054)
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