长链非编码RNA核富含丰富的转录本1对癫痫模型海马神经元凋亡的影响和作用机制

doi:10.11852/zgetbjzz2021-0402

摘要/Abstract

摘要： 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。

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

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.

Key words: lncRNA NEAT1, miR-29b-3p, epilepsy, hippocampal neuron apoptosis

中图分类号:

R742.1

屈会霞, 袁小锋, 屈昕. 长链非编码RNA核富含丰富的转录本1对癫痫模型海马神经元凋亡的影响和作用机制[J]. 中国儿童保健杂志, 2021, 29(11): 1193-1197.

QU Hui-xia, YUAN Xiao-feng, QU Xin. Effect and mechanism of long non-coding RNAnuclear-enriched abundant transcript 1 on hippocampal neuronal apoptosis in epilepsy model[J]. Chinese Journal of Child Health Care, 2021, 29(11): 1193-1197.

参考文献

[1] Bigelow MD,Kouzani AZ.Neural stimulation systems for the control of refractory epilepsy:a review[J].J Neuroeng Rehabil,2019, 16(1):126-142.
[2] Holtkamp M, Theodore WH.Generic antiepileptic drugs-safe or harmful in patients with epilepsy？[J].Epilepsia,2018, 59(7):1273-1281.
[3] Lu CW, Huang SK, Lin TY,et al.Echinacoside, an active constituent of Herba Cistanche, suppresses epileptiform activity in hippocampal CA3 pyramidal neurons[J].Korean J Physiol Pharmacol,2018, 22(3):249-255.
[4] Xiang Y, Zhang Y, Xia Y,et al.LncRNA MEG3 targeting miR-424-5p via MAPK signaling pathway mediates neuronal apoptosis in ischemic stroke[J].Aging (Albany NY),2020, 12(4):3156-3174.
[5] Hauser RM, Henshall DC,Lubin FD.The epigenetics of epilepsy and its progression[J].Neuroscientist,2018,24(2):186-200.
[6] Yu Q, Zhao MW, Yang P.LncRNA UCA1 suppresses the inflammation via modulating miR-203-mediated regulation of MEF2C/NF-κB signaling pathway in epilepsy[J].Neurochem Res,2020, 45(4):783-795.
[7] Hirose T, Yamazaki T, Nakagawa S.Molecular anatomy of the architectural NEAT1 noncoding RNA:The domains, interactors, and biogenesis pathway required to build phase-separated nuclear paraspeckles[J].Wiley Interdiscip Rev RNA,2019, 10(6):e1545-e1557.
[8] Villa C,Lavitrano M, Combi R.Long non-coding RNAs and related molecular pathways in the pathogenesis of epilepsy[J].Int J Mol Sci,2019, 20(19):4898-4911.
[9] 谢南昌, 李钰娟, 李英娇, 等.FAM134B介导的自噬在无镁外液致痫海马神经元内质网应激及凋亡中的作用[J].中风与神经疾病杂志, 2020, 37(6):519-522.
[10] Tolchin B, Hirsch LJ, LaFrance WC.Neuropsychiatric aspects of epilepsy[J].Psychiatr Clin North Am,2020, 43(2):275-290..
[11] He C, Su C, Zhang W, et al.Modulatory Potential of LncRNA Zfas1 forinflammation and neuronal apoptosis in temporal lobe epilepsy[J].Yonsei Med J,2021, 62(3):215-223.
[12] Cai X, Long L, Zeng C, et al.LncRNA ILF3-AS1 mediated the occurrence of epilepsy through suppressing hippocampal miR-212 expression[J].Aging (Albany NY),2020, 12(9):8413-8422.
[13] Han CL, Ge M, Liu YP, et al.LncRNA H19 contributes to hippocampal glial cell activation via JAK/STAT signaling in a rat model of temporal lobe epilepsy[J].J Neuroinflammation,2018, 15(1):103-111.
[14] Wan Y, Yang ZQ.LncRNA NEAT1 affects inflammatory response by targeting miR-129-5p and regulating Notch signaling pathway in epilepsy[J].Cell Cycle,2020, 19(4):419-431.
[15] An H, Williams NG,Shelkovnikova TA.NEAT1 and paraspeckles in neurodegenerative diseases:A missing lnc found?[J].Noncoding RNA Res,2018, 3(4):243-252.
[16] Simchovitz A, Hanan M, Niederhoffer N, et al.NEAT1 is overexpressed in Parkinson′s disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress[J].FASEB J,2019, 33(10):11223-11234.
[17] Raghav PK,Kumar R,Kumar V,et al.Docking-based approach for identification of mutations that disrupt binding between Bcl-2 and Bax proteins:Inducing apoptosis in cancer cells[J].Mol Genet Genomic Med,2019,7(11):e910-e925.
[18] Toscano ECB, Vieira ÉLM,Portela ACDC, et al.Bcl-2/Bax ratio increase does not prevent apoptosis of glia and granular neurons in patients with temporal lobe epilepsy[J].Neuropathology,2019, 39(5):348-357.
[19] Guo X, Wang J, Wang N, et al.Wogonin preventive impact on hippocampal neurodegeneration, inflammation and cognitive defects in temporal lobe epilepsy[J].Saudi J Biol Sci,2020, 27(8):2149-2156.
[20] Zhou Y, Xu Z, Yu Y, et al.Comprehensive analysis of the lncRNA-associated ceRNA network identifies neuroinflammation biomarkers for Alzheimer′s disease[J].Mol Omics,2019, 15(6):459-469.
[21] Shi Y, Zhang L, Teng J,et al.HMGB1 mediates microglia activation via the TLR4/NF-κB pathway in coriaria lactone induced epilepsy[J].Mol Med Rep,2018, 17(4):5125-5131.
[22] Yan Y, Xia H, Hu J,et al.MicroRNA-542-3p regulates p-glycoprotein expression in rat epilepsy via the toll-like receptor 4/nuclear factor-kappaB signaling pathway[J].Curr Neurovasc Res,2019, 16(5):433-440.