Please wait a minute...
Journal of Integrative Neuroscience  2019, Vol. 18 Issue (1): 71-77    DOI: 10.31083/j.jin.2019.01.13
Original Research Previous articles | Next articles
Molecular expression and functional analysis of genes in children with temporal lobe epilepsy
Xiaojuan Wu1, Yajie Wang1, Zhenrong Sun2, Shouchen Ren1, Weili Yang1, Yaxian Deng1, Chaoxia Tian1, Yazhen Yu1, Baoqin Gao1, *()
1 Department of Pediatrics, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100050, China
2 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100050, China
Download:  PDF(868KB)  ( 785 ) Full text   ( 50 )
Export:  BibTeX | EndNote (RIS)      
Abstract  

Temporal lobe epilepsy is the most common form of epilepsy. However, for this type of condition, antiseizure medication is not effective for children. As miRNAs are involved in the development of temporal lobe epilepsy in children, they may provide potential therapeutic approaches for treatment. The primary aim of this study was to explore the expression and function of miR-135a-5p in children with temporal lobe epilepsy. Hippocampal slices from either normal (control) children or children with temporal lobe epilepsy were used to detect the expression of miR-135a-5p and its target gene caspase activity and apoptosis inhibitor 1. To further explore the role of miR-135a-5p in the development of temporal lobe epilepsy in children, primary hippocampal neurons from newborn rats were cultured in vitro in a magnesium-free medium to mimic the temporal lobe epilepsy condition in children. The effect of transfection of miR-135a-5p inhibitor into cells was also assessed. Apoptosis and proliferation of hippocampus cells was respectively assessed by flow cytometry or 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The level of miR-135a-5p was significantly increased in both children with temporal lobe epilepsy and the epileptiform discharge model that employed newborn rat hippocampal neurons; whereas, the expression of caspase activity and apoptosis inhibitor 1 was downregulated by overexpression of miR-135a-5p. Moreover, miR-135a-5p mediated the pro-apoptotic effect of temporal lobe epilepsy via repressing caspase activity and apoptosis inhibitor 1 expression. Additionally, miR-135a-5p reduced cell survival in the temporal lobe epilepsy condition. Overexpression of miR-135a-5p induced cell apoptosis through inhibition of caspase activity and apoptosis inhibitor 1 expression and suppressed cell survival in children with temporal lobe epilepsy.

Key words:  miR-135a-5p      CAAP1      temporal lobe epilepsy      functional analysis     
Submitted:  09 November 2018      Accepted:  18 March 2019      Published:  30 March 2019     
*Corresponding Author(s):  Baoqin Gao     E-mail:  gaobaoqin@bjtth.org

Cite this article: 

Xiaojuan Wu, Yajie Wang, Zhenrong Sun, Shouchen Ren, Weili Yang, Yaxian Deng, Chaoxia Tian, Yazhen Yu, Baoqin Gao. Molecular expression and functional analysis of genes in children with temporal lobe epilepsy. Journal of Integrative Neuroscience, 2019, 18(1): 71-77.

URL: 

https://jin.imrpress.com/EN/10.31083/j.jin.2019.01.13     OR     https://jin.imrpress.com/EN/Y2019/V18/I1/71

Figure 1.  miR-135a-5p expression is increased in temporal lobe epilepsy. A: Quantitative qRT-PCR analyses of miR-135a-5p levels in hippocampus of children. B: Prediction of miR-135a-5p binding site in Caap1 miRNA 3’-UTR by microRNA website (www.microrna.org/microrna/home.do). C: mRNA level of Caap1 in hippocampus of children. D: Protein abundance of Caap1 in hippocampus of children. qRT-PCR was performed using 15 samples from control or TLE children respectively. Experiments repeated three times (n = 3). **p < 0.05 vs. Control group.

Table 1  Clinical characteristics of brain donors
IVUS FFR
Parameters TLE children Control children
Number 15 15
Age, (years), mean (SD) 11.2 (2.6) 10.6 (3.7)
Male gender, n (%) 7 (46.7) 8 (53.3)
Onset age, (years), mean (SD) 7.4 (1.3) -
Seizure type, n (%) CPS, 9(60) SGTC, 6 (40) -
Family history, n (%) 3 (20) -
Figure 2.  miR-135a-5p is upregulated in TLE model in newborn rat hippocampal neurons. A: Quantitative qRT-PCR analyses of miR-135a-5p levels in primary hippocampal neurons from new born rats. B: mRNA level of Caap1 in hippocampus neurons from newborn rats. C: Protein abundance of Caap1 in primary hippocampal neurons from newborn rats. Experiments repeated three times (n = 3). No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs Control group.

Figure 3.  miR-135a-5p mediates the pro-apoptotic effect of TLE. A and B: FACS analysis of primary rat hippocampal cells for apoptosis and quantification of apoptotic cells per group. Cells treated with miR-135a-5p inhibitor for 24 hours before being cultured in magnesium-free medium. Annexin V labeled with FITC and PI were used to stain cells. No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs. Control group; $^{\#\#}$p < 0.05 vs. Control+miR-135a-5p inhibitor group; $^{\Delta\Delta}$p < 0.05 vs No Mg$^{2+}$ group.

Figure 4.  miR-135a-5p regulates Caap1 expression negatively in TLE model. A: mRNA level of Caap1 in primary rat hippocampus neurons from newborn rats treated with or without miR-135a-5p inhibitor. B: Protein abundance of Caap1 in hippocampus neurons from newborn rats. Experiments repeated three times (n = 3). No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs. Control group; $^{\#\#}$p < 0.05 vs Control+miR-135a-5p inhibitor group; $^{\Delta\Delta}$p < 0.05 vs. No Mg$^{2+}$ group.

Figure 5.  Inhibition of miR-135a-5p promotes TLE-reduced cell survival. Quantification of cell survival indicated by MTT assay in primary rat hippocampal cells. Cells treated with miR-135a-5p inhibitor for 24 hours before being cultured in magnesium-free medium. Experiments repeated three times (n = 3). No Mg$^{2+}$: magnesium-free medium. **p < 0.05 vs. Control group; $^{\#\#}$p < 0.05 vs. Control+miR-135a-5p inhibitor group; $^{\Delta\Delta}$p < 0.05 vs. No Mg$^{2+}$ group.

[1] Alsharafi, W. and Xiao, B. (2015) Dynamic expression of microRNAs (183, 135a, 125b, 128, 30c and 27a) in the rat pilocarpine model and temporal lobe epilepsy patients. CNS & Neurological Disorders Drug Targets 14, 1096-1102.
[2] Alsharafi, W. A., Xiao, B., Abuhamed, M. M. and Luo, Z. (2015) MiRNAs: biological and clinical determinants in epilepsy. Frontiers in Molecular Neuroscience 8, 1-15.
[3] Aronica, E., Fluiter, K., Iyer, A., Zurolo, E., Vreijling, J., van Vliet, E. A., Baayen, J. C. and Gorter, J. A. (2010) Expression pattern of miR-146a, an inflammation-associated microRNA, in experimental and human temporal lobe epilepsy. The European Journal of Neuroscience 31, 1100-1107.
doi: 10.1111/j.1460-9568.2010.07122.x pmid: 20214679
[4] Ashhab, M., Omran, A., Gan, N., Kong, H., Peng, J. and Yin, F. (2013 a) MicroRNA s (9, 138, 181A, 221, and 222) and mesial temporal lobe epilepsy in developing brains. Translational Neuroscience 4, 357-362.
doi: 10.2478/s13380-013-0128-z
[5] Ashhab, M. U., Omran, A., Kong, H., Gan, N., He, F., Peng, J. and Yin, F. (2013 b) Expressions of tumor necrosis factor alpha and microRNA-155 in immature rat model of status epilepticus and children with mesial temporal lobe epilepsy. Journal of Molecular Neuroscience: MN 51, 950-958.
doi: 10.1007/s12031-013-0013-9 pmid: 23636891
[6] Bartel, D. P. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233.
doi: 10.1016/j.cell.2009.01.002 pmid: 19167326
[7] Chen, Y., Huang, X., Chen, W., Wang, N. and Li, L. (2012) Tenuigenin promotes proliferation and differentiation of hippocampal neural stem cells. Neurochemical Research 37, 771-777.
doi: 10.1007/s11064-011-0671-3 pmid: 22179853
[8] Danial, N. N. and Korsmeyer, S. J. (2004) Cell death: critical control points. Cell 116, 205-219.
[9] Gleissner, U., Sassen, R., Schramm, J., Elger, C. E. and Helmstaedter, C. (2005) Greater functional recovery after temporal lobe epilepsy surgery in children. Brain : A Journal of Neurology 128, 2822-2829.
doi: 10.1093/brain/awh597 pmid: 16014650
[10] He, F., Liu, B., Meng, Q., Sun, Y., Wang, W. and Wang, C. (2016) Modulation of miR-146a/complement factor H-mediated inflammatory responses in a rat model of temporal lobe epilepsy. Bioscience Reports 36, 1-12.
doi: 10.1042/BSR20160290 pmid: 27852797
[11] Henshall, D. C. (2014) MicroRNA and epilepsy: profiling, functions and potential clinical applications. Current Opinion in Neurology 27, 199-205.
doi: 10.1097/WCO.0000000000000079 pmid: 24553459
[12] Henshall, D. C. (2007) Apoptosis signalling pathways in seizure-induced neuronal death and epilepsy. Biochemical Society Transactions 35, 421-423.
doi: 10.1042/bst0350421 pmid: 17371290
[13] Henshall, D. C. and Simon, R. P. (2005) Epilepsy and apoptosis pathways. Journal of Cerebral Blood Flow and Metabolism 25, 1557-1572.
[14] Iizuka, M., Matsui, T., Takisawa, H. and Smith, M. M. (2006) Regulation of replication licensing by acetyltransferase Hbo1. Molecular and Cellular Biology 26, 1098-1108.
[15] Kan, A. A., van Erp, S., Derijck, A. A., de Wit, M., Hessel, E. V., O'Duibhir, E., de Jager, W., Van Rijen, P. C., Gosselaar, P. H., de Graan, P. N. and Pasterkamp, R. J. (2012) Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response. Cellular and Molecular Life Sciences 69, 3127-3145.
[16] Lewis, D. V., Jones, L. S. and Mott, D. D. (1990) Hippocampal epileptiform activity induced by magnesium-free medium: differences between areas CA1 and CA2-3. Epilepsy Research 6, 95-101.
doi: 10.1016/0920-1211(90)90083-8 pmid: 2387288
[17] Liu, N., Shi, Y. F., Diao, H. Y., Li, Y. X., Cui, Y., Song, X. J., Tian, X., Li, T. Y. and Liu, B. (2017) MicroRNA-135a regulates apoptosis induced by hydrogen peroxide in rat. Cardiomyoblast Cells International Journal of Biological Sciences 13, 13-21.
[18] McKiernan, R. C., Jimenez-Mateos, E. M., Bray, I., Engel, T., Brennan, G. P., Sano, T., Michalak, Z., Moran, C., Delanty, N., Farrell, M., O'Brien, D., Meller, R., Simon, R. P., Stallings, R. L. and Henshall, D. C. (2012) Reduced mature microRNA levels in association with dicer loss in human temporal lobe epilepsy with hippocampal sclerosis. PloS One 7, 1-9.
doi: 10.1371/journal.pone.0035921 pmid: 3352899
[19] Navarro, A., Diaz, T., Martinez, A., Gaya, A., Pons, A., Gel, B., Codony, C., Ferrer, G., Martinez, C., Montserrat, E. and Monzo, M. (2009) Regulation of JAK2 by miR-135a: prognostic impact in classic. Hodgkin Lymphoma Blood 114, 2945-2951.
[20] Newman, D. M., Voss, A. K., Thomas, T. and Allan, R. S. (2017) Essential role for the histone acetyltransferase KAT7 in T cell development, fitness, and survival. Journal of Leukocyte Biology 101, 887-892.
doi: 10.1189/jlb.1MA0816-338R pmid: 27733580
[21] Nickels, K. C., Wong-Kisiel, L. C., Moseley, B. D. and Wirrell, E. C. (2011) Temporal lobe epilepsy in children. Epilepsy Research and Treatment 2012, 657-668.
doi: 10.1155/2012/849540 pmid: 3420576
[22] Nunez, J. (2008) Primary culture of hippocampal neurons from P0 newborn rats. Journal of Visualized Experiments 19, 1-2.
[23] Pardo, M., Yu, L., Shen, S., Tate, P., Bode, D., Letney, B. L., Quelle, D. E., Skarnes, W. and Choudhary, J. S. (2017) Myst2/Kat7 histone acetyltransferase interaction proteomics reveals tumour-suppressor niam as a novel binding partner in embryonic stem cells. Scientific Reports 7, 1-14.
doi: 10.1038/s41598-017-08456-2 pmid: 5557939
[24] Santos, P. K. F., de Souza Araujo, N., Francoso, E., Zuntini, A. R. and Arias, M. C. (2018) Diapause in a tropical oil-collecting bee: molecular basis unveiled by RNA-Seq. BMC Genomics 19, 1-11.
doi: 10.1186/s12864-018-4694-x pmid: 29703143
[25] Sombati, S. and Delorenzo, R. J. (1995) Recurrent spontaneous seizure activity in hippocampal neuronal networks in culture. Journal of Neurophysiology 73, 1706-1711.
doi: 10.1152/jn.1995.73.4.1706 pmid: 7643176
[26] Stelzl, U., Worm, U., Lalowski, M., Haenig, C., Brembeck, F. H., Goehler, H., Stroedicke, M., Zenkner, M., Schoenherr, A., Koeppen, S., Timm, J., Mintzlaff, S., Abraham, C., Bock, N., Kietzmann, S., Goedde, A., Toksoz, E., Droege, A., Krobitsch, S., Korn, B., Birchmeier, W., Lehrach, H. and Wanker, E. E. (2005) A human protein-protein interaction network: a resource for annotating the proteome. Cell 122, 957-968.
doi: 10.1016/j.cell.2005.08.029 pmid: 16169070
[27] Tang, W. W., Wan, G. P., Wan, Y. C., Zhang, L. and Cheng, W. J. (2013) Effects of miR-135a on HOXA10 expression, proliferation and apoptosis of ovarian cancer cells. Zhonghua Fu Chan Ke Za Zhi 48, 364-369. (In Chinses)
pmid: 24016480
[28] Thom, M. (2004) Recent advances in the neuropathology of focal lesions in epilepsy. Expert Review of Neurotherapeutics 4, 973-984.
doi: 10.1586/14737175.4.6.973 pmid: 15853524
[29] Yamamoto, A., Murphy, N., Schindler, C. K., So, N. K., Stohr, S., Taki, W., Prehn, J. H. and Henshall, D. C. (2006) Endoplasmic reticulum stress and apoptosis signaling in human temporal lobe epilepsy. Journal of Neuropathology and Experimental Neurology 65, 217-225.
doi: 10.1097/01.jnen.0000202886.22082.2a pmid: 16651883
[30] Wu, S., Lin, Y., Xu, D., Chen, J., Shu, M., Zhou, Y., Zhu, W., Su, X., Zhou, Y., Qiu, P. and Yan, G. (2012) MiR-135a functions as a selective killer of malignant glioma. Oncogene 31, 3866-3874.
doi: 10.1038/onc.2011.551 pmid: 22139076
[31] Zhang, T., Shao, Y., Chu, T. Y., Huang, H. S., Liou, Y. L., Li, Q. and Zhou, H. (2016) MiR-135a and MRP1 play pivotal roles in the selective lethality of phenethyl isothiocyanate to malignant glioma cells. American Journal of Cancer Research 6, 957-972.
pmid: 27293991
[32] Zhang, Y., Johansson, E., Miller, M. L., Janicke, R. U., Ferguson, D. J., Plas, D., Meller, J. and Anderson, M. W. (2011) Identification of a conserved anti-apoptotic protein that modulates the mitochondrial apoptosis pathway. PloS One 6, 1-14.
doi: 10.1371/journal.pone.0025284 pmid: 21980415
[33] Zhao, J., Li, X., Zou, M., He, J., Han, Y., Wu, D., Yang, H. and Wu, J. (2014) miR-135a inhibition protects A549 cells from LPS-induced apoptosis by targeting Bcl-2. Biochemical and Biophysical Research Communications 452, 951-957.
doi: 10.1016/j.bbrc.2014.09.025 pmid: 25230140
[34] Zhong, J. X., Zhou, L., Li, Z., Wang, Y. and Gui, J. F. (2014) Zebrafish noxa promotes mitosis in early embryonic development and regulates apoptosis in subsequent embryogenesis. Cell Death and Differentiation 21, 1013-1024.
doi: 10.1038/cdd.2014.22 pmid: 24608793
[1] Jun Lu, Hongxing Huang, Qichang Zeng, Xinmei Zhang, Min Xu, Yi Cai, Qin Wang, Yahui Huang, Qiong Peng, Lanqiuzi Deng. Hippocampal neuron loss and astrogliosis in medial temporal lobe epileptic patients with mental disorders[J]. Journal of Integrative Neuroscience, 2019, 18(2): 127-132.
No Suggested Reading articles found!