Wang Ying, Yang Zhiquan, Zhang Kai, Wan Yi, Zhou Yu, Yang Zhuanyi
Department of Pathology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China.
Department of Pathology, School of Basic Medical Science, Central South University, P.R. China.
Exp Ther Med. 2021 May;21(5):431. doi: 10.3892/etm.2021.9848. Epub 2021 Feb 26.
Epilepsy is a common neurological disease that can induce severe physiological brain damage, including nerve cell apoptosis. MicroRNAs (miRs) have been widely investigated in epilepsy therapy. miR-135a-5p expression levels in children with temporal lobe epilepsy were found to be significantly increased. However, whether miR-135a-5p participates in epilepsy-induced cell apoptosis is not completely understood. In the present study, an model of epilepsy in BV2 microglia cells was induced using 6-µm kainic acid (KA). Reverse-transcription quantitative PCR was performed to analyze miR-135a-5p and sirtuin 1 (SIRT1) mRNA expression levels. Western blotting was performed to measure SIRT1 protein expression levels. BV2 cell proliferation and apoptosis were assessed by performing MTT assays and flow cytometry, respectively. A BCA protein assay kit was used to detect caspase-3 and caspase-9 activities. TargetScan and dual luciferase reporter assays were performed to investigate the interaction between miR-135a-5p and the 3'-untranslated region (UTR) of SIRT1. miR-135a-5p expression was significantly increased in the KA-induced model of epilepsy in BV2 microglia. miR-135a-5p inhibitor effectively promoted BV2 microglia proliferation and inhibited microglia apoptosis, whereas small interfering RNA targeting SIRT1 significantly repressed BV2 microglia proliferation and induced microglia apoptosis. In addition, the results demonstrated that the 3'-UTR of SIRT1 mRNA was targeted by miR-135a-5p, and SIRT1 knockdown attenuated miR-135a-5p inhibitor-mediated effects on epilepsy. In summary, the results of the present study identified the role of miR-135a-5p inhibitor pretreatment in protecting nerve cells against epilepsy-induced apoptosis and provided a novel strategy for the treatment of neural damage in seizures.
癫痫是一种常见的神经系统疾病，可导致严重的生理性脑损伤，包括神经细胞凋亡。微小RNA（miR）在癫痫治疗中已得到广泛研究。研究发现，颞叶癫痫患儿的miR-135a-5p表达水平显著升高。然而，miR-135a-5p是否参与癫痫诱导的细胞凋亡尚未完全明确。在本研究中，使用6-μm海藻酸（KA）诱导BV2小胶质细胞建立癫痫模型。采用逆转录定量PCR分析miR-135a-5p和沉默调节蛋白1（SIRT1）mRNA表达水平。进行蛋白质免疫印迹法检测SIRT1蛋白表达水平。分别通过MTT法和流式细胞术评估BV2细胞增殖和凋亡情况。使用BCA蛋白检测试剂盒检测半胱天冬酶-3和半胱天冬酶-9活性。采用TargetScan和双荧光素酶报告基因检测法研究miR-135a-5p与SIRT1的3'-非翻译区（UTR）之间的相互作用。在KA诱导的BV2小胶质细胞癫痫模型中，miR-135a-5p表达显著增加。miR-135a-5p抑制剂有效促进BV2小胶质细胞增殖并抑制小胶质细胞凋亡，而靶向SIRT1的小干扰RNA显著抑制BV2小胶质细胞增殖并诱导小胶质细胞凋亡。此外，结果表明SIRT1 mRNA的3'-UTR是miR-135a-5p的靶点，敲低SIRT1可减弱miR-135a-5p抑制剂介导的抗癫痫作用。总之，本研究结果确定了miR-135a-5p抑制剂预处理在保护神经细胞免受癫痫诱导的凋亡中的作用，并为治疗癫痫性神经损伤提供了一种新策略。
