Wu Zhenggang, Liu Ying, Huang Jing, Huang Yujing, Fan Lin
Department of Neurology, Taizhou People's Hospital, 366 Taihu Road, Taizhou Medicine High-tech Zone, Taizhou City, 225300, Jiangsu Province, China.
Cytotechnology. 2019 Jun 26;71(4):809-818. doi: 10.1007/s10616-019-00324-3.
To determine the function of miR-206 in epilepsy. Epileptic rat model was established by intra-amygdala injection of kainic acid (KA). Expression levels of miR-206, C-C Motif Chemokine Ligand 2 (CCL2) and interleukin-1β (Il-1β) in hippocampus tissues was measured by reverse transcription-quantitative PCR (RT-qPCR) and western blot. Dual luciferase reporter assay was performed to determine the binding of miR-206 to 3' untranslated region (UTR) of CCL2. Finally, brain waves were recorded and Hematoxylin and eosin (HE) staining and Nissl's staining were performed on the epileptic rat injected with LPS, miR-206 agomir, adeno-associated virus (AAV) expressed CCL2 alone or in combination. Expression of miR-206 was specially decreased in hippocampus tissues compared to cortex in response to KA induced pathologic brain activity. Enforced expression of miR-206 by injection miR-206 agomir not only decreased seizure activity, but also protected KA-induced neuronal loss. And enforced expression of miR-206 suppressed increase of C-C Motif Chemokine Ligand 2 (CCL2) and interleukin-1β (Il-1β) which were induced by injection of KA or KA combined with lipopolysaccharide (LPS). Further more, results of dual luciferase reporter assay confirmed CCL2 was a target of miR-206. Finally, co-injection adeno-associated virus (AAV) expressed CCL2 with miR-206 agomir abolished the function of miR-206 agomir. Taken together, our results showed that expression of miR-206 could inhibit seizure-induced brain injury by targeting CCL2. Our results showed that expression of miR-206 could inhibit seizure-induced brain injury by targeting CCL2.
为确定miR-206在癫痫中的作用。通过杏仁核内注射海藻酸（KA）建立癫痫大鼠模型。采用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测海马组织中miR-206、C-C基序趋化因子配体2（CCL2）和白细胞介素-1β（Il-1β）的表达水平。进行双荧光素酶报告基因检测以确定miR-206与CCL2的3'非翻译区（UTR）的结合情况。最后，记录脑电波，并对注射了脂多糖（LPS）、miR-206激动剂、单独或联合表达CCL2的腺相关病毒（AAV）的癫痫大鼠进行苏木精-伊红（HE）染色和尼氏染色。与皮质相比，在KA诱导的病理性脑活动中，海马组织中miR-206的表达特异性降低。通过注射miR-206激动剂增强miR-206的表达不仅降低了癫痫发作活动，还保护了KA诱导的神经元丢失。并且增强miR-206的表达抑制了由注射KA或KA与脂多糖（LPS）联合诱导的C-C基序趋化因子配体2（CCL2）和白细胞介素-1β（Il-1β）的增加。此外，双荧光素酶报告基因检测结果证实CCL2是miR-206的靶标。最后，将表达CCL2的腺相关病毒（AAV）与miR-206激动剂共同注射消除了miR-206激动剂的功能。综上所述，我们的结果表明miR-206的表达可通过靶向CCL2抑制癫痫发作诱导的脑损伤。我们的结果表明miR-206的表达可通过靶向CCL2抑制癫痫发作诱导的脑损伤。
