miR-195对小鼠癫痫发作及Clusterin蛋白表达的影响

The Effect of miR-195 on Epileptic Seizure and Clusterin Expression in Mice

目的:观察miR-195对小鼠癫痫发作频率及小鼠脑皮层丛生蛋白(Clusterin)的影响。方法:将40只C57BL/6小鼠随机分为匹罗卡品组和生理盐水组,Racine评分评估癫痫发作的严重程度,RT-PCR法检测miR-195在两组小鼠脑皮质组织中的表达含量。通过Target scan及miR Base网站,预测miR-195靶基因;Western blot检测miR-195靶蛋白在两组小鼠脑皮质中的表达。miR-195 mimics或miR-mimics-NC立体定向下注射入生理盐水组小鼠侧脑室,miR-195 inhibitor或miR-inhibitor-NC注射入匹罗卡品组小鼠侧脑室,免疫荧光染色观察靶蛋白的表达情况,Racine评分记录小鼠癫痫发作次数。结果:与生理盐水组相比,匹罗卡品组小鼠Racine评分高,miR-195含量增加(均P<0.05)。Target scan及miR Base预测Clusterin是miR-195靶基因之一,匹罗卡品组小鼠脑皮质Clusterin蛋白表达含量低于生理盐水组(P<0.05)。miR-195 mimics组小鼠脑皮质Clusterin中的表达量低于miR-mimics-NC组(P<0.05),Racine评分高于miR-mimics-NC组(P<0.05);miR-195 inhibitor组小鼠脑皮质Clusterin表达含量高于miR-inhibitor-NC组(P<0.05),小鼠Racine评分低于miR-inhibitor-NC组(P<0.05)。结论:miR-195可通过抑制脑皮层Clusterin蛋白表达,增加小鼠[JP]癫痫发作频率。

Objective:To determine the effect of miR-195 on Clusterin expression in cerebral cortex and seizure frequency in mice.Methods:There were 40 C57BL/6 mice divided into the pilocarpine group and the normal saline group.The severity of epileptic seizure was evaluated by Racine score.RT-PCR was used to detect the level of miR-195 in the cerebral cortex.Target scan and miR Base websites were used to predict miR-195 target genes.Western blot was used to verify target protein of miR-195 in the cerebral cortex.Finally,miR-195 mimics or miR-mimics-NC were stereotactically injected into the lateral ventricle for the saline group,while miR-195 inhibitor or miR-inhibitor-NC were for the pilocarpine group,respectively.The protein level was evaluated using immunofluorescence staining method,and the frequency of epileptic seizures was recorded by Racine score.Results:Compared to the normal saline group,Racine score and miR-195 level were both higher in the pilocarpine group(both P<0.05).Target Scan and miR Base predicted that Clusterin was one of the target genes of miR-195,which was decreased in cerebral cortex by pilocarpine than normal saline(P<0.05).Clusterin level was lower and Racine score was higher in the miR-195 mimics group than those in the miR-mimics-NC group(both P<0.05).However,miR-195 inhibitor performed with an increase of Clusterin and a decrease of Racine score when compared with the miR-inhibitor-NC group(both P<0.05).Conclusion:miR-195 could induce epileptic seizure in mice via inhibition of Clusterin in the cerebral cortex.