蛛网膜下腔出血后血管平滑肌细胞凋亡的microRNA芯片检测及分析

MicroRNA microarray analysis for vascular smooth muscle cell apoptosis after subarachnoid hemorrhage

目的:采用microRNA芯片技术筛选蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后血管平滑肌细胞(vascular smooth muscle cell,VSMC)凋亡中起重要作用的microRNA。方法:首先建立SAH后VSMC凋亡模型。然后采用microRNA芯片筛选差异表达microRNA。最后采用qRT-PCR方法对筛选出来的microRNA145进行验证,并通过生物信息数据库预测其可能的靶基因。结果:通过200μmol/L氧合血红蛋白(oxyhemoglobin,OxyHb)作用24 h,成功建立SAH后VSMC凋亡模型。MTT检测显示200μmol/L OxyHb组作用24 h后吸光度(absorbance,A)值为0.197±0.131,流式细胞仪检测凋亡率为30.233±7.231,与其他组相比差异明显(P<0.05)。然后通过microRNA芯片检测,获得差异表达microRNA 22个,其中显著上调的16个,显著下调的6个。其中microRNA145差异变化最大。经qRT-PCR验证,SAH后VSMC中microRNA145异常高表达。预测结果显示,Bcl-2/腺病毒E1B19000相互作用蛋白3(Bcl-2/E1B 19 kDa-interacting protein 3,BNIP3)为其可能的靶基因。结论:microRNA145可能是调控SAH后VSMC凋亡的分子靶点。microRNA145可能通过BNIP3相关信号途径,导致了SAH后VSMC凋亡,诱发并加重了脑血管痉挛。

Objective ：To select microRNA,which plays an important role in the apoptosis of vascular smooth muscle cells（VSMC） after subarachnoid hemorrhage（SAH） by microRNA microarray. Methods：Firstly,the model of VSMC apoptosis after SAH was established,then,microRNA microarray was used to screen differentially expressed microRNA. Finally,the screened out microRNA145 was validated by qRT-PCR and biological information database was used to predict the possible target genes. Results ： Model of VSMC apoptosis after SAH was established successfully. MTT assay showed that A value of 200 μmol/L OxyHb group was 0.197 ± 0.131 after 24 h and apoptosis rate was 30.233± 7.23 ,with significant differences compared with those of other groups（P〈0.05）. There were 22 differently expressed micreRNAs, 16 micreRNAs of which were up-regulated,6 microRNAs were down-regulated obviously and microRNA145 was the greatest change one among them. MicroRNA145 expressed highly in VSMC after SAH,which was validated by qRT-PCR. It was suggested that Bcl-2/E1B 19 kDa-interacting protein 3 （BNIP3） may be the target gene for it. Cottclusions：microRNA145 may be the molecular target regulating VSMC apoptosis after SAH. MicreRNAlg5 causes apoptosis of VSMC after SAH, induce and increase cerebral vasospasm possibly through BNIP3 related signaling pathways.