小鼠蛛网膜下腔出血后大脑皮层长链非编码RNA及mRNA的表达谱研究  

Altered expressions of long non-coding RNA and mRNA in mouse cortex after subarachnoid hemorrhage

作　　者：彭建华 陈义天 吴越[3] 庞金伟 曹芳[4] 田晓翠[5] 秦兴虎 郐莉 陈礼刚 孙晓川[3] 江涌 

机构地区：[1]西南医科大学附属医院神经外科,泸州646000 [2]苏州大学医学部2014级临床医学专业,苏州215006 [3]重庆医科大学附属第一医院神经外科,重庆400016 [4]遵义医学院附属医院脑血管病科,遵义563000 [5]重庆市生物化学与分子药理学重点实验室,400016 [6]西南医科大学附属医院眼科,泸州646000

出　　处：《中华神经医学杂志》2016年第11期1111-1117,共7页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81371319、81571159）;新世纪优秀人才支持计划（NCET-12-1057）;四川省科技厅杰出青年项目（2014JQ0022）

摘　　要：目的探讨小鼠蛛网膜下腔出血（SAH）后早期大脑皮层长链非编码RNA（lncRNAs）及mRNAs的差异表达情况，寻求调控SAH后早期脑损伤（EBI）的新机制。方法6只C57BL／6J雄性基因野生型小鼠按随机数字表法分为假手术组及SAH组（n=3），后者采用颈动脉内穿刺法构建SAH模型。造模后24h断头取脑，提取皮层总RNA。经质量检测合格后，行PCR扩增建立cRNA文库，采用Illumina HiSeq^TM2500测序。运用软件Tophat2将转录本比对至小鼠基因组，运用RPKM法分别计算lncRNAs及mRNAs的表达量。通过数据库KEGG、GO富集对有差异表达的mRNAs进行通路分析，得到mRNAs参与的生物学途径。运用实时定量荧光PCR（qRT-PCR）技术对部分有差异表达的lncRNAs（fantom3_C730003K16、fantom3_I830129C17、fantom3_A430024L20、fantom3_C330006P03）进行验证。结果成功得到假手术组及SAH组小鼠大脑皮层lncRNAs及mRNAs的表达谱。3对样本中均有差异表达的lncRNAs共617种，其中103种上调、514种下调。3对样本中均有差异表达的mRNAs共444种，其中387种上调、54种下调。GO富集及KEGG通路分析提示，有差异表达的mRNAs涉及到包括炎症在内的多种生物学途径。随机选取的4条lncRNAs的变化趋势（fantom3_C730003K16、fantom3_I830129C17表达上调，fantom3_A430024L20、fantom3_C330006P03表达下调）经qRT-PCR技术得到验证。结论小鼠SAH后大脑皮层lncRNAs较正常脑组织存在明显差异，lncRNAs也许可以成为SAH后EBI病情及预后的判断标志以及潜在的治疗靶点。Objective To investigate the differential expressions of long non-coding RNAs （lncRNAs） and mRNAs in early phases of subarachnoid hemorrhage （SAH） and explore the possible new mechanisms which may regulate early brain injury （EBI）. Methods A total of 6 C57BL/6J male wild-type mice were divided into sham-operated group and SAH group （n=3）. SAH models were induced by endovascular perforation. Twenty-four hours after operation, all animals were sacrificed, brain samples were removed rapidly, and total RNAs were extracted. After quality control, total RNAs were amplified and transcribed into fluorescent cRNA, and then performed RNA-seq by an Illumina HiSeq^TM2500. Reads were aligned to the mouse transcriptome with Tophat2 software. The whole sample expression levels were presented by RPKM （expected number of Reads Per Kilobase of transcript sequence per Millions base pairs Sequenced）. The differential expressions of mRNAs were analyzed by biological pathways, and all the differentially expressed mRNAs were selected for Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway. Quantitative real-time （qRT）-PCR was used to confirm some differentially expressed mRNAs. Results The expression profiles of lncRNAs and mRNAs of sham-operated and SAH mice brain tissues were successfully analyzed by RNA-Seq. In these 6 samples, 617 lncRNAs （103 being upregulated and 514 being downregulated） and 444 mRNAs （387 being upregulated and 54 being downregulated） were differentially expressed in three pair brain tissues. GO and KEGG analysis indicated that differentially expressed mRNAs were involved in a variety of biological processes, including inflammation. The qRT-PCR analysis of 4 randomly selected lncRNAs （fantom3_C730003K16 and fantom3_I830129C17 up-regulation expressions; fantom3_A430024L20 and fantom3_C330006P03 down-regulation expressions） confirmed the accuracy of RNA-seq. Conclusion LncRNAs significantly differentially express in the SAH brain tissues and the

关 键 词：蛛网膜下腔出血 早期脑损伤 长链非编码RNA 炎症 二代测序 

分 类 号：R743.35[医药卫生—神经病学与精神病学]