血管平滑肌细胞泡沫化进程差异LncRNA的筛选与分析

Screen and analysis of differential LncRNAs in vascular smooth muscle cell frothing process

目的通过高通量测序获得血管平滑肌细胞(VSMCs)转录谱,筛选VSMCs泡沫化进程中差异表达的主要长链非编码RNA(LncRNA)转录本,并对筛选得到的差异表达LncRNA进行功能预测及分析。方法经氧化低密度脂蛋白(ox-LDL)处理的家兔平滑肌细胞(浓度为80 mg/mL,24 h)为实验组,对照组无处理。收集实验组和对照组细胞,提取RNA进行质检并进行RNA-seq测序。收集测序结果中差异表达水平显著的LncRNA转录本,利用生物信息学工具预测差异表达LncRNA的潜在靶基因,并通过在线数据库对靶基因功能基因预测以探究筛选到的LncRNA与动脉粥样硬化(AS)相关性。结果RNA提取的浓度和质量符合LncRNA测序要求。通过测序质量评估确定LncRNA在细胞中单个碱基位置测序错误率均<0.1%,测序的结果合格可靠。经筛选得到28个差异显著表达LncRNA,其中显著上调的转录本包括以下7个:TCONS_01701109、TCONS_02288701、TCONS_02636927、TCONS_00846803、TCONS_02499900、TCONS_02236116、TCONS_02225105;表达显著下调的包括以下5个:TCONS_01803508、TCONS_02523114、TCONS_00489746、TCONS_02443383、TCONS_01645487。结论ox-LDL对VSMCs中的LncRNA表达水平有显著影响,本研究得到的差异表达LncRNA转录本可能参与了AS相关疾病的形成和发展,并且可能为后续研究提供了重要备选靶基因。

Objective To analyze obtain the vascular smooth muscle cell(VSMCs)transcription profile by high-throughput sequencing,screen the main differentially expressed long nocoding RNA(LncRNA)transcription profile in VSMCs frothing process,and perform functional prediction and analysis of the screened differentially expressed LncRNAs.Methods Rabbit smooth muscle cells were treated with oxidized low density lipoprotein(ox-LDL)(concentration:80 mg/mL 24 h)were the experimental group,while those in the control group were not treated.The cells of the experimental group and control group were cdlected,extracting RNAS,and quality inspection and RNA-seq sequencing were performed on the extracted RNA;LncRNA transcription profiles with significant differential expression levels were collected,potential target genes of differentially expressed LncRNAs were predicted by bioinformatics tools,and the relationship between screened LncRNAs and atherosclerosis(AS)was explored by predicting the functional genes of target genes through online database.Results The concentration and quality of RNA were in line with the requirements of LncRNA sequencing.By sequencing quality assessment,the error rate of LncRNA sequencing in single base position in cells was determined to be lower than 0.1%,the sequencing results were qualified and reliable.After screening,28 LncRNAs with significant differences were obtained,those seven significantly transcription profiles were upregulated:TCONS_01701109,TCONS_02288701,TCONS_02636927,TCONS_00846803,TCONS_02499900,TCONS_02236116,TCONS_02225105;and five transcription profiles were significantly down-regulated:TCONS_01803508,TCONS_02523114,TCONS_00489746,TCONS_02443383,TCONS_01645487.Conclusion Ox-LDL has a significant effect on the expression level of LncRNA in VSMCs.The differentially expressed LncRNA transcripts obtained in this paper may be involved in the formation and development of atherosclerotic diseases,these differentially expressed LncRNA transcription profile may provide important target genes for subsequent studies.