Chromatin Capture Identifies SCARB1-LAG3 Proinflammatory Cardiovascular Disease Gene Networks [RNA-Seq]

Purpose: Utilized Next-generation sequencing (NGS) to profile transcriptional differences between two populations, a selected carrier (C) alele of SNP (rs10846744) associated with cardiovascular disease (CVD) and a selected non-carrier (G) allele. Methods: RNA was isolated from three subjects homozygous for the rs10846744 reference (G) allele and three subjects homozygous for the rs10846744 risk (C) allele and then subjected to full transcriptome sequencing. Triplicates of one sample from each group were also sequences to overlay with chromatin data from each group. Knockdown of NR2F2 using lentiviral particles assessed differential expression between the treated and untreated risk replicates. The Illumina NextSeq 500 next gen sequencing platform (UCONN CGI, Storrs, CT) was used for RNA-Seq. HiSeq 2000 was used for biological replicates at Perkin Elmer. RNA targets of interest were validated by qPCR using TaqMan assays and proteins were blotted using standard western methodologies. Results: We normalized the raw read count by FPKM and using Cufflinks, identified differential transcripts. RNA-seq data confirmed differential expression of NR2F2 and LAG3, validated by qPCR. Conclusions: Our study represents the first detailed analysis of significant changes in gene expression by an altered chromatin interaction network from SCARB1 to NR2F2 and LAG3, contributing to CAD proinflammatory gene networks. Transcriptional profiles from 3 carriers of the risk (C) allele and 3 non-carriers of the (G) reference allele, were generated by NGS, in triplicate, using Illumina platform technology. Replicates of one sample from each group and knockdown of NR2F2 in the risk replicate were also sequenced (n=3) to overlay with chromatin interaction data.

研究目的：采用下一代测序技术（Next-generation sequencing, NGS），对与心血管疾病（cardiovascular disease, CVD）相关的单核苷酸多态性（single nucleotide polymorphism, SNP）rs10846744的风险等位基因（C等位基因）携带者与非风险等位基因（G等位基因）携带者两个群体间的转录组差异进行分析。
研究方法：分别从3例rs10846744参考等位基因（G）纯合子受试者与3例rs10846744风险等位基因（C）纯合子受试者中提取RNA，随后进行全转录组测序。同时，对两组各1份样本进行三次生物学重复测序，以与两组的染色质数据进行整合分析。通过慢病毒颗粒介导NR2F2基因敲低，检测敲低组与未处理风险组重复样本间的差异表达基因。本研究的RNA测序采用Illumina NextSeq 500下一代测序平台（美国康涅狄格大学CGI中心，斯托尔斯，康涅狄格州）；生物学重复样本的测序则在珀金埃尔默公司采用HiSeq 2000平台完成。通过TaqMan探针法进行实时定量PCR（quantitative real-time polymerase chain reaction, qPCR）验证目标RNA靶点，并采用标准蛋白质免疫印迹实验对蛋白进行检测。
研究结果：采用每百万映射读数每千碱基片段数（fragments per kilobase of transcript per million mapped reads, FPKM）对原始读长计数进行标准化，并通过Cufflinks软件鉴定差异转录本。RNA测序数据证实NR2F2与LAG3的差异表达，该结果经qPCR验证。
研究结论：本研究首次详细分析了SCARB1至NR2F2、LAG3的染色质相互作用网络改变所导致的基因表达显著变化，该变化参与促冠状动脉疾病（coronary artery disease, CAD）炎症基因网络的调控。本研究通过NGS技术，采用Illumina平台完成了3例风险（C）等位基因携带者与3例参考（G）等位基因非携带者的转录组谱分析，所有样本均设置三次生物学重复。同时，对两组各1份样本的重复样本以及风险组中NR2F2敲低样本进行测序（n=3），以与染色质相互作用数据进行整合分析。