Polyadenylated RNA Sequencing of C57BL/6J Embryonic, Adult and Pressure-Overloaded Hearts

A few reports have implicated specific lncRNAs in cardiac development or failure, but precise details of lncRNAs expressed in hearts and how their expression may be altered during embryonic heart development or by adult heart disease is unknown. By comparing lncRNA profiles of normal embryonic (~E14), normal adult, and hypertrophied adult hearts we defined a distinct fetal lncRNA abundance signature that includes 157 lncRNAs differentially expressed compared to adults (fold-change> = 50%, FDR=0.02), and which was only poorly recapitulated in hypertrophied hearts (17 differentially expressed lncRNAs; 13 of these observed in embryonic hearts). Analysis of protein-coding mRNAs from the same samples identified 22 concordantly and 11 reciprocally regulated mRNAs within 10 kb of dynamically expressed lncRNAs, reciprocal relationships of lncRNA and mRNA levels was validated for the Mccc1 and Relb genes using in vitro lncRNA knockdown in C2C12 cells. Network analysis suggested a central role for lncRNAs in modulating NFkappaB- and CREB1-regulated genes during embryonic heart growth and identified multiple mRNAs within these pathways that are also regulated, but independently of lncRNAs. Cardiac polyadenylated RNA (mRNA and lncRNA) profiles were generated from C57BL/6J mouse hearts were generated on Illumina HiSeq 2000 instruments. 7 independent E13.5 hearts, 12 adult hearts (6 at 6 weeks of age, 6 at 16 weeks of age), 4 sham-operated hearts at 12 weeks of age, and 4 hearts after 4 weeks of pressure overload (TAC) at 12 weeks of age.已有多项研究表明特定长链非编码RNA（lncRNAs）与心脏发育及心力衰竭存在关联，但目前对于心脏中表达的lncRNAs的精准特征，以及其在胚胎心脏发育过程或成年心脏疾病状态下的表达变化机制仍不明晰。本研究通过对比正常胚胎（约E14）、正常成年及肥厚型成年小鼠心脏的lncRNA表达谱，鉴定出一类独特的胎儿源性lncRNA丰度特征：相较于成年心脏，共有157个lncRNA呈现显著差异表达（倍数变化≥50%，错误发现率FDR=0.02）；而该特征在肥厚型心脏中仅能得到微弱重现——仅17个差异表达lncRNA，其中13个亦在胚胎心脏中被检测到。对同一样本中编码蛋白的mRNA进行分析后发现，在动态表达的lncRNAs上下游10kb范围内，共有22个mRNA呈协同调控模式，11个呈反向调控模式；通过在C2C12细胞中开展体外lncRNA敲低实验，验证了Mccc1与Relb基因对应的lncRNA与mRNA水平的反向调控关系。网络分析结果显示，lncRNAs在胚胎心脏发育过程中，于核因子κB（NFkappaB）与CREB1调控的基因通路中发挥核心调控作用，并鉴定出这些通路中多个不依赖lncRNAs的受调控mRNA。本研究从C57BL/6J小鼠心脏中提取聚腺苷酸化RNA（包括mRNA与lncRNA），并在Illumina HiSeq 2000测序平台上完成表达谱构建。所用样本包含7个独立的E13.5胚胎心脏、12个成年心脏（6个为6周龄，6个为16周龄）、4个12周龄假手术对照心脏，以及4只12周龄小鼠经压力超负荷（TAC）造模4周后的心脏。