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.

已有少数研究报道特定长链非编码RNA（lncRNA）与心脏发育或心力衰竭相关，但心脏中表达的lncRNA的精准细节，以及其在胚胎心脏发育过程中或成人心脏疾病时的表达调控机制仍未明确。本研究通过对比正常胚胎（约E14时期）、正常成年以及肥厚型成年小鼠心脏的lncRNA表达谱，鉴定出一组独特的胎儿期lncRNA丰度特征：相较于成年心脏，共有157个lncRNA存在差异表达（倍数变化≥50%，错误发现率FDR=0.02）；而肥厚型心脏仅能微弱重现该特征，仅存在17个差异表达lncRNA，其中13个也在胚胎心脏中被检测到。对同一样本中的编码蛋白mRNA进行分析后发现，在动态表达的lncRNA上下游10kb区域内，共有22个mRNA呈协同调控、11个呈反向调控。通过在C2C12细胞中进行lncRNA体外敲低实验，验证了Mccc1与Relb基因的lncRNA-mRNA表达反向调控关系。网络分析结果表明，lncRNAs在胚胎心脏发育过程中调控NF-κB与CREB1靶基因方面发挥核心作用，并鉴定出这些通路中多个不依赖lncRNAs的独立调控mRNA。本研究的心脏多聚腺苷酸化RNA（包含mRNA与lncRNA）表达谱均基于C57BL/6J小鼠心脏样本，通过Illumina HiSeq 2000测序平台完成构建。样本包括7个独立的E13.5时期胚胎心脏、12个成年心脏（6只6周龄、6只16周龄）、4只12周龄假手术对照心脏，以及4只12周龄时接受4周压力超负荷（TAC）造模的心脏。