Genome-wide transcriptional profiling of YAP target genes in human endothelial cells. Homo sapiens

Endothelial cell is the major cell type that senses and transduces mechanosignal generated by shear stress. We have recently shown that Hippo/YAP pathway is a mechanosensitive pathway that is critical for maintaining endothelial cell homeostasis. However, the transcritpional targets and biological functions of YAP in endothelial cells remain largely unknown. To evaluate YAP-dependent gene expression in endothelial cells, we performed RNA-sequencing in YAP depleted (by transfection with by YAP siRNA) and overexpressed (by infection with YAP-S127A catalytically active adenovirus) human endothelial cells. We observed that YAP critically regulates endothelial function by modulating multiple atherosclerosis-related genes. Our study provides mechanistic insights into the question how YAP regulates endothelial function and atherosclerosis by modulating endothelial transcriptional profile. Overall design: Human Umbilical Vein Endothelial Cells (HUVEC, passage 3 to passage 5) were seeded in 0.2% gelatin-coated dishes the day before experiment. The next day, 80% confluent HUVECs were transfected with non-targetting siRNA pool (siNC, GE Dharmacon, #D-001810-10) or YAP1 siRNA pool (siYAP1, GE Dharmacon, #M-012200) for 48 hours. In the overexpression experiment, 80% confluent HUVECs were infected with control adenovirus (Ad-NC) or YAP-S127A catalytically active adenovirus (Ad-YAP-S127A, gifted by Prof. Junichi Sadoshima, New Jersey Medical School, NJ) for 48 hours. After treatment, total RNA was isolated using the RNA-Easy Mini Plus kit (QIAGEN). High quality RNA samples (pre-assessed by Nanodrop measurements) were further processed in the Genome Research Center of the University of Rochester Medical Center. The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3’ adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt are generated for each sample and aligned to the organism specific reference genome. Sequenced reads were cleaned using Trimmomatic-0.32 before mapping some of to the human reference genome (GRCh38.p2) with STAR-2.4.2a. Raw read counts were obtained using HTSeq and gencode 23 human gene annotations. DESeq2-1.10.1 was used to perform data normalization and differential expression analysis with an adjusted p-value threshold of 0.05. Then, Cufflinks-2.0.2 Software was used with the gencode 23 human gene annotations to perform differential expression analysis with an FDR cutoff of 0.05.

内皮细胞是感知并转导剪切应力所产生机械信号的主要细胞类型。本团队近期研究表明，Hippo/YAP通路（Hippo/YAP pathway）是一类机械敏感通路，对维持内皮细胞稳态至关重要。然而，目前对于YAP在内皮细胞中的转录靶标及其生物学功能仍知之甚少。为评估内皮细胞中YAP依赖的基因表达谱，我们分别对YAP敲低（通过转染YAP小干扰RNA，siRNA）与过表达（通过感染具有催化活性的YAP-S127A腺病毒）的人内皮细胞开展了RNA测序（RNA-sequencing）实验。结果发现，YAP可通过调控多个动脉粥样硬化相关基因，关键调控内皮细胞功能。本研究为解析YAP如何通过调控内皮细胞转录组进而调控内皮功能与动脉粥样硬化提供了机制层面的见解。

实验整体设计：所用细胞为传代3至5代的人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVEC），实验前一日接种于0.2%明胶包被的培养皿中。次日，当细胞汇合度达到80%时，分别用非靶向siRNA混合物（siNC，GE Dharmacon，货号#D-001810-10）或YAP1 siRNA混合物（siYAP1，GE Dharmacon，货号#M-012200）转染细胞，处理48小时。在过表达实验组中，80%汇合度的HUVEC分别感染对照腺病毒（Ad-NC）或具有催化活性的YAP-S127A腺病毒（Ad-YAP-S127A，由新泽西医学院Junichi Sadoshima教授惠赠），处理48小时。

处理结束后，使用RNA-Easy Mini Plus试剂盒（QIAGEN）提取总RNA。经Nanodrop检测确认质量合格的RNA样品，被送至罗切斯特大学医学中心基因组研究中心进行后续实验。按照制造商说明书，使用TruSeq RNA Sample Preparation Kit V2（Illumina，加州圣地亚哥）构建下一代测序文库。简要流程如下：从100ng总RNA中通过寡聚dT磁珠纯化mRNA并进行片段化；采用随机六聚体引物完成第一链cDNA合成，随后合成第二链cDNA；对双链cDNA进行末端修复及3'端腺苷酸化；在cDNA两端连接Illumina接头，经凝胶电泳纯化后，使用针对接头序列的PCR引物进行扩增，得到长度约200-500bp的扩增产物。将扩增后的文库与Illumina单端流动槽杂交，使用cBot系统（Illumina，加州圣地亚哥）以每泳道8pM的浓度进行桥式扩增。每个样本生成100nt的单端读段，并比对至对应物种的参考基因组。

测序读段在比对前使用Trimmomatic-0.32进行质量修剪，随后使用STAR-2.4.2a将读段比对至人类参考基因组GRCh38.p2。使用HTSeq及gencode 23人类基因注释文件获取原始读段计数。使用DESeq2-1.10.1进行数据标准化及差异表达分析，校正后P值阈值设为0.05。此外，使用Cufflinks-2.0.2软件结合gencode 23人类基因注释文件开展差异表达分析，错误发现率（FDR） cutoff设为0.05。