PBEF Knockdown in HMVEC-LBI. Homo sapiens

This study employed Affymetrix GeneChips to profile transcriptome of human pulmonary microvascular endothelial cells (HMVEC-L) treated with PBEFsiRNA to gain insight into transcriptional regulations of PBEF on the endothelial function. We isolated and labeled mRNAs from PBEF siRNA transfected HMVEC-L and hybridized them to Affymetrix GeneChip HG-U133 plus 2. Differentially expressed genes and canonical pathways were analyzed. Expressions of selected genes were validated by RT-PCR or western blotting. Several important themes are emerged from this study. First, PBEF induces the upregulation and downregulation of multiple genes in the endothelium. Expression of 373 genes were increased and 64 genes decreased by at least 1.3 fold in the PBEFsiRNA treated group compared to the control group of PBEFscRNA treated HMVEC-L. Second, the microarray results confirmed some previous reports of PBEF mediated gene expressions in some pathways but provided a more complete repertoire of molecules in those pathways. Third, most of affected canonical pathways or differentially expressed genes in PBEF siRNA treated HMVEC-L over their controls have not previously been reported to be PBEF-responsive. Our first transcriptome analysis of human pulmonary microvascular endothelial cells treated with PBEFsiRNA has provided important insights into the transcriptional regulation of gene expression in HMVEC-L cells by PBEF. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular mechanisms underlying PBEF mediated endothelial functions and dysfunctions in the physiology and the pathogenesis of inflammatory conditions, cancer, diabetes, coronary heart disease and provide new leads of therapeutic targets to those diseases. Overall design: 6 sample microarray study - 3 replicates of the PBEF knockdown and 3 replicates of the SC RNA.

本研究采用Affymetrix基因芯片（Affymetrix GeneChips）对经PBEF小干扰RNA（PBEFsiRNA）处理的人肺微血管内皮细胞（human pulmonary microvascular endothelial cells, HMVEC-L）的转录组进行谱分析，以解析PBEF对内皮细胞功能的转录调控机制。我们从转染PBEFsiRNA的HMVEC-L中分离并标记mRNA，将其与Affymetrix GeneChip HG-U133 plus 2芯片进行杂交，随后对差异表达基因与经典通路展开分析。通过逆转录PCR（RT-PCR）及蛋白质印迹（western blotting）验证了部分候选基因的表达水平。本研究得出多项重要发现：其一，PBEF可调控内皮细胞内多组基因的表达水平，使其出现上调或下调。与转染PBEF对照小RNA（PBEFscRNA）的对照组HMVEC-L相比，PBEFsiRNA处理组中373个基因的表达量上调至少1.3倍，64个基因的表达量下调至少1.3倍。其二，本次芯片实验结果验证了此前关于PBEF介导部分通路基因表达的相关研究，并补充了这些通路中更为完整的分子谱系。其三，相较于对照组，PBEFsiRNA处理的HMVEC-L中受影响的经典通路与差异表达基因，多数此前未被报道与PBEF的调控作用相关。本研究首次针对经PBEFsiRNA处理的人肺微血管内皮细胞开展转录组分析，为解析PBEF对HMVEC-L细胞基因表达的转录调控提供了重要依据。后续针对本研究中报道的转录调控机制开展深入追踪分析，有望阐明PBEF介导的内皮细胞功能异常在炎症性疾病、癌症、糖尿病、冠心病等生理状态及发病过程中的分子机制，并为上述疾病提供全新的治疗靶点线索。实验设计：本芯片研究共包含6个样本，其中PBEF敲低组与对照小RNA处理组各设3个生物学重复。