RNAseq of Extracellular vesicle RNAs and cellular RNAs from HPASMCs with or without TGF-b1 or BMP4 treatment

Excessive TGF-ß signalling has been shown to underlie pulmonary hypertension (PAH). Human pulmonary artery smooth muscle cells (HPASMCs) can release extracellular vesicles (EVs) but their content and significance have not yet been studied. Here, we analysed the content and biological relevance of HPASMC-derived EVs. We used low-input RNA-Seq to analyse the RNAs of EVs released by HPASMC under basal conditions. This data was compared to cellular RNAs. The same experiments (RNA-seq of EV and cellular RNAs) were carried out on HPASMC treated with TGF-b1 and BMP4, recapitulating pathological conditions. Overall design: Extracellular vesicles were isolated from HPASMC with or without TGF-b1 or BMP4 treatment. Low-input RNA-seq was performed on EV and cellular RNAs by BGI. Three replicates were carried out and sequenced for most conditions except for EV from untreated and BMP4 treated HPASMC with 2 replicates due to failure of the library preparation of one replicate.

过量的转化生长因子β（Transforming Growth Factor-β, TGF-β）信号通路已被证实是肺动脉高压（PAH）的致病基础。人肺动脉平滑肌细胞（HPASMCs）能够释放细胞外囊泡（EVs），但目前尚未有研究对这类囊泡的内容物及其生物学意义进行探究。本研究针对人肺动脉平滑肌细胞来源的细胞外囊泡的内容物与生物学相关性展开分析。我们采用低起始量RNA测序（low-input RNA-Seq）技术，对基础培养条件下人肺动脉平滑肌细胞释放的细胞外囊泡中的RNA进行检测分析，并将其与对应细胞内的RNA进行比对。此外，我们对经转化生长因子β1（TGF-β1）与骨形态发生蛋白4（BMP4）处理的人肺动脉平滑肌细胞开展了相同的实验（即细胞外囊泡与细胞内RNA的RNA测序），以此模拟病理状态下的细胞环境。整体实验设计如下：从经或未经转化生长因子β1、骨形态发生蛋白4处理的人肺动脉平滑肌细胞中分离细胞外囊泡；由华大基因（BGI）对细胞外囊泡与细胞内RNA进行低起始量RNA测序。多数实验条件设置3次生物学重复并完成测序，但未处理组与骨形态发生蛋白4处理组的细胞外囊泡样本，因其中1次重复的文库制备失败，仅完成2次重复的测序。