Non-invasive approach for evaluation of CTEPH using extracellular vesicle-associated small non-coding RNA. Non-invasive approach for evaluation of CTEPH using extracellular vesicle-associated small non-coding RNA

The aim of this study was to determine whether extracellular vesicle (EV)-associated small non-coding RNAs (sncRNAs) have potential as biomarkers for chronic thromboembolic pulmonary hypertension (CTEPH). EVs were isolated using different methods from serum of 23 CTEPH patients and 23 controls. EV-associated RNAs were analysed by next-generation sequencing using the TrueQuant method for molecular barcoding, and differentially expressed sncRNAs were validated by qRT-PCR. We identified 18 miRNAs and 21 piRNAs or piRNA clusters that were differentially expressed in CTEPH patients compared with the control group. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodelling. Furthermore, the expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. In summary, EV-associated piRNA DQ593039 shows promise as biomarker and may be a potential therapeutic target for CTEPH. Overall design: Small RNA profiles of CTEPH patients and age- and sex-matched healthy controls were generated by deep sequencing, in triplicate, using Illumina NextSeq 500.

本研究旨在明确细胞外囊泡（extracellular vesicle, EV）结合的小非编码RNA（small non-coding RNAs, sncRNAs）是否可作为慢性血栓栓塞性肺动脉高压（chronic thromboembolic pulmonary hypertension, CTEPH）的潜在生物标志物。

本研究采用不同分离方法，从23例CTEPH患者与23例对照者的血清中分离EV；通过搭载分子条形码技术的TrueQuant方法开展下一代测序，分析EV结合RNA的表达谱，并通过qRT-PCR验证差异表达的sncRNAs。

本研究鉴定出18种微小RNA（miRNA）及21种piwi相互作用RNA（piRNA）或piRNA簇，在CTEPH患者组与对照组中存在差异表达。

生物信息学分析预测，这些piRNA可能参与心脏与血管重构的进展过程。

此外，DQ593039的表达水平与多项临床关键参数显著相关，包括平均肺动脉压、肺血管阻力、右心室收缩压及N末端B型利钠肽原水平。

综上，EV结合的piRNA DQ593039有望成为CTEPH的生物标志物及潜在治疗靶点。

整体实验设计：采用Illumina NextSeq 500测序平台，对CTEPH患者与年龄、性别匹配的健康对照者的小RNA表达谱进行三次重复深度测序。