Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.
Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of California San Diego, 3020 Children's Way, MC 5030, San Diego, CA, 92103-8450, USA.
Sci Rep. 2022 Feb 22;12(1):3027. doi: 10.1038/s41598-022-06809-0.
Primary sclerosing cholangitis (PSC) is an idiopathic and heterogenous cholestatic liver disease characterized by chronic inflammation and fibrosis of the biliary tree. Currently, no effective therapies are available for this condition, whose incidence is rising. At present, specificity and sensitivity of current serum markers used to diagnose PSC are limited and often unreliable. In this study, we characterize circulating extracellular vesicles and provide supporting data on their potential use as novel surrogate biomarkers for PSC. EVs are membrane surrounded structures, 100-1000 nm in size, released by cells under various conditions and which carry a variety of bioactive molecules, including small non-coding RNAs, lipids and proteins. In recent years, a large body of evidence has pointed to diagnostic implications of EVs and relative cargo in various human diseases. We isolated EVs from serum of well-characterized patients with PSC or control subjects by differential centrifugation and size-exclusion chromatography. A complete characterization identified elevated levels of circulating EVs in PSC patients compared to healthy control subjects (2000 vs. 500 Calcein-FITC + EVs/μL). Tissue and cell specificity of circulating EVs was assessed by identification of liver-specific markers and cholangiocyte marker CK-19. Further molecular characterization identified 282 proteins that were differentially regulated in PSC-derived compared to healthy control-EVs. Among those, IL-13Ra1 was the most significantly and differentially expressed protein in PSC-derived EVs and correlated with the degree of liver fibrosis. In addition to protein profiling, we performed a miRNA-sequencing analysis which identified 11 among established, liver-specific (e.g., miR-122 and miR-192) and novel miRNAs. One of the newly identified miRNAs, miR-4645-3p, was significantly up-regulated fourfold in PSC-derived EVs compared to circulating EVs isolated from healthy controls. This study provides supporting evidence of the potential role of circulating EVs and associated protein and miRNA cargo as surrogate noninvasive and reliable biomarker for PSC.
原发性硬化性胆管炎（PSC）是一种特发性和异质性胆汁淤积性肝病，其特征为胆道的慢性炎症和纤维化。目前，对于这种发病率不断上升的疾病，尚无有效的治疗方法。目前，用于诊断 PSC 的现有血清标志物的特异性和敏感性有限，且往往不可靠。在这项研究中，我们对循环细胞外囊泡进行了特征描述，并提供了支持其作为 PSC 新型替代生物标志物的潜在用途的数据。细胞外囊泡是由细胞在各种条件下释放的、具有各种生物活性分子（包括小非编码 RNA、脂质和蛋白质）的、大小为 100-1000nm 的被膜包围的结构。近年来，大量证据表明 EVs 及其相对货物在各种人类疾病中有诊断意义。我们通过差速离心和大小排阻色谱法从特征明确的 PSC 患者或对照者的血清中分离 EVs。全面的特征描述表明，与健康对照者相比，PSC 患者的循环 EVs 水平升高（2000 比 500Calcein-FITC+EVs/μL）。通过鉴定肝特异性标志物和胆管细胞标志物 CK-19，评估了循环 EVs 的组织和细胞特异性。进一步的分子特征鉴定表明，与健康对照 EVs 相比，PSC 衍生的 EVs 中有 282 种蛋白表达差异。其中，IL-13Ra1 是 PSC 衍生 EVs 中表达最显著和差异最大的蛋白，与肝纤维化程度相关。除了蛋白谱分析外，我们还进行了 miRNA 测序分析，鉴定出了 11 种已建立的、肝特异性（如 miR-122 和 miR-192）和新型 miRNA。新鉴定的 miRNA 之一，miR-4645-3p，在 PSC 衍生的 EVs 中比从健康对照者分离的循环 EVs 高 4 倍。这项研究提供了支持循环 EVs 及其相关蛋白和 miRNA 货物作为 PSC 替代无创和可靠生物标志物的潜在作用的证据。
