Circulating serum miRNAs as potential diagnostic biomarkers for oesophageal adenocarcinoma

Exosomes were purified from 250 ul serum using ExoQuickTm. The presence of particles consistent in size with exosomes (60-150nm) was confirmed using a Nanosight LM10. miRNA was extracted from exosomes using an miRNeasy Serum/Plasma kit (Qiagen, #217184). miRNA was reversed transcribed using a TaqMan® microRNA Reverse Transcription Kit (Life technologies, #4366596). miRNA profiling was performed with a high throughput TaqMan® OpenArray® Human microRNA panel (Life technologies, #4461104). The panel consisted of probes for 754 human miRNAs that are based on miRNA sequences derived from Sanger miRBase v14. MegaplexTM Primer Human Pool A v2.1 and Human Pool B v2.0 or v3.0 The poor prognosis and rising incidence of oesophageal adenocarcinoma highlight the need for improved methods for detection of this cancer. Molecular biomarkers offer potential for this. The potential for circulating miRNAs as biomarkers in some other cancers has been shown, but circulating miRNAs have not been well characterized in oesophageal adenocarcinoma. This study investigated whether circulating miRNAs could be used to detect oesophageal adenocarcinoma. OpenArray high throughput real time PCR of microRNAs -- 19 patients without oesophageal disease, 10 patients with non-dysplastic Barrett's Oesophagus, 18 patients with locally advanced Oesophageal Adenocarcinoma

采用ExoQuick™试剂从250 μL血清中纯化外泌体（exosomes）。使用Nanosight LM10仪器验证了粒径符合外泌体特征（60~150 nm）的颗粒存在。采用miRNeasy血清/血浆试剂盒（Qiagen，货号#217184）从外泌体中提取微小RNA（miRNA）；使用TaqMan®微小RNA逆转录试剂盒（Life Technologies，货号#4366596）完成miRNA的逆转录反应。采用高通量TaqMan® OpenArray®人类微小RNA检测面板（Life Technologies，货号#4461104）进行miRNA表达谱分析，该检测面板包含754个人类miRNA的探针，其序列来源于Sanger miRBase v14数据库，所用引物为Megaplex™引物人类混合池A v2.1以及人类混合池B v2.0或v3.0。

食管腺癌（oesophageal adenocarcinoma）预后不佳且发病率持续攀升，凸显了开发该癌症优化检测手段的迫切需求。分子生物标志物为此提供了潜在解决方案。已有研究证实循环miRNA可作为部分其他癌症的生物标志物，但食管腺癌患者体内的循环miRNA尚未得到充分表征。本研究旨在探究循环miRNA能否用于食管腺癌的检测。

本研究采用OpenArray高通量实时荧光定量PCR对miRNA进行表达谱分析，受试者分为3组：19名无食管疾病者、10名伴非发育异常性巴雷特食管者以及18名局部晚期食管腺癌患者。