Hitachi Chemical Co. America, Ltd. R and D Center, 1003 Health Sciences Rd, Irvine, CA, 92617, USA.
Department of Obstetrics, Gynecology and Reproductive Biology, Gynecologic Oncology Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA, 02115, USA.
J Ovarian Res. 2018 Mar 2;11(1):20. doi: 10.1186/s13048-018-0391-2.
Extracellular vesicles (EVs) are considered as a new class of resources for potential biomarkers. We analyzed expression of specific mRNA and miRNA in EVs derived from ovarian cancer ascites and the ideal controls, peritoneal fluids from benign patients for potential early detection and prognostic biomarkers.
Fluids were collected from subjects with benign cysts or endometrioma (n = 10), or low/high grade serous ovarian carcinoma (n = 8). EV particles were captured using primarily ExoComplete filterplate or ultracentrifugation and analyzed by nanoparticle tracking analysis, ELISA, and scanning electron microscopy. EV RNAs extracted from two ascites and three peritoneal fluids were submitted for next-generation sequencing. The expression of 34 mRNA and 18 miRNAs in the EVs isolated from patient fluids and cell line media was determined using qPCR.
EVs isolated from patient samples had concentrations greater than 10 EV particles/mL and 30% were EpCAM-positive based on ELISA. EV particle sizes averaged 113 ± 11.5 nm. The qPCR studies identified five mRNA (CA11, MEDAG, LAMA4, SPINT2, NANOG) and six miRNA (let-7b, miR23b, miR29a, miR30d, miR205, miR720) that were significantly differentially expressed between cancer ascites and peritoneal fluids. In addition, CA11 mRNA was decreased to 0.5-fold and SPINT2 and NANOG mRNA were significantly increased up to 100-fold in conditioned media of cancer cells compared to immortalized ovarian surface and fallopian tube epithelial cell lines, the hypothesized cells of origin for ovarian cancer development.
This study indicates that EV mRNA profiles can reflect the disease stage and may provide a potentially novel source for discovery of biomarkers in ovarian cancer.
细胞外囊泡 (EV) 被认为是具有潜在应用价值的生物标志物的新型资源。本研究分析了来源于卵巢癌腹水和理想对照良性患者腹腔液的 EV 中特定 mRNA 和 miRNA 的表达，旨在寻找潜在的早期检测和预后生物标志物。
采集良性囊肿或子宫内膜异位症患者（n=10）或低/高级别浆液性卵巢癌患者（n=8）的腹水和腹腔液。使用 ExoComplete 滤板或超速离心法捕获 EV 颗粒，通过纳米颗粒跟踪分析、ELISA 和扫描电子显微镜进行分析。从两份腹水和三份腹腔液中提取 EV RNA，并进行下一代测序。使用 qPCR 检测从患者体液和细胞系培养基中分离的 EV 中 34 个 mRNA 和 18 个 miRNA 的表达。
患者样本中分离的 EV 浓度大于 10 EV 颗粒/mL，ELISA 检测 30%为 EpCAM 阳性。EV 颗粒平均粒径为 113±11.5nm。qPCR 研究发现，在癌症腹水和腹腔液之间，有 5 个 mRNA（CA11、MEDAG、LAMA4、SPINT2、NANOG）和 6 个 miRNA（let-7b、miR23b、miR29a、miR30d、miR205、miR720）的表达存在显著差异。此外，与永生化卵巢表面和输卵管上皮细胞系相比，癌细胞条件培养基中 CA11 mRNA 降低至 0.5 倍，SPINT2 和 NANOG mRNA 分别显著增加至 100 倍。
本研究表明，EV mRNA 谱可反映疾病阶段，可能为卵巢癌生物标志物的发现提供新的潜在来源。
