结直肠癌患者血浆外泌体中miR?590?3p的表达及其临床意义

目的 探讨结直肠癌（colorectal cancer，CRC）患者血浆外泌体中miR-590-3p的表达水平及其临床预后价值。方法 收集2015年1月至2015年12月于利川市人民医院、武汉大学中南医院住院并接受手术治疗的97例CRC患者以及20名健康对照者的血浆样本，采用ExoQuick Exosome Precipitation Solution试剂盒从患者血浆中分离外泌体，分别用透射电镜、NanoSight纳米颗粒分析仪鉴定其形态和粒径，Western blot检测外泌体特异性标记蛋白CD63和TSG101的表达；采用RT-qPCR检测血浆外泌体中miR-590-3p的表达水平，并分析其与CRC患者临床病理特征的关系；采用多因素Cox回归模型分析影响CRC患者预后的因素，Kaplan-Meier法分析miR-590-3p高表达组和低表达组患者的总生存期。结果 从97例CRC患者和20名健康对照者血浆分离的外泌体粒径主要集中在100 nm左右，Western blot检测可见CD63和TSG101表达，且CRC患者血浆中的外泌体含量高于健康对照组。RT-qPCR检测结果显示，CRC患者血浆外泌体中miR-590-3p的表达水平高于健康对照组（7.163±0.147 vs 1.339±0.161，P<0.001），其表达水平与CRC患者的肿瘤分化程度、淋巴结转移、淋巴脉管浸润及TNM分期有关（均P<0.05）。生存分析显示，血浆外泌体miR-590-3p高表达组患者的总生存期较低表达组短（P=0.003）。多因素Cox回归模型分析显示，血浆外泌体miR-590-3p高表达是CRC患者预后的独立危险因素（HR=1.954，95%CI：1.271~3.524，P=0.032）。结论 miR-590-3p在结直肠癌患者血浆外泌体中表达上调，且与肿瘤进展和不良预后密切相关，可能作为独立标志物用于CRC预后评估。

Expression and clinical significance of plasma exosomal miR?590?3p in patients with colorectal cancer

Objective To investigate the expression level of miR-590-3p in plasma exosomes of patients with colorectal cancer (CRC) and its clinical prognostic value. Methods The plasma samples of 97 CRC patients, hospitalized and received surgical treatment in Lichuan People's Hospital or Zhongnan Hospital of Wuhan University from January 2015 to December 2015, and 20 healthy controls were collected. Exosome were extracted from the plasma by ExoQuick Exosome Precipitation Solution kit, its morphology and particle size were identified by transmission electron microscopy and NanoSight nanoparticle analyzer, and the expression of exosome specific marker proteins CD63 and TSG101 were detected by Western blot. RT-qPCR was used to detect the expression of miR-590-3p in plasma exosome, and its relationship with clinicopathological features of CRC patients was analyzed. The prognostic factors of CRC patients were analyzed by multivariable Cox regression model, and the overall survival of patients in the miR-590-3p high expression group and the low expression group was analyzed by Kaplan-Meier method. Results The particle size of exosome isolated from plasma of 97 CRC patients and 20 healthy controls was about 100 nm. The expression of CD63 and TSG101 could be detected by Western blot, and the content of exosomes in the plasma of CRC patients was higher than that of healthy controls. RT-qPCR showed that the expression level of miR-590-3p in the plasma exosomes of CRC patients was higher than that of healthy controls (7.163±0.147 vs 1.339±0.161, P<0.001), and its expression level was related to tumor differentiation, lymph node metastasis, lymph vascular invasion and TNM stage of CRC patients(all P<0.05). Survival analysis showed that the overall survival of patients with high miR-590-3p expression in plasma exosomes was shorter than those with low expression (P=0.003). Furthermore, the multivariable Cox regression model analysis showed that high expression of miR-590-3p in plasma exosomes was an independent risk factor for prognosis in patients with CRC (HR=1.954, 95%CI: 1.271-3.524, P=0.032). Conclusions The expression of miR-590-3p is up-regulated in the plasma exosomes of patients with CRC, and closely related to tumor progression and poor prognosis, which may be an independent biomarker for CRC prognosis assessment.