非小细胞肺癌血清外泌体中miRNA–99b–5p的表达及对脑转移的诊断和预后价值

杨学智; 刘远波; 刘远友

非小细胞肺癌血清外泌体中miRNA–99b–5p的表达及对脑转移的诊断和预后价值

Expression of miRNA-99b-5p in serum exosomes of non-small cell lung cancer and its diagnostic and prognostic value for brain metastasis

摘要

摘要:
目的： 探讨非小细胞肺癌（NSCLC）病人血清外泌体中miRNA–99b–5p表达水平及对病人脑转移的诊断和预后价值。
方法： 选取145例NSCLC病人为研究对象，其中合并脑转移的55例病人作为转移组，未发生脑转移的90例病人作为未转移组；选取60名同期健康体检者作为对照组。分离3组受试者血清外泌体，RT–PCR检测外泌体中miRNA–99b–5p表达水平。分析miRNA–99b–5p表达水平与NSCLC病人临床病理特征的关系，受试者工作特征（ROC）曲线分析血清外泌体中miRNA–99b–5p表达水平对NSCLC病人发生脑转移的诊断价值，COX回归分析血清外泌体中miRNA–99b–5p表达水平与NSCLC病人预后之间的关系。
结果： 分离的外泌体在电镜下表现为具有脂质双层膜结构的圆形颗粒。Western blotting结果显示，外泌体表面蛋白CD63和TSG101蛋白呈高表达。转移组和未转移组血清外泌体中miRNA–99b–5p表达水平均低于对照组（P ＜ 0.05），转移组miRNA–99b–5p表达水平低于未转移组（P ＜ 0.05）。血清外泌体中miRNA–99b–5p表达水平与NSCLC肿瘤大小及TNM分期相关（P ＜ 0.01），与年龄、肿瘤位置、病理类型及吸烟无关（P ＞ 0.05）。ROC曲线分析显示，当截断值为0.35时，血清外泌体中miRNA–99b–5p表达水平诊断NSCLC病人脑转移的曲线下面积为0.925（95%CI：0.835 ~ 0.956，P ＜ 0.01），灵敏度和特异度分别为89.74%和94.39%。COX回归分析显示，TNM分期、脑转移、血清外泌体中miRNA–99b–5p表达水平是影响NSCLC预后的危险因素（P ＜ 0.01）。
结论： NSCLC病人血清外泌体中miRNA–99b–5p表达下调，且与恶性临床病理特征和脑转移相关，可作为影响NSCLC预后的潜在分子标志物。

Abstract:
Objective To investigate the expression level of miRNA-99b-5p in serum exosomes of patients with non-small cell lung cancer (NSCLC) and its diagnostic and prognostic value for brain metastasis.
Methods One hundred and forty-five patients with NSCLC were selected as the study subjects, including 55 patients with brain metastasis as the metastasis group, and 90 patients without brain metastasis as the non-metastasis group, and 60 health examinees in the same period were set as the control group. The serum exosomes of subjects in the three groups were isolated, and the expression level of miRNA-99b-5p in the exosomes was detected by RT-PCR. The relationship between the expression level of miRNA-99b-5p and the clinicopathological characteristics of NSCLC patients was analyzed, the diagnostic value of the expression level of miRNA-99b-5p in serum exosomes for brain metastasis in NSCLC patients was analyzed by receiver operating characteristic (ROC) curve, and the relationship between the expression level of miRNA-99b-5p in serum exosomes and the prognosis of NSCLC patients was analyzed by COX regression.
Results The isolated exosomes were round particles with lipid-bilayer membrane under electron microscope. The results of Western blotting showed that the surface proteins CD63 and TSG101 were highly expressed in the exosomes. The expression level of miRNA-99b-5p in serum exosomes in the metastasis group and the non-metastasis group was lower than that in the control group (P ＜ 0.05), and the expression level of miRNA-99b-5p in the metastasis group was lower than that in the non-metastasis group (P ＜ 0.05). The expression level of miRNA-99b-5p in serum exosomes was correlated with tumor size and TNM stage of NSCLC (P ＜ 0.01), but not correlated with age, tumor location, pathological type and smoking (P ＞ 0.05). ROC curve analysis showed that when the cut-off value was 0.35, the area under the curve of miRNA-99b-5p expression level in serum exosomes for the diagnosis of brain metastasis in NSCLC patients was 0.925 (95%CI: 0.835–0.956, P ＜ 0.01), and the sensitivity and specificity were 89.74% and 94.39%, respectively. COX regression analysis showed that TNM stage, brain metastasis and miRNA-99b-5p expression level in serum exosomes were risk factors affecting the prognosis of NSCLC (P ＜ 0.01).
Conclusions The expression of miRNA-99b-5p in serum exosomes of patients with NSCLC is down-regulated, which is related to malignant clinicopathological features and brain metastasis, and can be used as a potential molecular marker for the prognosis of NSCLC.

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