非小细胞肺癌脑转移患者血清外泌体中miR-21、miR-330水平及临床意义

Levels of miRNA-21 and miRNA-330 in serum exosomes of non-small cell lung cancer patients with brain metastases and their clinical significances

目的探讨非小细胞肺癌(NSCLC)脑转移患者血清外泌体中miRNA-21(miR-21)、miRNA-330(miR-330)的表达水平及二者与患者预后的关系。方法前瞻性队列研究。前瞻性选择2021年3月至2022年9月就诊于山东第一医科大学附属人民医院的125例NSCLC患者,经CT、头部增强磁共振成像或手术病理检查判断是否脑转移。将NSCLC患者按照是否合并脑转移分为转移组(58例)和未转移组(67例),选择同期收治的50例肺部良性疾病患者及同期体检的50名体检健康者分别为良性组和健康对照组。收集所有研究对象血清样本(其中患者为治疗前样本),提取外泌体,采用实时荧光定量聚合酶链反应法测定外泌体中miR-21、miR-330转录水平相对表达量;采用电化学发光免疫法测定血清肿瘤标志物[神经元特异性烯醇化酶(NSE)、癌胚抗原(CEA)、鳞状细胞癌抗原(SCCA)]水平。比较4组血清外泌体中miR-21、miR-330及血清肿瘤标志物水平,采用Pearson法分析NSCLC脑转移患者治疗前血清外泌体miR-21、miR-330间相关性及二者与血清肿瘤标志物间的相关性。以CT、头部增强磁共振成像或手术病理确定的脑转移为金标准,绘制依据治疗前血清外泌体中miR-21、miR-330水平单独及二者联合判断NSCLC患者脑转移发生的受试者工作特征(ROC)曲线。根据NSCLC患者随访期间是否因肿瘤病死,将NSCLC患者分为预后不良组和预后良好组,对比两组临床特征及治疗前血清外泌体中miR-21、miR-330水平。采用多因素logistic回归分析NSCLC患者因肿瘤病死的独立影响因素。结果125例NSCLC患者中,男性68例(54.4%),女性57例(45.6%);年龄(63±5)岁,范围:49~82岁;腺癌89例(71.2%),鳞状细胞癌36例(28.8%)。健康对照组、良性组、未转移组、转移组血清外泌体中miR-21转录水平相对表达量依次升高,miR-330转录水平相对表达量依次下降,血清NSE、CEA、SCCA蛋白浓度依次升高,两两组间比较,差异均有统计学意义(均P<0.001)。Pearson相关性分析显示,NSCLC脑转移患者治疗前血清外泌体中miR-21与血清NSE、CEA、SCCA水平呈正相关性(r值分别为0.641、0.785、0.612,P值分别为0.015、0.011、0.019),治疗前血清外泌体中miR-330与血清NSE、CEA、SCCA水平呈负相关性(r值分别为-0.612、-0.689、-0.587,P值分别为0.016、0.021、0.013),治疗前血清外泌体中miR-21与miR-330呈负相关性(r=-0.529,P=0.023)。ROC曲线分析显示,治疗前血清外泌体中miR-21、miR-330单独及二者联合判断NSCLC患者脑转移发生的曲线下面积分别为0.861(95%CI:0.792~0.931)、0.894(95%CI:0.840~0.947)、0.906(95%CI:0.849~0.963),差异均有统计学意义(均P<0.001);miR-21相对表达量最佳临界值为1.625,相应的灵敏度、特异度分别为77.4%、71.5%,miR-330相对表达量最佳临界值为0.611,相应的灵敏度、特异度分别为81.1%、74.9%,二者联合时达最佳临界值时的灵敏度和特异度分别为84.5%、73.8%。NSCLC患者中位随访19个月(95%CI:17~21个月),随访期间因肿瘤病死23例(18.4%)。预后不良组中年龄≥60岁、临床分期Ⅲ~Ⅳ期、脑转移患者的比例及治疗前血清外泌体中miR-21相对表达量均高于预后良好组,治疗前血清外泌体中miR-330相对表达量低于预后良好组,差异均有统计学意义(均P<0.05)。多因素logistic回归分析显示,年龄高(≥60岁比<60岁,OR=3.750,95%CI:1.191~11.806,P=0.024)、临床分期晚(Ⅲ~Ⅳ期比Ⅰ~Ⅱ期,OR=4.667,95%CI:1.303~16.716,P=0.018)、脑转移(转移比未转移,OR=2.573,95%CI:1.008~6.611,P=0.049)、治疗前血清外泌体中miR-21相对表达量升高(OR=2.585,95%CI:1.198~6.152,P=0.008)是NSCLC患者因肿瘤病死的独立危险因素,治疗前血清外泌体中miR-330相对表达量升高是因肿瘤病死的独立保护因素(OR=0.821,95%CI:0.715~0.954,P<0.001)。结论NSCLC脑转移患者治疗前血清外泌体中miR-21水平高,miR-330水平低,二者间呈负相关,且二者与NSCLC脑转移患者血清多种肿瘤标志物及NSCLC患者预后密切相关;二者联合可能预测NSCLC脑转移发生情况。

Objective To investigate the expression levels ofmiRNA-21(miR-21)andmiRNA-330(miR-330)in serum exosomes of non-small cell lung cancer(NSCLC)patients with brain metastases,and the correlation of the two with the prognosis of patients.Methods A prospective cohort study was conducted.A total of 125 NSCLC patients who were admitted to the Affiliated People's Hospital of Shandong First Medical University from March 2021 to September 2022 were prospectively selected,and the brain metastasis was determined by CT,contrast-enhanced magnetic resonance imaging of the head,or surgical pathology.The NSCLC patients were divided into the metastatic group(58 cases)and the non-metastatic group(67 cases)according to whether they had brain metastases,and 50 patients with benign lung diseases and 50 healthy subjects who underwent physical examination in the same period were selected as benign group and healthy control group respectively.Serum samples were collected from all subjects(including patients'pre-treatment samples),the exosomes were extracted,and real-time fluorescence quantitative polymerase chain reaction was used to determine the relative expression of miR-21 and miR-330 in exosomes at the transcriptional level,and electrochemiluminescence immunoassay was used to detect the levels of serum tumor markers[neuron-specific enolase(NSE),carcinoembryonic antigen(CEA),squamous cell carcinoma antigen(SCCA)].The levels of miR-21 and miR-330 in serum exosomes and serum tumor markers in the 4 groups were compared,and the correlation between miR-21 and miR-330 in serum exosomes of NSCLC patients with brain metastases before treatment and the correlation between miR-21,miR-330 and serum tumor markers were analyzed by Pearson method.Using brain metastases identified by CT,contrast-enhanced magnetic resonance imaging of the head or surgical pathology as the gold standard,the receiver operating characteristic(ROC)curves were drawn to determine the occurrence of brain metastases in NSCLC patients based on the levels of miR-21,miR-330 and their combination in the serum exosomes before treatment.NSCLC patients were divided into the poor prognosis group and the good prognosis group according to whether or not they died of tumor during the follow-up period,and the clinical characteristics and levels of miR-21 and miR-330 in serum exosomes before treatment were compared between the two groups.The independent influencing factors of death due to tumor in NSCLC patients were analyzed by multivariate logistic regression.Results Among 125 NSCLC patients,68(54.4%)were male and 57(45.6%)were female;the age was(63±5)years old,ranging from 49 to 82 years old;89 patients(71.2%)were adenocarcinoma and 36 patients(28.8%)were squamous cell carcinoma.The transcriptional level relative expression of miR-21 in serum exosomes of healthy control group,benign group,non-metastatic group and metastatic group increased sequentially,the transcriptional level relative expression of miR-330 decreased sequentially,the protein concentrations of NSE,CEA and SCCA increased sequentially,and the differences between each two groups were statistically significant(all P<0.001).Pearson correlation analysis showed that in the serum exosomes of NSCLC patients with brain metastases before treatment,miR-21 was positively correlated with serum NSE,CEA and SCCA levels(r values were 0.641,0.785 and 0.612,respectively;P values were 0.015,0.011 and 0.019,respectively),miR-330 in the serum exosomes before treatment was negatively correlated with serum NSE,CEA,and SCCA levels(r values were-0.612,-0.689 and-0.587,respectively;P values were 0.016,0.021 and 0.013,respectively),and miR-21 was positively correlated with miR-330 in the serum exosomes before treatment(r=-0.529,P=0.023).ROC curve analysis showed that the area under the curve of miR-21,miR-330 and their combination in serum exosomes before treatment for determining the occurrence of brain metastases in NSCLC patients were 0.861(95%CI:0.792-0.931),0.894(95%CI:0.840-0.947)and 0.906(95%CI:0.849-0.963),and the differences were statistically significant(all P<0.001).The optimal cut-off value of miR-21 relative expression was 1.625,and the corresponding sensitivity and specificity were 77.4% and 71.5%,respectively;the optimal cut-off value of miR-330 was 0.611,and the corresponding sensitivity and specificity were 81.1% and 74.9%,respectively;the sensitivity and specificity when the two were combined to reach the optimal cut-off value were 84.5% and 73.8%,respectively.NSCLC patients were followed up for a median time of 19 months(95%CI:17-21 months),and 23 cases(18.4%)died due to the tumor during the follow-up period.The proportions of patients with age≥60 years old,clinical stage Ⅲ-Ⅳ and brain metastases and the relative expression of miR-21 in serum exosomes before treatment in the poor prognosis group were higher than those in the good prognosis group,the relative expression of miR-330 in the serum exosomes before treatment was lower than that in the good prognosis group,and the differences were all statistically significant(all P<0.05).Multivariate logistic regression analysis showed that the high age(≥60 years old vs.<60 years old,OR=3.750,95%CI:1.191-11.806,P=0.024),late clinical stage(stage Ⅲ-Ⅳvs.stage Ⅰ-Ⅱ,OR=4.667,95%CI:1.303-16.716,P=0.018),brain metastasis(with metastasis vs.non-metastasis,OR=2.573,95%CI:1.008-6.611,P=0.049),and elevated relative expression of miR-21 in serum exosomes before treatment(OR=2.585,95%CI:1.198-6.152,P=0.008)were the independent risk factors for death due to tumor in NSCLC patients,and elevated relative expression of miR-330 in serum exosomes before treatment was an independent protective factor for death due to tumor(OR=0.821,95%CI:0.715-0.954,P<0.001).Conclusions miR-21 level is high and miR-330 level is low in serum exosomes of NSCLC patients with brain metastases before treatment,and there is a negative correlation between them,and they are closely related to various serum tumor markers of NSCLC patients with brain metastases and NSCLC patients'prognosis;the combination of the two may predict the occurrence status of brain metastases in NSCLC.