非小细胞肺癌尿液游离miRNA液态活检及其临床意义

发布时间：2018-05-13 14:09

本文选题：非小细胞肺癌 + 尿液　；参考：《宁波大学》2017年硕士论文

【摘要】：非小细胞肺癌(non-small cell lung cancer,NSCLC)的发病率及死亡率位居在中国及世界首位,复发转移是患者死亡的主要因素。NSCLC恶性程度高,目前临床上缺乏针对其有效的早期筛查及诊断方法,大部分患者就诊时就已经为晚期。因此,寻找有效的肺癌相关的早期诊断的标志物对于国民健康具有重要意义。Micro RNAs(mi RNAs)是一类具有调控基因表达功能的生物小分子,其中一些mi RNAs在肿瘤发生发展过程中扮演癌基因或抑癌基因的角色,对肿瘤的早期诊断、治疗、预后判断具有重要的临床意义。液态活检(liquid biopsy)是通过应用灵敏的分子生物学技术检测体液(血浆、血清、尿液、唾液、胸水、腹水)中与疾病发生、发展高度相关的特异性分子标志物,为疾病的筛查、早期诊断、药物选择、疗效监测、预后评估等方面提供无创性的方法。近年来,液态活检在肿瘤的筛查、诊断和疗效监测方面的应用取得巨大进展。液态活检在循环mi RNA作为肿瘤生物标志物的研究应用受到广泛关注。作为肿瘤标志物,循环mi RNA具有稳定、易检测及重复性好等优点,能为肿瘤的诊断和治疗提供帮助。但是,血液成分复杂,循环mi RNA易受到全血RNA的污染,会使疾病诊断的准确性受到明显影响。尿液也是液态活检的重要研究对象;与血清或血浆相比,尿液具有组成简单、取样方便、无创、稳定、可反复取样等优点。目前,已有研究表明,尿液mi RNA可作为肿瘤标志物应用于胰腺癌、乳腺癌、膀胱癌及肝癌等的早期诊断及预后评估,但对于NSCLC尚缺乏研究。因此,本研究欲通过应用新一代RNA深度测序技术(next-generation RNA sequencing)来探索性地寻找NSCLC患者尿液中与肺癌相关的游离mi RNA,应用逆转录-实时荧光定量PCR(RT-q PCR)验证筛选结果,并初步探讨尿液游离mi RNA在NSCLC早期诊断中的意义。本研究首先应用小RNA深度测序的方法对4例NSCLC患者和2例健康人尿液进行探索性研究,筛选出10种表达水平显著高于对照组的尿液游离mi RNA(mi R10a,mi R-10b,mi R-155,mi R-191,mi R-192,mi R-196a,mi R-21,mi R-224,mi R-26a,mi R-98)。随后,我们选取43例NSCLC首诊患者和26例健康人尿液样本用RT-q PCR方法对上述10种尿液游离mi RNA进行验证。结果显示,NSCLC患者尿液中6种mi RNA(mi R-10a,mi R-10b,mi R-191,mi R-192,mi R-21,mi R-26a)的水平显著高于健康人(p0.01);而NSCLC患者尿液mi R-155的水平显著低于正常组(p0.01);NSCLC患者尿液中mi R-196a,mi R-224和mi R-98在NSCLC患者和健康组中无显著性差异。受试者工作特征(receiver operating characteristic,ROC)曲线显示mi R-26a,mi R-21,mi R-191,mi R-192,mi R-10a,mi R-10b,mi R-155及mi R-21:mi R-155比值的曲线下面积(area under the curve,AUC)分别是0.901(0.829,0.974),0.842(0.755,0.929),0.753(0.633,0.873),0.746(0.621,0.870),0.679(0.551,0.807),0.735(0.615,0.855),0.699(0.568,0.831)和0.872(0.787,0.956);其中,mi R-21:mi R-155比值用于NSCLC诊断的灵敏度和特异性最高,分别为86%和84.6%。二元Logistic回归分析显示联合7种尿液游离mi RNA(mi R-10a,mi R-191,mi R-192,mi R21,mi R26a,mi R-10b,mi R-155)检测对NSCLC的诊断效率明显高于单个mi RNA,其AUC为0.927(0.864,0.989),灵敏度和特异性分别为92.3%和83.7%。综上,本研究结果表明联合7种尿液游离mi RNA(mi R-10a,mi R-191,mi R-192,mi R21,mi R26a,mi R-155,mi R-10b)检测可作为生物标志物用于NSCLC的辅助诊断。
[Abstract]:The incidence and mortality of non small cell lung cancer (non-small cell lung cancer, NSCLC) are in the first place in China and the world. The recurrence and metastasis is the main factor of death of the patients,.NSCLC with high degree of malignancy. At present, there is a lack of effective early screening and diagnosis methods for the patients. The marker of early diagnosis of lung cancer is of great significance to national health..Micro RNAs (MI RNAs) is a kind of small biomarker which has the function of regulating gene expression, some of which MI RNAs plays the role of oncogene or tumor suppressor gene in the process of tumor development, and the early diagnosis, treatment, and prognosis of the tumor are judged to have Important clinical significance. Liquid biopsy is a specific molecular marker that is highly related to the development of body fluid (plasma, serum, urine, saliva, saliva, hydrothorax, and ascites) by the application of sensitive molecular biology techniques. It is provided for screening, early diagnosis, drug selection, curative effect monitoring, prognosis evaluation and so on. Noninvasive methods. In recent years, liquid biopsy has made great progress in the application of tumor screening, diagnosis and therapeutic monitoring. Liquid biopsy is widely concerned in the research and application of circulating mi RNA as a tumor biomarker. As a tumor marker, circulating mi RNA has the advantages of stable, easy to detect and reproducible, and can be used as a tumor Diagnosis and treatment provide help. However, the blood components are complex, the circulating mi RNA is easily contaminated by the whole blood RNA, and the accuracy of the diagnosis of the disease is obviously affected. Urine is also an important object of liquid biopsy. Compared with the serum or plasma, urine has the advantages of simple composition, convenient sampling, noninvasive, stable, and repeated sampling. Studies have shown that urine mi RNA can be used as a tumor marker in the early diagnosis and prognosis evaluation of pancreatic cancer, breast cancer, bladder cancer and liver cancer, but it is still lack of research on NSCLC. Therefore, this study seeks to explore the urine of NSCLC patients by using a new generation of RNA deep sequencing technology (next-generation RNA sequencing). The free mi RNA associated with lung cancer was used to verify the screening results by reverse transcriptase real-time quantitative PCR (RT-q PCR), and the significance of urine free mi RNA in the early diagnosis of NSCLC was preliminarily investigated. First, 4 cases of NSCLC and 2 healthy human urine were studied by small RNA depth sequencing, and 10 kinds of expression water were screened. It was significantly higher than the urine free mi RNA (MI R10a, MI R-10b, MI R-155, MI R-191, MI R-192). Then, we selected 43 cases of first diagnosed patients and 26 healthy human urine samples to verify the above 10 urine free radicals. The level of the 6 kinds of MI RNA (MI R-10a, MI R-10b, MI R-191, MI R-192, MI R-21) is significantly higher than that of the healthy person. Perating characteristic, ROC) curves show mi R-26a, MI R-21, MI R-191, MI R-192, MI, 0.746, 0.679, 0.735, 0.699, 0.699 (0.699), 0.699 (0.699), 0.699 (0.699), 0.699 (0.735), 0.699 (0.699). 0.568,0.831 and 0.872 (0.787,0.956), among which, the sensitivity and specificity of MI R-21:mi R-155 ratio for NSCLC diagnosis were the highest, and 86% and 84.6%. two yuan Logistic regression analysis showed that the diagnostic efficiency of the combined urine mi RNA was obviously higher than that of the single NSCLC. Mi RNA, whose AUC is 0.927 (0.864,0.989), and the sensitivity and specificity are 92.3% and 83.7%., respectively. The results of this study show that the combination of 7 kinds of urine free mi RNA (MI R-10a, MI R-191, MI) can be used as a biomarker for auxiliary diagnosis.

【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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