血清microRNA在非小细胞肺癌辅助诊断和化疗效果评估中的应用研究

发布时间：2017-12-28 19:35

本文关键词：血清microRNA在非小细胞肺癌辅助诊断和化疗效果评估中的应用研究　出处：《北京市结核病胸部肿瘤研究所》2015年博士论文　论文类型：学位论文

【摘要】：第一部分血清micro RNA在非小细胞肺癌辅助诊断中的临床应用研究研究背景肺癌是当今世界最主要的癌症之一,每年大概有130万人死于肺癌,其死亡率居各种恶性肿瘤之首,并且75-80%的肺癌患者为非小细胞肺癌(NSCLC)。由于早期NSCLC患者无明显的临床症状,因此大约75%的患者被确诊时已经属于晚期NSCLC,失去了手术治疗的最佳时机。缺乏有效的NSCLC早期诊断手段是NSCLC患者高死亡率的主要原因,早发现、早治疗对于NSCLC患者是非常必要的。血液肿瘤标志物在肿瘤筛查中有着重要的临床价值,并且具有取材方便、创伤性小、价格相对低廉、容易复检等优点。理想的肿瘤标志物在临床诊断、预后评估和疗效监测等方面有着非常好的临床应用价值。血清微小RNA(micro RNA,mi RNA)作为新型的肿瘤标志物是当今研究的热点。mi RNA是一类内源性高度保守的非编码小RNA,长度约18-25个核苷酸,主要通过转录后机制调控靶基因的翻译或表达。有大量的研究表明,mi RNA参与各种类型肿瘤基因的表达和癌症的发生、发展,包括肺癌、乳腺癌、胃癌和直肠癌等。检测mi RNA的表达水平在癌症的诊断、治疗和预后判断中有非常大的潜在临床应用价值。已经有研究证明,mi RNA非常稳定地存在于人类血浆和血清中,并且不受RNA内切酶活性的影响,非常适合作为癌症诊断或预后判断的血液肿瘤标志物。研究目的本研究通过检测NSCLC患者、肺部良性疾病患者和健康人对照血清中mi RNA的表达情况,探讨血清mi RNA表达水平变化在NSCLC辅助诊断中的临床应用价值。研究方法1.收集2012年8月至2013年10月首都医科大学附属北京胸科医院120例NSCLC患者、45例肺部良性疾病患者和45名健康对照者血清5ml(所有血清样本采血前均未进行任何治疗),然后应用micro RNA提取试剂盒提取血清总micro RNA。2.选取NSCLC患者和健康对照者血清标本各3例,采用人类血清micro RNA One Array#174;基因芯片进行mi RNA表达谱分析,筛选在NSCLC患者与对照者血清中差异表达的mi RNA分子。3.所有血清mi RNA样本先进行逆转录反应,然后采用实时荧光定量聚合酶链反应(q RT-PCR)技术在120例NSCLC患者、45例肺部良性疾病患者和45名健康对照者样本中对差异表达的血清mi RNA分子进行独立验证。应用mi R-103做为内部参考基因,并对所有血清mi RNA样本的q RT-PCR实验数据进行标准化。4.应用受试者工作曲线(ROC曲线)分析血清mi RNA在NSCLC中的敏感性和特异性,并与其它临床常用肿瘤标志物,如癌胚抗原(CEA)以及细胞角质蛋白(CK)-3A9进行比较,评价血清mi RNA在NSCLC中的临床诊断价值。研究结果1.血清micro RNA基因芯片表达谱分析结果显示,在NSCLC血清中上调的mi RNA有26个,下调的mi RNA有24个。应用q RT-PCR方法对其中10个候选血清mi RNA进行了验证,包括5个上调mi RNA(mi R-22、mi R-125b、mi R-197、mi R-19b、mi R-27b)和5个下调mi RNA(mi R-15b、mi R-16、mi R-25、mi R-205、mi R-155)。2.q RT-PCR实验结果显示,10个候选血清mi RNA均可检测到有效表达。NSCLC患者血清中mi R-125b的表达水平显著高于正常人和肺部良性疾病组,差异有统计学意义(Z=2.238,P=0.025);NSCLC患者血清中mi R-22的表达水平显著高于正常人和肺部良性疾病组,差异有统计学意义(Z=2.129,P=0.035);NSCLC患者血清中mi R-15b的表达水平显著低于正常人和肺部良性疾病组,差异有统计学意义(Z=2.590,P=0.010)。血清mi R-125b在Ⅲ期+Ⅳ期中的表达水平显著高于Ⅰ期+Ⅱ期,差异有统计学意义(Z=2.891,P=0.004);血清mi R-15b在Ⅲ期+Ⅳ期中的表达水平显著低于Ⅰ期+Ⅱ期,差异有统计学意义(Z=2.081,P=0.040)。3.血清mi R-22对早期NSCLC(Ⅰ期+Ⅱ期)检测的敏感性(43.5%)显著高于CEA(21.7%),差异有统计学意义(χ2=4.946,P=0.026);血清mi R-15b在Ⅰ期+Ⅱ期NSCLC中检测的敏感性(41.3%)显著高于CEA,差异有统计学意义(χ2=4.079,P=0.043)。血清mi R-125b在Ⅲ期+Ⅳ期NSCLC中检测的敏感性(58.1%)显著高于Ⅰ期+Ⅱ期(30.4%),差异有统计学意义(χ2=5.875,P=0.015)。4.ROC曲线分析显示,血清mi R-22、mi R-125b和mi R-15b诊断NSCLC的ROC曲线下面积(AUC)分别为0.725(95%CI=0.623-0.827)、0.704(95%CI=0.601-0.808)、0.619(95%CI=0.536-0.702),这3种血清mi RNA对NSCLC的诊断价值均高于血清CEA(AUC=0.594,95%CI=0.482-0.707)。结论1.本研究通过血清micro RNA One Array基因芯片筛选出的10种差异表达的mi RNA分子,又经过q RT-PCR方法的验证,结果表明应用micro RNA One Array基因芯片筛选血清mi RNA差异表达谱是一种可行、可靠的方法。2.本研究结果显示,NSCLC患者血清中mi R-125b和mi R-22表达显著上调,mi R-15b表达显著下调,表明血清mi RNA作为筛查NSCLC的肿瘤标志物具有潜在的临床应用价值。3.本研究发现血清mi R-22和mi R-15b在早期NSCLC(Ⅰ期+Ⅱ期)中的敏感性显著高于CEA,表明血清mi R-22和mi R-15b有可能成为NSCLC早期诊断的参考指标。4.NSCLC患者血清中mi RNA差异性表达可能与NSCLC的形成和发展有关,血清mi RNA的检测对于NSCLC的辅助诊断和早期诊断有一定的临床应用价值。第二部分血清micro RNA在非小细胞肺癌化疗效果评估中的临床应用研究研究背景由于早期非小细胞肺癌(NSCLC)患者无明显的临床症状,大多数NSCLC患者在临床确诊时就已经属于晚期,失去了手术治疗的最佳时机。目前,针对晚期NSCLC患者的治疗主要有化学治疗或放射治疗。实践证明,联合化疗能有效地提高晚期肺癌患者的生存率。但是,一线化疗方案治疗的有效率只有30%左右,NSCLC患者对化疗药物的耐药性不仅会导致化疗失败,并且严重损害了患者的免疫系统,使患者不能及时地得到其它更好的治疗方案。因此,对化疗患者进行疗效监测是非常必要的。目前,临床上通常在NSCLC患者进行3-4个周期后采用实体瘤评价法对化疗效果进行评估,根据肿瘤体积或直径的改变来评估化疗效果的好坏。但是,实体瘤评价法不仅评价时间长,并且需要费用较高的影像学资料支持。实体瘤评价法在实际应用中还有其它的一些局限性,比如有胸腔积液、弥漫性结节以及肿瘤边界不清的NSCLC患者用这种方法容易导致不正确的评价结果。因此,目前迫切需要价格低廉、操作简便的用于评价NSCLC化疗效果的工具,这对于早期评估疗效、及时改变治疗方案以及NSCLC的个体化治疗具有非常大临床价值。目前,血液肿瘤标志物已经广泛应用于NSCLC的预后评估和疗效监测,比如癌胚抗原(CEA)、特异性神经云烯醇化酶(NSE)、细胞角质蛋白(Cyfra21-1)等。血清微小RNA(micro RNA,mi RNA)作为新的肿瘤标志物,在NSCLC预后评估和疗效监测中临床应用方面的研究刚刚起步。有大量的研究表明,mi RNA参与各种类型肿瘤基因的表达和癌症的发生、发展,检测mi RNA的表达水平在癌症的治疗和预后判断中有非常大的潜在临床应用价值。研究目的本研究通过检测晚期(Ⅲ期+Ⅳ期)NSCLC患者化疗前和化疗后血清中miRNA的表达量变化情况,分析血清mi RNA浓度变化与化疗效果是否存在关联,探讨血清mi RNA表达水平变化在NSCLC化疗效果评估中的临床应用价值。研究方法1.收集2012年8月至2013年10月首都医科大学附属北京胸科医院74例晚期NSCLC患者化疗前和化疗后2周期血清各5ml,然后应用mi RNA提取试剂盒提取血清总mi RNA。2.总计144例血清mi RNA样本先进行逆转录反应,然后采用实时荧光定量聚合酶链反应(q RT-PCR)技术检测在本课题第一部分筛选并验证的在NSCLC患者中异常表达的血清mi RNA分子(mi R-125b、mi R-22、mi R-15b)的表达量。应用mi R-103做为内部参考基因,并对144例血清mi RNA样本的q RT-PCR实验数据进行标准化。3.化疗效果评估采用世界卫生组织(WHO)推荐的实体瘤评价法,评价结果分为:完全缓解(CR),部分缓解(PR),疾病稳定(SD)和疾病进展(PD)。4.分析晚期NSCLC患者化疗前和化疗后血清中mi RNA的表达水平变化,评价血清mi RNA在晚期NSCLC化疗效果评估中的临床应用价值。研究结果1.74例晚期NSCLC患者经过2个周期的化疗后,应用实体瘤疗效评价法(RECIST)对化疗效果进行评估:完全缓解0例,部分缓解34例(占45.9%),疾病稳定33例(占44.6%)和疾病进展7例(占9.5%)。2.与化疗前比较,晚期(Ⅲ期+Ⅳ期)NSCLC患者经过2个周期的化疗后,血清中mi R-125b的表达量明显降低,差异有统计学意义(Z=2.196,P=0.028);而血清中mi R-15b的表达量明显升高,差异有统计学意义(Z=2.650,P=0.008);血清中mi R-22的表达量无明显变化,差异无统计学意义(P0.05)。3.与化疗前比较,晚期NSCLC患者经过2个周期的化疗后,化疗敏感组患者(CR+PR)血清中mi R-125b的表达量明显降低,差异有统计学意义(Z=2.128,P=0.033);化疗敏感组患者血清中mi R-15b的表达量明显升高,差异有统计学意义(Z=2.794,P=0.005);而化疗不敏感组患者(SD+PD)血清中mi R-125b和mi R-15b的表达量无明显变化,差异无统计学意义(P0.05)。4.研究结果显示,晚期NSCLC患者化疗效果与化疗前血清中mi RNA的表达量密切相关联。在血清mi R-125b呈高表达的NSCLC患者中,有34.9%的患者对化疗敏感(CR+PR);而在血清mi R-125b呈低表达的NSCLC患者中,有51.6%的患者对化疗敏感,这种差异具有统计学意义(χ2=5.879,P=0.015)。在血清mi R-15b呈低表达的NSCLC患者中,有37.5%的患者对化疗敏感;而在血清mi R-15b呈高表达的NSCLC患者中,有52.9%的患者对化疗敏感,这种差异有统计学意义(χ2=4.795,P=0.029)。结论1.本研究结果显示,晚期NSCLC患者化疗前和化疗后血清中mi R-125b和mi R-15b的表达水平有显著的变化,并且与化疗效果评价相关联,表明血清mi RNA的检测对于晚期NSCLC的化疗疗效评价具有潜在的临床应用价值。2.晚期NSCLC患者化疗前血清mi RNA的表达水平与疗效评价密切相关,根据晚期NSCLC患者化疗前血清mi R-125b和mi R-15b的表达水平,可以用来判断化疗患者的预后情况。
[Abstract]:The first part of the clinical application of serum micro RNA background lung cancer in non small cell lung cancer diagnosis is one of the most important cancer of the world today, there are about 1 million 300 thousand people die of lung cancer each year, the mortality rate among all malignant tumors, and 75-80% lung cancer patients for non-small cell lung cancer (NSCLC). Since early NSCLC patients have no obvious clinical symptoms, about 75% of the patients were diagnosed as late NSCLC and lost the best time for surgical treatment. The lack of effective early diagnosis of NSCLC is the main cause of high mortality in NSCLC patients. Early detection and early treatment are essential for patients with NSCLC. Blood tumor markers have important clinical value in tumor screening, and have the advantages of convenient material, small trauma, relatively low price and easy reexamination. The ideal tumor markers are of great clinical value in clinical diagnosis, prognosis evaluation and therapeutic monitoring. As a new tumor marker, the serum RNA (micro RNA, MI RNA) is a hot spot of research. Mi RNA is a class of endogenous highly conserved non coded small RNA, with a length of about 18-25 nucleotides, which mainly regulate the translation or expression of target genes through post transcriptional mechanism. A large number of studies have shown that MI RNA participates in the expression of various types of tumor genes and the occurrence and development of cancer, including lung cancer, breast cancer, gastric cancer and rectal cancer. The detection of the expression level of MI RNA has a great potential clinical value in the diagnosis, treatment and prognosis of cancer. Studies have shown that MI RNA is very stable in human plasma and serum, and is not affected by the activity of RNA endonuclease. It is very suitable for cancer diagnosis or prognosis of blood tumor markers. The purpose of this study is to detect the expression of MI RNA in serum of patients with NSCLC, pulmonary benign diseases and healthy controls, and to explore the clinical application value of serum mi RNA expression level in NSCLC aided diagnosis. Methods 1. from August 2012 to October 2013 in Beijing Thoracic Hospital of Capital Medical University from 120 patients with NSCLC, 45 patients with benign lung disease and 45 healthy controls serum 5ml (all serum samples from the blood before them were without any treatment), and then apply the micro RNA extraction kit to extract serum total micro RNA. 2. select 3 serum samples from NSCLC patients and healthy controls. We used human serum micro RNA One Array#174, gene chip to analyze mi RNA expression profiles, and screen mi RNA molecules expressed in serum of NSCLC patients and controls. All 3. serum mi RNA samples before reverse transcription reaction, then by real-time fluorescence quantitative polymerase chain reaction (Q RT-PCR) in 120 NSCLC patients, 45 patients with benign lung disease and 45 healthy controls serum mi RNA expression of differences in the sample were validated independently. Mi R-103 was used as an internal reference gene and the Q RT-PCR experimental data of all serum mi RNA samples were standardized. 4. application of receiver operating characteristic curve (ROC curve) analysis of the sensitivity and specificity of serum mi RNA in NSCLC, and markers and other clinical common tumors, such as carcinoembryonic antigen (CEA) and cytokeratin (CK) -3A9 were compared to evaluate the clinical diagnostic value of serum mi RNA in NSCLC. Results 1. serum micro RNA gene chip expression profiles showed that there were 26 mi RNA up-regulated in NSCLC serum, and 24 down-regulated mi RNA. 10 candidate serum mi RNA were verified by Q RT-PCR method, including 5 upregulated mi RNA (MI R-22, MI R-125b, MI R-125b, MI, 5). The results of 2.q RT-PCR experiment showed that 10 candidate serum mi RNA could be effectively expressed. The expression level in the serum of patients with NSCLC mi R-125b was significantly higher than that in normal and benign lung disease group, the difference was statistically significant (Z=2.238, P=0.025); the expression level in the serum of patients with NSCLC mi R-22 was significantly higher than that in normal and benign lung disease group, the difference was statistically significant (Z=2.129, P=0.035); the expression level of serum NSCLC mi R-15b was significantly lower than that in normal and benign lung disease group, the difference was statistically significant (Z=2.590, P=0.010). The expression level of serum mi R-125b in stage III + IV is significantly higher than that in stage I + phase II (Z=2.891, P=0.004). The expression level of serum mi R-15b in stage III + IV is significantly lower than that in stage I + phase II, with statistically significant difference (Z=2.081, P=0.040). 3. serum mi R-22 in early NSCLC (stage I + II) detection sensitivity (43.5%) was significantly higher than that of CEA (21.7%), the difference was statistically significant (2=4.946, P=0.026); the sensitivity of serum mi R-15b in stage I + II detection in NSCLC (41.3%) was significantly higher than that of CEA, the difference was statistically meaning (x 2=4.079, P=0.043). The sensitivity of serum mi R-125b in stage III + IV NSCLC (58.1%) was significantly higher than that in phase I + II (30.4%), and the difference was statistically significant (x 2=5.875, P=0.015). 4.ROC curve analysis showed that the area of MI R-22, the MI curve of serum ROC R-125b and MI R-15b NSCLC (AUC) at diagnosis were 0.725 (95%CI=0.623-0.827), 0.704 (95%CI=0.601-0.808), 0.619 (95%CI=0.536-0.702), the value of these 3 kinds of serum mi RNA for diagnosis of NSCLC were higher in serum CEA (AUC=0.594,95%CI=0.482-0.707). Conclusion Mi expression of RNA 10 between the 1. through the study of serum micro RNA One Array gene chips were screened, and validated by Q RT-PCR method. The results show that the application of micro RNA One Array gene chips to screen the differences of serum Mi expression profile of RNA is a feasible and reliable method. 2., the results of this study showed that the expressions of MI R-125b and MI R-22 in NSCLC patients were significantly up-regulated, and the expression of MI R-15b was significantly down regulated, indicating that serum mi RNA was used as a tumor marker for screening NSCLC.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

【参考文献】