MicroRNA-320c、MicroRNA-451a、MicroRNA-486在食管鳞癌及癌前病变患者血清中的表达及其

发布时间：2019-06-20 07:47

【摘要】：研究目的研究微小RNA(microRNA,miRNA)在食管鳞癌及其癌前病变患者血清中的表达情况,探索其作为初筛标志物的可行性。材料和方法1.筛选阶段:收集食管鳞癌患者血清,开展血清差异miRNAs筛选,miRNAs芯片检测及结果分析由上海生物芯片有限公司完成。芯片杂交原始数据经均一化和对数转换后进行差异显著性分析,选取差异倍数(Fold Change,FC)绝对值2(上调)或者0.5(下调)的miRNAs,结合文献报道有生物学功能的作为候选差异miRNAs。2.验证阶段:选取食管鳞癌患者及癌前病变患者血清开展验证,同时收集危险因素暴露及临床相关信息。采用qRT-PCR技术检测miRNAs的表达水平。将原始数据取3次重复的平均值,以miR-1228为内参,进行归一化,使用定量PCR中的相对定量法,得出△Ct,miRNAs表达量用2-△Ct表示。3.实时定量PCR数据用SPSS 22.0软件进行统计分析。首先进行描述性分析,计算一般人口学资料主要指标,对其进行均衡性检验,对于计数资料对比采用χ2检验,正态性计量资料采用t检验或方差分析。miRNAs表达量数据为非正态数据,通过SPSS正态得分对miRNAs表达量数据进行正态转化,正态转化前miRNAs表达水平以“p50(p25,p75)”形式置于表中,正态转化后数据以“均数±标准差”形式置于表中。应用多元logistics回归分析方法分析组间miRNAs的差异表达,以“组别”为因变量,“组间差异因素”及各miRNAs表达量正态转换值为自变量,采用逐步向前法,分析组间miRNAs差异表达,同时筛选自变量。所筛选的差异自变量应用于后续构建多变量观察值ROC曲线。应用二元logistics回归模型及ROC曲线进行多变量(多项测量指标)观察值的ROC曲线分析。采用逐步向前法,将多元logistics回归分析阶段筛选的差异自变量纳入二元logistic回归模型,计算求出预测值概率,将此概率纳入ROC模型,绘制受试者工作特征曲线(ROC),计算曲线下面积(AUC),AUC:0.5-0.7,较低准确性;AUC:0.7-0.9,有一定准确性;AUC0.9,较高准确性;AUC=0.5,无诊断价值。根据ROC曲线确定临界值,并根据临界值确定其灵敏度(Sen)、特异度(Spe)。比较单项miRNA指标与联合miRNAs指标的诊断效能。同时,将总体分为试验组与验证组,分别计算并对比其诊断价值,评估miRNAs诊断价值稳定性。采用双侧检验,检验水准为 α=0.05。研究结果1.miRNAs 芯片筛选出 7 个候选差异miRNAs:miR-451a、miR-638、miR-486-5p、miR-16-5p、miR-92a-3p、miR-197-5p、miR-320c。本研究在人群验证 miR-320c、miR-451a、miR-486。2.食管鳞癌组、异型增生组及对照组两两组间血清miR-320c均差异表达,miR-320c在食管鳞癌组及异型增生组血清中高表达,且异型增生组中miR-320c表达水平高于食管癌组;异型增生组血清miR-451a、miR-486分别与食管鳞癌组、对照组间差异表达,且miR-451a、miR-486在异型增生组血清中高表达。食管癌组、异型增生组组内比较结果:食管癌患者不同发病部位、TNM分期、分化程度、病变分型组间血清miR-320c、miR-451a、miR-486差异表达无统计学意义;各级别异型增生组间血清miR-320c、miR-451a、miR-486表达也无统计学差异。3.总体血清miR-320c在食管癌组与对照组间诊断食管癌患者的ROC曲线下面积AUC为0.867(0.813-0.922)。当临界值取0.56时,灵敏度为81.3%,特异度为80.8%。总体血清miR-320c、miR-451a、miR-486单项或联合应用均可在异型增生组与对照组间诊断异型增生患者,血清miR-320c与三种miRNAs联合诊断价值相对较高,血清miR-320c诊断ROC曲线下面积AUC为0.785(0.716-0.854),当临界值取0.56时,灵敏度为70.7%,特异度为79.5%。三种miRNAs联合诊断ROC曲线下面积AUC为0.782(0.713-0.852)。当临界值取0.56时,灵敏度为70.7%,特异度为78.2%。总体血清miR-320c、miR-451a、miR-486单项或联合应用均可在食管癌组与异型增生组间诊断食管癌患者,三种miRNAs联合诊断ROC曲线下面积最高,诊断准确性最好,且具有较高诊断价值,其ROC曲线下面积AUC为0.813(0.753-0.872)。当临界值取0.49时,灵敏度为78.1%,特异度为70.7%。结论1.血清miR-320c、miR-451a、miR-486单项或联合应用可区分食管鳞癌组、异型增生组与对照组;2.血清miR-320c、miR-451a、miR-486单项或联合应用对食管癌及其癌前病变具有较好诊断价值,准确性较高,且具有稳定性,可作为初筛的潜在应用指标;3.未发现血清miR-320c、miR-451a、miR-486差异表达与肿瘤患者发病部位、TNM分期、分化程度、病变分型及各级别异型增生之间的关联,仍需更大样本量的深入探讨;
[Abstract]:Objective To study the expression of microRNA (miRNA) in the serum of esophageal squamous cell carcinoma and its precancerous lesions, and to explore its feasibility as a primary screen marker. Materials and methods 1. Screening stage: collecting serum of esophageal squamous cell carcinoma, screening the serum difference miRNAs, detecting and analyzing the miRNAs chip, and analyzing the results to be completed by the Shanghai Biochip Co., Ltd. After homogenization and log-conversion of the hybrid raw data of the chip, the difference significance analysis was carried out, and the miRNAs of the difference multiple (fold change, FC) absolute value 2 (up-regulation) or 0.5 (down-regulation) were selected, and the biological function as the candidate difference miRNAs was reported in the binding document. Validation phase: The serum of patients with esophageal squamous cell carcinoma and pre-cancerous lesions was selected for verification, and the exposure of risk factors and the relevant clinical information were also collected. The expression level of miRNAs was detected by qRT-PCR. The raw data were averaged three times, and the miR-1228 was used as the internal reference for normalization. Using the relative quantitative method in the quantitative PCR, the expression quantity of miRNAs and miRNAs was expressed by 2-BCt. The real-time quantitative PCR data was analyzed by SPSS 22.0 software. First, the descriptive analysis was carried out to calculate the main indexes of general demographic data, and the balanced test was carried out. For the comparison of the counting data, the second test was adopted, and the positive and negative measurement data were t-test or variance analysis. The data of miRNAs expression is non-normal data, and the data of miRNAs expression is normal transformed by using the SPSS normal score, and the expression level of the miRNAs before normal transformation is placed in a table in a "p50(p25,p75)" form, and the normal transformed data is placed in a table in a "mean square standard deviation" form. The differential expression of miRNAs between the two groups was analyzed by the multi-factor logistic regression analysis method. The "Group" was the independent variable and the normal conversion value of the "Inter-group difference factor" and each of the miRNAs was the independent variable. The differential expression of miRNAs between the groups was analyzed by a step-by-step method, and the independent variables were also selected. The selected difference argument is applied to the subsequent construction of the multi-variable observation value ROC curve. The ROC curve of the multi-variable (multiple measurement index) observation was analyzed by using the binary logistic regression model and the ROC curve. The method comprises the following steps: a step-by-step forward method is adopted, the difference independent variable screened by the multivariate logistic regression analysis stage is included in a binary logistic regression model, the probability of the predicted value is calculated, the probability is included in the ROC model, the working characteristic curve (ROC) of the subject is drawn, the area under the curve (AUC) is calculated, the AUC is 0.5 to 0.7, Lower accuracy; AUC: 0.7-0.9, with certain accuracy; AUC0-9, higher accuracy; AUC = 0.5, no diagnostic value. The critical value is determined according to the ROC curve and its sensitivity (Sen), specificity (Spe) are determined according to the critical value. To compare the diagnostic efficacy of the single miRNA index and the combined miRNAs index. At the same time, it is divided into the test group and the verification group, and the diagnostic value of the test group is calculated and compared, and the diagnostic value stability of the miRNAs is evaluated. Two-sided test was used, and the test level was 1 = 0.05. The results of the study 1. miRNAs chip screened seven candidate difference miRNAs: miR-451a, miR-638, miR-486-5p, miR-16-5p, miR-92a-3p, miR-197-5p, and miR-320c. The study demonstrated miR-320c, miR-451a, and miR-486.2 in the population. The expression of miR-320c in the two groups of the esophageal squamous cell carcinoma group, the special-shaped hyperplasia group and the control group is different, and the miR-320c is high in the serum of the esophageal squamous cell carcinoma group and the special-shaped hyperplasia group, and the expression level of the miR-320c in the special-shaped hyperplasia group is higher than that of the esophageal cancer group, and the serum miR-451a and the miR-486 in the special-shaped hyperplasia group are respectively connected with the esophageal squamous cell carcinoma group, The difference was expressed in the control group, and the miR-451a and the miR-486 were expressed in the serum of the abnormal hyperplasia group. There was no significant difference in the expression of miR-320c, miR-451a and miR-486 in the different parts of esophageal carcinoma, TNM stage and degree of differentiation. The overall serum miR-320c had an area under the ROC curve of 0.867 (0.813-0.922) in the patients with esophageal cancer between the esophageal cancer group and the control group. When the critical value was 0.56, the sensitivity was 81.3% and the specificity was 80.8%. The total serum miR-320c, miR-451a, and miR-486 single or combined application can be used for diagnosing the abnormal hyperplasia in the abnormal hyperplasia group and the control group, the combined diagnosis value of the serum miR-320c and the three miRNAs is relatively high, the area under the ROC curve of the serum miR-320c is 0.785 (0.716-0.854), when the critical value is 0.56, the sensitivity is 70.7 percent, The specificity is 79.5%. The area under the ROC curve for three miRNAs was 0.782 (0.713-0.852). When the critical value was 0.56, the sensitivity was 70.7% and the specificity was 78.2%. The overall serum miR-320c, miR-451a, and miR-486 single or combined application can be used for the diagnosis of esophageal cancer patients in the esophageal cancer group and the abnormal hyperplasia group, the area of the three miRNAs in the joint diagnosis ROC curve is the highest, the diagnosis accuracy is the best, and the diagnostic value is high, and the area under the ROC curve of the three miRNAs is 0.813 (0.753-0.872). When the critical value was 0.49, the sensitivity was 78.1% and the specificity was 70.7%. Conclusion 1. Serum miR-320c, miR-451a and miR-486 can be used for distinguishing esophageal squamous cell carcinoma group, abnormal hyperplasia group and control group. The serum miR-320c, miR-451a and miR-486 can be used for single or combined application to have good diagnostic value for esophageal cancer and precancerous lesions, and has high accuracy and stability, and can be used as a potential application index of a primary screen; and 3. The relationship between the differential expression of miR-320c, miR-451a, miR-486 and the incidence of tumor, TNM stage, degree of differentiation, type of lesion and heterotypic proliferation at all levels was not found.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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