MicroRNA-320c、MicroRNA-451a、MicroRNA-486在食管鱗癌及癌前病變患者血清中的表達(dá)及其
發(fā)布時(shí)間：2019-06-20 07:47
【摘要】：研究目的研究微小RNA(microRNA,miRNA)在食管鱗癌及其癌前病變患者血清中的表達(dá)情況,探索其作為初篩標(biāo)志物的可行性。材料和方法1.篩選階段:收集食管鱗癌患者血清,開(kāi)展血清差異miRNAs篩選,miRNAs芯片檢測(cè)及結(jié)果分析由上海生物芯片有限公司完成。芯片雜交原始數(shù)據(jù)經(jīng)均一化和對(duì)數(shù)轉(zhuǎn)換后進(jìn)行差異顯著性分析,選取差異倍數(shù)(Fold Change,FC)絕對(duì)值2(上調(diào))或者0.5(下調(diào))的miRNAs,結(jié)合文獻(xiàn)報(bào)道有生物學(xué)功能的作為候選差異miRNAs。2.驗(yàn)證階段:選取食管鱗癌患者及癌前病變患者血清開(kāi)展驗(yàn)證,同時(shí)收集危險(xiǎn)因素暴露及臨床相關(guān)信息。采用qRT-PCR技術(shù)檢測(cè)miRNAs的表達(dá)水平。將原始數(shù)據(jù)取3次重復(fù)的平均值,以miR-1228為內(nèi)參,進(jìn)行歸一化,使用定量PCR中的相對(duì)定量法,得出△Ct,miRNAs表達(dá)量用2-△Ct表示。3.實(shí)時(shí)定量PCR數(shù)據(jù)用SPSS 22.0軟件進(jìn)行統(tǒng)計(jì)分析。首先進(jìn)行描述性分析,計(jì)算一般人口學(xué)資料主要指標(biāo),對(duì)其進(jìn)行均衡性檢驗(yàn),對(duì)于計(jì)數(shù)資料對(duì)比采用χ2檢驗(yàn),正態(tài)性計(jì)量資料采用t檢驗(yàn)或方差分析。miRNAs表達(dá)量數(shù)據(jù)為非正態(tài)數(shù)據(jù),通過(guò)SPSS正態(tài)得分對(duì)miRNAs表達(dá)量數(shù)據(jù)進(jìn)行正態(tài)轉(zhuǎn)化,正態(tài)轉(zhuǎn)化前miRNAs表達(dá)水平以“p50(p25,p75)”形式置于表中,正態(tài)轉(zhuǎn)化后數(shù)據(jù)以“均數(shù)±標(biāo)準(zhǔn)差”形式置于表中。應(yīng)用多元logistics回歸分析方法分析組間miRNAs的差異表達(dá),以“組別”為因變量,“組間差異因素”及各miRNAs表達(dá)量正態(tài)轉(zhuǎn)換值為自變量,采用逐步向前法,分析組間miRNAs差異表達(dá),同時(shí)篩選自變量。所篩選的差異自變量應(yīng)用于后續(xù)構(gòu)建多變量觀察值ROC曲線。應(yīng)用二元logistics回歸模型及ROC曲線進(jìn)行多變量(多項(xiàng)測(cè)量指標(biāo))觀察值的ROC曲線分析。采用逐步向前法,將多元logistics回歸分析階段篩選的差異自變量納入二元logistic回歸模型,計(jì)算求出預(yù)測(cè)值概率,將此概率納入ROC模型,繪制受試者工作特征曲線(ROC),計(jì)算曲線下面積(AUC),AUC:0.5-0.7,較低準(zhǔn)確性;AUC:0.7-0.9,有一定準(zhǔn)確性;AUC0.9,較高準(zhǔn)確性;AUC=0.5,無(wú)診斷價(jià)值。根據(jù)ROC曲線確定臨界值,并根據(jù)臨界值確定其靈敏度(Sen)、特異度(Spe)。比較單項(xiàng)miRNA指標(biāo)與聯(lián)合miRNAs指標(biāo)的診斷效能。同時(shí),將總體分為試驗(yàn)組與驗(yàn)證組,分別計(jì)算并對(duì)比其診斷價(jià)值,評(píng)估m(xù)iRNAs診斷價(jià)值穩(wěn)定性。采用雙側(cè)檢驗(yàn),檢驗(yàn)水準(zhǔn)為 α=0.05。研究結(jié)果1.miRNAs 芯片篩選出 7 個(gè)候選差異miRNAs:miR-451a、miR-638、miR-486-5p、miR-16-5p、miR-92a-3p、miR-197-5p、miR-320c。本研究在人群驗(yàn)證 miR-320c、miR-451a、miR-486。2.食管鱗癌組、異型增生組及對(duì)照組兩兩組間血清miR-320c均差異表達(dá),miR-320c在食管鱗癌組及異型增生組血清中高表達(dá),且異型增生組中miR-320c表達(dá)水平高于食管癌組;異型增生組血清miR-451a、miR-486分別與食管鱗癌組、對(duì)照組間差異表達(dá),且miR-451a、miR-486在異型增生組血清中高表達(dá)。食管癌組、異型增生組組內(nèi)比較結(jié)果:食管癌患者不同發(fā)病部位、TNM分期、分化程度、病變分型組間血清miR-320c、miR-451a、miR-486差異表達(dá)無(wú)統(tǒng)計(jì)學(xué)意義;各級(jí)別異型增生組間血清miR-320c、miR-451a、miR-486表達(dá)也無(wú)統(tǒng)計(jì)學(xué)差異。3.總體血清miR-320c在食管癌組與對(duì)照組間診斷食管癌患者的ROC曲線下面積AUC為0.867(0.813-0.922)。當(dāng)臨界值取0.56時(shí),靈敏度為81.3%,特異度為80.8%�？傮w血清miR-320c、miR-451a、miR-486單項(xiàng)或聯(lián)合應(yīng)用均可在異型增生組與對(duì)照組間診斷異型增生患者,血清miR-320c與三種miRNAs聯(lián)合診斷價(jià)值相對(duì)較高,血清miR-320c診斷ROC曲線下面積AUC為0.785(0.716-0.854),當(dāng)臨界值取0.56時(shí),靈敏度為70.7%,特異度為79.5%。三種miRNAs聯(lián)合診斷ROC曲線下面積AUC為0.782(0.713-0.852)。當(dāng)臨界值取0.56時(shí),靈敏度為70.7%,特異度為78.2%�？傮w血清miR-320c、miR-451a、miR-486單項(xiàng)或聯(lián)合應(yīng)用均可在食管癌組與異型增生組間診斷食管癌患者,三種miRNAs聯(lián)合診斷ROC曲線下面積最高,診斷準(zhǔn)確性最好,且具有較高診斷價(jià)值,其ROC曲線下面積AUC為0.813(0.753-0.872)。當(dāng)臨界值取0.49時(shí),靈敏度為78.1%,特異度為70.7%。結(jié)論1.血清miR-320c、miR-451a、miR-486單項(xiàng)或聯(lián)合應(yīng)用可區(qū)分食管鱗癌組、異型增生組與對(duì)照組;2.血清miR-320c、miR-451a、miR-486單項(xiàng)或聯(lián)合應(yīng)用對(duì)食管癌及其癌前病變具有較好診斷價(jià)值,準(zhǔn)確性較高,且具有穩(wěn)定性,可作為初篩的潛在應(yīng)用指標(biāo);3.未發(fā)現(xiàn)血清miR-320c、miR-451a、miR-486差異表達(dá)與腫瘤患者發(fā)病部位、TNM分期、分化程度、病變分型及各級(jí)別異型增生之間的關(guān)聯(lián),仍需更大樣本量的深入探討;
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1
本文編號(hào)：2503054
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1
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