任艷 高軍 王小瑋 劉建強(qiáng) 顧俊駿 黃浩杰 金晶 龔燕芳 李兆申

·論著·

糞便microRNAs檢測(cè)用于胰腺癌篩查診斷的價(jià)值評(píng)價(jià)

任艷 高軍 王小瑋 劉建強(qiáng) 顧俊駿 黃浩杰 金晶 龔燕芳 李兆申

目的檢測(cè)胰腺癌患者糞便microRNAs，評(píng)價(jià)其診斷價(jià)值。方法收集29例胰腺癌患者、22例慢性胰腺炎(CP)患者以及13例健康志愿者的糞便標(biāo)本，抽提糞便總RNA，應(yīng)用實(shí)時(shí)定量PCR法檢測(cè)各組樣本miR-21、miR-155、miR-181a、miR-181b、miR-196a、miR-210的表達(dá)量，以miR-16作為內(nèi)參基因。應(yīng)用接受者操作特征(ROC)曲線(AUC)評(píng)估m(xù)icroRNAs對(duì)胰腺癌的診斷價(jià)值。結(jié)果糞便總RNA抽提及microRNAs檢測(cè)方法具有穩(wěn)定及可重復(fù)性。胰腺癌組miR-181b、miR-196a、miR-210的表達(dá)量分別為2.22±0.64、2.78±0.14、5.55±0.38；CP組為1.42±0.39、3.88±0.85、5.39±0.69；對(duì)照組為0.32±0.40、1.14±0.98、4.23±0.99。胰腺癌組和CP組均較對(duì)照組顯著增加(P值均<0.05)；而胰腺癌組與CP組間無顯著差異。胰腺癌組對(duì)對(duì)照組的miR-181b AUC為0.745(95%CI0.597～0.894)，診斷胰腺癌的敏感性和特異性分別為84.6%和51.7%；miR-210的AUC為0.772(95%CI0.629～0.914)，對(duì)胰腺癌的診斷敏感性和特異性分別為84.6%和65.5%。兩組比較差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.05)。miR-196a對(duì)胰腺癌無診斷意義，但胰腺癌患者糞便miR-196a的表達(dá)與腫瘤直徑相關(guān)(r=0.516,P=0.041)。結(jié)論糞便RNA的抽提和microRNAs檢測(cè)為無創(chuàng)性，且具有可重復(fù)性。miR-181b和 miR-210在胰腺癌患者糞便中的表達(dá)增高，有可能是胰腺癌潛在的分子標(biāo)志物。

胰腺腫瘤； 微RNAs； 糞便； 診斷

胰腺癌的發(fā)病率在國內(nèi)外均逐年升高，但由于解剖位置關(guān)系以及缺乏特異有效的檢查手段，胰腺癌的早期診斷仍相當(dāng)困難[1]。 近年來，大量的研究結(jié)果表明microRNA(miRNA)與腫瘤的發(fā)生和發(fā)展存在密切的聯(lián)系[2]，miRNA已成為近幾年來分子生物學(xué)和遺傳學(xué)領(lǐng)域的研究熱點(diǎn)。本實(shí)驗(yàn)旨在尋找一種新的、采用無創(chuàng)手段檢測(cè)的胰腺癌分子標(biāo)志物。

材料與方法

一、材料

收集長海醫(yī)院2008年1月至2010年3月住院的29例胰腺癌患者、22例慢性胰腺炎(CP)患者糞便，另取13例健康志愿者糞便作為對(duì)照。胰腺癌根據(jù)病理診斷或者臨床診斷[3]。所有患者均簽屬知情同意書。糞便標(biāo)本收集后立即置-80℃冰箱保存。

二、糞便RNA提取

使用E.Z.N.A糞便提取試劑盒(Omega，美國)抽提糞便總RNA(包含miRNA)，按說明書操作。抽提的RNA用分光光度計(jì)測(cè)濃度及鑒定純度，1%瓊脂糖凝膠電泳分離觀察條帶。樣本置-80℃保存。

三、miRNA檢測(cè)

根據(jù)以往文獻(xiàn)報(bào)道[4-5]，我們選擇miR-21、miR-155、miR-181a、 miR-181b、miR-196a、miR-210 6個(gè)miRNA 進(jìn)行檢測(cè)。依據(jù)Genorm軟件以及Link等[6]報(bào)道，選擇miR-16作為內(nèi)參。

miRNAs的定量檢測(cè)采用Taqman MicroRNAs檢測(cè)試劑盒(Applied Biosystems公司),引物由ABI公司設(shè)計(jì)。RT反應(yīng)條件：16℃ 30 min，42℃ 30 min；80℃ 5 min。實(shí)時(shí)PCR反應(yīng)條件：95℃ 10 min， 95℃ 15 s，55個(gè)循環(huán)，60℃ 15 s。利用ABI 7500 Real Time PCR儀SDS 2.1 software(Applied Biosystems, USA)得到各個(gè)miRNA的Ct值，△△Ct=(Ct樣本-Ct樣本 miR-16)-(Ct對(duì)照-Ct對(duì)照miR-16)，相對(duì)表達(dá)豐度值(RQ)=2-△△Ct。應(yīng)用ROC曲線(AUC)評(píng)估m(xù)iRNA對(duì)胰腺癌的診斷價(jià)值。

四、統(tǒng)計(jì)分析

結(jié) 果

一、糞便RNA抽提的重復(fù)性及miRNA檢測(cè)的重復(fù)性

200 g糞便可抽提到總RNA量為19 315～69 995 ng，對(duì)6個(gè)樣本進(jìn)行重復(fù)抽提，兩次miRNAs檢測(cè)結(jié)果均呈線性關(guān)系(R2=0.998及R2=0.988,P<0.001，圖1)。

圖1 RNA抽提(a)及miRNA檢測(cè)(b)可重復(fù)性檢測(cè)線性圖

二、糞便miRNAs表達(dá)量

胰腺癌組miR-181b、miR-196a、miR-210的表達(dá)量分別為2.22±0.64、2.78±0.14、5.55±0.38；CP組為1.42±0.39、3.88±0.85、5.39±0.69；對(duì)照組為0.32±0.40、1.14±0.98、4.23±0.99。胰腺癌組較對(duì)照組顯著增加(P值均<0.05)；CP組也較對(duì)照組顯著增加(P值均<0.05)；而胰腺癌組與CP組間無顯著差異。三組miR-155、miR-181a、miR-21的表達(dá)量均無顯著差異。

三、糞便miRNAs的診斷價(jià)值

miR-181b、miR-196a、miR-210對(duì)胰腺癌的診斷價(jià)值見表1。

表1 糞便miR-181b、miR-196a、miR-210的AUC和診斷敏感性、特異性

四、胰腺癌miRNAs表達(dá)與臨床參數(shù)的關(guān)系

胰腺癌miR-181b、miR-210的表達(dá)與患者年齡、性別、體重、吸煙指數(shù)、胰管直徑、TNM分期以及血清CEA、CA19-9、ALT、AST、總膽紅素、血糖等均無關(guān)，僅miR-196a表達(dá)與腫瘤最大直徑呈正相關(guān)(r=0.736，P=0.041)。

討 論

近年研究認(rèn)為，miRNAs在人類惡性腫瘤中表達(dá)異常[2]，miRNAs的表達(dá)譜可以較為準(zhǔn)確地反映腫瘤的組織來源和分化狀態(tài)，并可以根據(jù)miRNA的表達(dá)譜對(duì)多種低分化腫瘤進(jìn)行分類，在腫瘤診斷和預(yù)后評(píng)估中具有重要價(jià)值[7]。目前已有乳腺、肺、食管、前列腺等腫瘤miRNAs表達(dá)譜的報(bào)道，并且認(rèn)為miRNAs的表達(dá)與腫瘤分期、血管轉(zhuǎn)移等組織學(xué)特性有關(guān)。

Lee等[4]報(bào)道，miR-21、miR-155、miR-181a在胰腺癌組織中表達(dá)高于正常胰腺組織，Dillhoff等[5]報(bào)道，miR-21是一個(gè)潛在的分子標(biāo)志物，并且與患者生存期有關(guān)。Bloomston等[8]等報(bào)道，胰腺癌組織miR-21、miR-155、miR-181a、miR-181b、miR-210表達(dá)高于相應(yīng)癌旁組織。我們先前檢測(cè)了胰腺癌患者和CP患者血清中miR-16、miR-155、miR-181a、miR-181b、miR-21、miR-196a、miR-221、miR-222的表達(dá)，結(jié)果顯示miR-21、miR-155、miR-196a在胰腺疾病中高表達(dá)。

本實(shí)驗(yàn)檢測(cè)糞便中miRNAs表達(dá)。糞便易獲取，無創(chuàng)性，抽提的總RNA重復(fù)性好，同時(shí)，miRNAs檢測(cè)的重復(fù)性也好，因此是腫瘤篩查的理想標(biāo)本。

本實(shí)驗(yàn)結(jié)果顯示，胰腺癌患者糞便中miR-181b、miR-210、miR-196a表達(dá)較對(duì)照組顯著增加。這一結(jié)果與以往研究的血清標(biāo)本結(jié)果不完全一致，可能與糞便中miRNAs的來源與血液中不同相關(guān)。第一，脫落細(xì)胞來源于腫瘤組織，混合在胰液內(nèi)分泌入消化道。第二，腫瘤來源的分泌小泡進(jìn)入血液循環(huán)，經(jīng)由消化道進(jìn)入糞便[9]。

[1] Real FX.A "catastrophic hypothesis" for pancreas cancer progression.Gastroenterology,2003,124:1958-1964.

[2] Volinia S,Calin GA,Liu CG,et al.A microRNA expression signature of human solid tumors defines cancer gene targets.Proc Natl Acad Sci USA,2006,103:2257-2261.

[3] Kawarada Y.New classification of pancreatic carcinoma-Japan Pancreas Society.Nippon Shokakibyo Gakkai Zasshi,2003,100:974-80.

[4] Lee EJ,Guser Y, Jiang J,et al.Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer,2007,120:1046-1054.

[5] Dillhoff M,Liu J,Frankel W,et al. MicroRNA-21 is overexpressed in pancreatic cancer and a potential predictor of survival. J Gastrointest Surg,2008,12:2171-2176.

[6] Link A,Balaguer F,Shen Y,et al.Fecal microRNAs as novel biomarkers for colon cancer screening. Cancer Epidemiol Biomarkers Prev,2010,19:1766-1774.

[7] Lu J,Getz G,Miska EA,et al.MicroRNA expression profiles classify human cancers.Nature, 2005,435:834-838.

[8] Bloomston M,Frankel WL,Petrocca F,et al.MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA,2007,297:1901-1908.

[9] Taylor DD,Gercel-Taylor C.MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol,2008,110:13-21.

2010-11-11)

(本文編輯：屠振興)

ExpressionofmicroRNAsinfecalofpatientsforpancreaticcancerscreeninganddetection

RENYan,GAOJun,WANGXiao-wei,LIUJian-qiang,GUJun-jun,HUANGHao-jie,JINJing,GONGYan-fang,LIZhao-shen.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

LIZhao-shen,Email:lizhaoshen111@gmail.com

ObjectiveTo detect the microRNAs in fecal with patients of pancreatic cancer, and evaluate its diagnostic value.MethodsStool samples were collected from three group persons including 29 pancreatic cancer, 22 chronic pancreatitis and 13 normal controls. The total fecal microRNAs were extracted. The quantity of miR-16, miR-21, miR-155, miR-181a, miR-181b, miR-196a, and miR-210 were detected by using real-time PCR, and miR-16 was used as reference gene. ROC AUC was used to evaluate the diagnostic value for pancreatic cancer.ResultsMicroRNAs were efficiently obtained from stools, and independent experiments showed high reproducibility for microRNAs extraction and detection. The expression of miR-181b, miR-196a, miR-210 in fecal was 2.22±0.64, 2.78±0.14, 5.55±0.38 in pancreatic cancer; 1.42±0.39,3.88±0.85,5.39±0.69 in chronic pancreatitis; 0.32±0.40, 1.14±0.98,4.23±0.99 in normal controls; the three microRNA expressions in pancreatic cancer were group and CP group significantly higher than those in normal controls (P<0.05). But there was no significant difference between pancreatic cancer group and chronic pancreatitis group. AUC of pancreatic cancer / normal controls miR 181b was 0.745(95%CI0.597-0.894), the sentivity, specificity for pancreatic cancer was 84.6% and 51.7%. AUC of miR-210 was 0.772(95%CI0.629-0.914), the sentivity, specificity for pancreatic cancer was 84.6% and 65.5%, and the difference was statistically significant (P<0.05). miR-196a was no significant for the diagnosis of pancreatic cancer, but the expression of miR-196a was correlated with the tumor size (r=0.516,P=0.041).ConclusionsThe extraction and detection of the fecal microRNAs were non-invasive and reproducible. The expression of miR-181b and miR-210 was increased in stool of patients with pancreatic cancer, and may be potential biomarker for pancreatic cancer.

Pancreatic neoplasms; MicroRNAs; Feces; Diagnosis

10.3760/cma.j.issn.1674-1935.2011.02.009

國家科技支撐計(jì)劃資助項(xiàng)目(2006BAI02A12)

200433 上海，第二軍醫(yī)大學(xué)長海醫(yī)院消化內(nèi)科

李兆申，Email:lizhaoshen111@gmail.com