粪便microRNAs检测用于胰腺癌筛查诊断的价值评价

任艳 高军 王小玮 刘建强 顾俊骏 黄浩杰 金晶 龚燕芳 李兆申

·论著·

粪便microRNAs检测用于胰腺癌筛查诊断的价值评价

任艳 高军 王小玮 刘建强 顾俊骏 黄浩杰 金晶 龚燕芳 李兆申

目的检测胰腺癌患者粪便microRNAs，评价其诊断价值。方法收集29例胰腺癌患者、22例慢性胰腺炎(CP)患者以及13例健康志愿者的粪便标本，抽提粪便总RNA，应用实时定量PCR法检测各组样本miR-21、miR-155、miR-181a、miR-181b、miR-196a、miR-210的表达量，以miR-16作为内参基因。应用接受者操作特征(ROC)曲线(AUC)评估microRNAs对胰腺癌的诊断价值。结果粪便总RNA抽提及microRNAs检测方法具有稳定及可重复性。胰腺癌组miR-181b、miR-196a、miR-210的表达量分别为2.22±0.64、2.78±0.14、5.55±0.38；CP组为1.42±0.39、3.88±0.85、5.39±0.69；对照组为0.32±0.40、1.14±0.98、4.23±0.99。胰腺癌组和CP组均较对照组显著增加(P值均<0.05)；而胰腺癌组与CP组间无显著差异。胰腺癌组对对照组的miR-181b AUC为0.745(95%CI0.597～0.894)，诊断胰腺癌的敏感性和特异性分别为84.6%和51.7%；miR-210的AUC为0.772(95%CI0.629～0.914)，对胰腺癌的诊断敏感性和特异性分别为84.6%和65.5%。两组比较差异均有统计学意义(P值均<0.05)。miR-196a对胰腺癌无诊断意义，但胰腺癌患者粪便miR-196a的表达与肿瘤直径相关(r=0.516,P=0.041)。结论粪便RNA的抽提和microRNAs检测为无创性，且具有可重复性。miR-181b和 miR-210在胰腺癌患者粪便中的表达增高，有可能是胰腺癌潜在的分子标志物。

胰腺肿瘤； 微RNAs； 粪便； 诊断

胰腺癌的发病率在国内外均逐年升高，但由于解剖位置关系以及缺乏特异有效的检查手段，胰腺癌的早期诊断仍相当困难[1]。 近年来，大量的研究结果表明microRNA(miRNA)与肿瘤的发生和发展存在密切的联系[2]，miRNA已成为近几年来分子生物学和遗传学领域的研究热点。本实验旨在寻找一种新的、采用无创手段检测的胰腺癌分子标志物。

材料与方法

一、材料

收集长海医院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)患者粪便，另取13例健康志愿者粪便作为对照。胰腺癌根据病理诊断或者临床诊断[3]。所有患者均签属知情同意书。粪便标本收集后立即置-80℃冰箱保存。

二、粪便RNA提取

使用E.Z.N.A粪便提取试剂盒(Omega，美国)抽提粪便总RNA(包含miRNA)，按说明书操作。抽提的RNA用分光光度计测浓度及鉴定纯度，1%琼脂糖凝胶电泳分离观察条带。样本置-80℃保存。

三、miRNA检测

根据以往文献报道[4-5]，我们选择miR-21、miR-155、miR-181a、 miR-181b、miR-196a、miR-210 6个miRNA 进行检测。依据Genorm软件以及Link等[6]报道，选择miR-16作为内参。

miRNAs的定量检测采用Taqman MicroRNAs检测试剂盒(Applied Biosystems公司),引物由ABI公司设计。RT反应条件：16℃ 30 min，42℃ 30 min；80℃ 5 min。实时PCR反应条件：95℃ 10 min， 95℃ 15 s，55个循环，60℃ 15 s。利用ABI 7500 Real Time PCR仪SDS 2.1 software(Applied Biosystems, USA)得到各个miRNA的Ct值，△△Ct=(Ct样本-Ct样本 miR-16)-(Ct对照-Ct对照miR-16)，相对表达丰度值(RQ)=2-△△Ct。应用ROC曲线(AUC)评估miRNA对胰腺癌的诊断价值。

四、统计分析

结 果

一、粪便RNA抽提的重复性及miRNA检测的重复性

200 g粪便可抽提到总RNA量为19 315～69 995 ng，对6个样本进行重复抽提，两次miRNAs检测结果均呈线性关系(R2=0.998及R2=0.988,P<0.001，图1)。

图1 RNA抽提(a)及miRNA检测(b)可重复性检测线性图

二、粪便miRNAs表达量

胰腺癌组miR-181b、miR-196a、miR-210的表达量分别为2.22±0.64、2.78±0.14、5.55±0.38；CP组为1.42±0.39、3.88±0.85、5.39±0.69；对照组为0.32±0.40、1.14±0.98、4.23±0.99。胰腺癌组较对照组显著增加(P值均<0.05)；CP组也较对照组显著增加(P值均<0.05)；而胰腺癌组与CP组间无显著差异。三组miR-155、miR-181a、miR-21的表达量均无显著差异。

三、粪便miRNAs的诊断价值

miR-181b、miR-196a、miR-210对胰腺癌的诊断价值见表1。

表1 粪便miR-181b、miR-196a、miR-210的AUC和诊断敏感性、特异性

四、胰腺癌miRNAs表达与临床参数的关系

胰腺癌miR-181b、miR-210的表达与患者年龄、性别、体重、吸烟指数、胰管直径、TNM分期以及血清CEA、CA19-9、ALT、AST、总胆红素、血糖等均无关，仅miR-196a表达与肿瘤最大直径呈正相关(r=0.736，P=0.041)。

讨 论

近年研究认为，miRNAs在人类恶性肿瘤中表达异常[2]，miRNAs的表达谱可以较为准确地反映肿瘤的组织来源和分化状态，并可以根据miRNA的表达谱对多种低分化肿瘤进行分类，在肿瘤诊断和预后评估中具有重要价值[7]。目前已有乳腺、肺、食管、前列腺等肿瘤miRNAs表达谱的报道，并且认为miRNAs的表达与肿瘤分期、血管转移等组织学特性有关。

Lee等[4]报道，miR-21、miR-155、miR-181a在胰腺癌组织中表达高于正常胰腺组织，Dillhoff等[5]报道，miR-21是一个潜在的分子标志物，并且与患者生存期有关。Bloomston等[8]等报道，胰腺癌组织miR-21、miR-155、miR-181a、miR-181b、miR-210表达高于相应癌旁组织。我们先前检测了胰腺癌患者和CP患者血清中miR-16、miR-155、miR-181a、miR-181b、miR-21、miR-196a、miR-221、miR-222的表达，结果显示miR-21、miR-155、miR-196a在胰腺疾病中高表达。

本实验检测粪便中miRNAs表达。粪便易获取，无创性，抽提的总RNA重复性好，同时，miRNAs检测的重复性也好，因此是肿瘤筛查的理想标本。

本实验结果显示，胰腺癌患者粪便中miR-181b、miR-210、miR-196a表达较对照组显著增加。这一结果与以往研究的血清标本结果不完全一致，可能与粪便中miRNAs的来源与血液中不同相关。第一，脱落细胞来源于肿瘤组织，混合在胰液内分泌入消化道。第二，肿瘤来源的分泌小泡进入血液循环，经由消化道进入粪便[9]。

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[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.

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2010-11-11)

(本文编辑：屠振兴)

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

国家科技支撑计划资助项目(2006BAI02A12)

200433 上海，第二军医大学长海医院消化内科

李兆申，Email:lizhaoshen111@gmail.com