胰腺癌患者血浆miR-155检测及其诊断价值

刘建强 高军 李兆申 任艳 王小玮 王卫卫 路华

·论著·

胰腺癌患者血浆miR-155检测及其诊断价值

刘建强 高军 李兆申 任艳 王小玮 王卫卫 路华

目的检测胰腺癌患者血浆miR-155表达量，评价其对胰腺癌的诊断价值。方法收集62例胰腺癌、61例慢性胰腺炎(CP)及36例正常对照者的血标本，抽提血浆RNA，应用实时PCR检测miR-155表达量，并分析其与胰腺癌临床参数的关系。应用接受者操作特征(ROC)曲线下面积(AUC)评价血浆miR-155表达量对胰腺癌的诊断价值。结果胰腺癌、CP和正常对照组的血浆miR-155表达量分别为5.41±3.14、2.59±2.49和0.77±1.17，胰腺癌血浆miR-155表达量显著高于CP及正常对照组(P<0.01 )。胰腺癌血浆miR-155表达量与患者年龄、性别、肿瘤大小等均无显著相关性，而与肿瘤TNM分期呈显著负相关(r=-0.323，P=0.01)。经ROC分析，胰腺癌对正常对照组的AUC为0.943(95%CI0.902～0.985)，敏感性和特异性分别为87.1%和83.3%；胰腺癌对CP的AUC为0.762(95%CI0.678～0.846)，敏感性和特异性分别为64.5%和73.8%；胰腺癌对正常对照组+CP组的AUC为0.829(95%CI0.767～0.892)，敏感性和特异性分别为62.9%和84.5%。结论胰腺癌患者血浆miR-155表达量显著升高，对胰腺癌的诊断可能有一定的应用价值。

胰腺肿瘤； 微RNA； 诊断

血清CA19-9对胰腺癌的诊断敏感性为70%～80%[1]，而特异性不到50%[2]，这就需要发现新的胰腺癌肿瘤标志物。众多研究结果表明，microRNA(miRNA)与肿瘤的发病及预后相关，其中miR-155在胰腺癌组织中显著高表达[3]。本研究检测胰腺癌患者血浆miR-155表达量，评价其对胰腺癌的诊断价值。

资料和方法

一、临床资料

收集2008年1月至2009年12月第二军医大学附属长海医院收治的62例胰腺癌患者和61例慢性胰腺炎(CP)患者的血浆标本。所有胰腺癌患者均经手术及病理证实。CP患者通过影像学诊断，并随访半年以上无肿瘤发生者。以36例年龄匹配、无任何肿瘤病史的健康体检者作为正常对照组。

二、血浆miR-155检测

采集2 ml外周静脉血，置EDTA试管抗凝，4℃离心取上层血浆至1.5 ml Eppendorf管， 4℃ 12 000 g离心10 min后置-80℃保存。取200 μl血浆，加入5 μl作为内参的50 pmol/L的线虫miR-39(Qiagen公司)，再加入20 μl 5 mol/L醋酸和750 μl TRI Regent BD(Molecular Research Center)，按TRI Regent BD说明书抽提RNA。取2 μl RNA用TaqMan MicroRNA 逆转录试剂盒(Applied Biosystems公司)获取cDNA，反应条件：16℃ 30 min，42℃ 30 min，85℃ 5 min，4℃ 维持。取2 μl cDNA作为模板，用TaqMan Universal Master Mix Ⅱ试剂盒进行实时 PCR，miR-155和miR-39探针为TaqMan公司提供。PCR条件：95℃ 1 min，95℃ 15 s，60℃ 30 s，50个循环。每份标本设3个复孔。采用ABI 7500实时定量PCR仪进行检测，用SDS 2.1软件计算标本Ct值。miR-155表达量=-△△Ct=-[(Ct患者-Ct患者miR-39)-(Ct对照标本-Ct对照miR-39)]。

三、统计学处理

结 果

一、一般情况

62例胰腺癌患者中，男42例，女20例，中位年龄62岁(36～79岁)。其中胰头癌43例，胰体尾癌19例。TNM分期Ⅰ期10例，Ⅱ期15例，Ⅲ期14例，Ⅳ期23例。61例CP患者中，男44例，女17例，中位年龄48岁(19～76岁)。36例对照者中，男20例，女16例，中位年龄49岁(25～68岁)。

二、血浆miR-155表达量

胰腺癌、CP、对照组血浆miR-155表达量分别为5.41±3.14、2.59±2.49和0.77±1.17，胰腺癌组显著高于CP组及对照组(P<0.01)。CP组血浆miRNA表达量虽高于对照组，但无统计学意义。

三、血浆miR-155水平与胰腺癌临床病理参数的关系

血浆miR-155水平与胰腺癌患者年龄、性别、CA19-9水平、肿瘤最大直径、局部淋巴结和远处转移均无显著相关性，但与T分期和临床TNM分期呈显著负相关(P<0.05，表1)。

表1 血浆miRNA表达量与胰腺癌临床参数的关系

注：a：pearson相关分析,其他采用spearman秩相关分析

四、血浆miR-155表达量对胰腺癌的诊断价值

根据logistic回归模型，血浆miR-155诊断胰腺癌的AUC及诊断敏感性、特异性、准确性、阳性预测值、阴性预测值见表2。血浆miR-155表达量在鉴别胰腺癌与对照组时的敏感性和特异性较高。

表2 血浆miR-155对胰腺癌的诊断价值

讨 论

miRNA是一类内源性的非编码调控单链小分子RNA，长度约18～25个核苷酸，通过对mRNA的调控，参与细胞增殖、分化、凋亡和代谢等过程的调节[4]，目前已可预测哺乳动物约30%基因表达的miRNA调控靶点[5]。通常情况下，miRNA通过沉默基因表达而调控细胞功能，其表达异常可导致肿瘤等疾病的发生[6-7]。已经证实，miRNA在结肠癌、乳腺癌和肺癌等肿瘤中起负调控作用[8-9]，在血液系统淋巴瘤和白血病中miRNA异常表达[10-11]。

MiR-155在甲状腺癌[12]、乳腺癌[2]、结肠癌[10]和胰腺导管腺癌[13-14]等肿瘤中高表达，并在肿瘤发生中起癌基因的作用[15]。研究表明，在胰腺癌早期肿瘤蛋白53诱导核蛋白1(TP53INP1)缺失，而miR-155参与了TP53INP1的转录后调控。胰腺癌细胞株转染miR-155后伴有TP53INP1缺失[16]。本结果显示，胰腺癌患者血浆miR-155表达量显著高于CP患者和对照组，CP患者血浆miR-155表达量也显著高于对照组，与正常胰腺到CP，再发展为胰腺癌的规律相符，提示miR-155具有胰腺癌的肿瘤特异性。血浆miR-155水平与胰腺癌大小、是否局部淋巴结和远处转移等临床参数之间无显著相关性，而与肿瘤的T分期和临床TNM分期呈负相关，这是否表示肿瘤浸润胰腺周边组织及血管者伴随血浆miR-155的降低还需进一步研究证实。通过logistic回归及ROC曲线分析，血浆miR-155对胰腺癌诊断的敏感性和特异性较理想，有一定的临床应用价值。

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(本文编辑：吕芳萍)

DiagnosticvalueofplasmamiR55forpancreaticcancer

LIUJian-qiang,GAOJun,LIZhao-shen,RENYan,WANGXiao-wei,WANGWei-wei,LUHua.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

LIZhao-shen,lizhaoshen@yahoo.com

ObjectiveTo detect the level of plasma miR-155 in patients with pancreatic cancer and evaluate its diagnostic value for pancreatic cancer.MethodsSixty-two cases of pancreatic cancer patients, 61 patients of chronic pancreatitis and 36 normal controls were included in the study. miR-155 in the total RNA extracted from the plasma was measured using real time PCR. The diagnostic parameters and relationship of the miR-155 with clinical characteristics in pancreatic cancer patients were analyzed.ResultsThe relative abundance of plasma miR-155 in pancreatic cancer, chronic pancreatitis and normal controls were 5.41±3.14,2.59±2.49 and 0.77±1.17, and the value was significantly higher in pancreatic cancer group compared with those of chronic pancreatitis and normal group (P<0.01). No significant correlation between the level of plasma miR-155 and age, sex, tumor size in pancreatic cancer patients was found. But there was a negative correlation between the level of plasma miR-155 and TNM staging (r=-0.323,P=0.01). For discriminating pancreatic cancer from normal control, the AUC ROC of miR-155 was 0.943 (95%CI0.902-0.985), and the sensitivity and specificity were87.1% and 83.3%, respectively. For discriminating pancreatic cancer from chronic pancreatitis, the AUC ROC of miR-155 was 0.762 (95%CI0.678-0.846), and the sensitivity and specificity were 64.5% and 73.8%, respectively. For discriminating pancreatic cancer from chronic pancreatitis and normal, the AUC-ROC of miR-155 was 0.829(95%CI0.767-0.892), and the sensitivity and specificity were 62.9% and 84.5%, respectively.ConclusionsPlasma miR-155 was

共同第一作者：高军

significantly increased in patients with pancreatic cancer, and can be used for diagnosis of pancreatic cancer.

Pancreatic neoplasms； MicroRNAs； Diagnosis

10.3760/cma.j.issn.1674-1935.2011.02.001

国家科技支撑计划资助项目(2006BAI02A12)

200433 上海，第二军医大学附属长海医院消化内科

李兆申，Email：lizhaoshen@yahoo.com

2010-12-29)