劉建強 高軍 李兆申 任艷 王小瑋 王衛(wèi)衛(wèi) 路華

·論著·

胰腺癌患者血漿miR-155檢測及其診斷價值

劉建強 高軍 李兆申 任艷 王小瑋 王衛(wèi)衛(wèi) 路華

目的檢測胰腺癌患者血漿miR-155表達(dá)量，評價其對胰腺癌的診斷價值。方法收集62例胰腺癌、61例慢性胰腺炎(CP)及36例正常對照者的血標(biāo)本，抽提血漿RNA，應(yīng)用實時PCR檢測miR-155表達(dá)量，并分析其與胰腺癌臨床參數(shù)的關(guān)系。應(yīng)用接受者操作特征(ROC)曲線下面積(AUC)評價血漿miR-155表達(dá)量對胰腺癌的診斷價值。結(jié)果胰腺癌、CP和正常對照組的血漿miR-155表達(dá)量分別為5.41±3.14、2.59±2.49和0.77±1.17，胰腺癌血漿miR-155表達(dá)量顯著高于CP及正常對照組(P<0.01 )。胰腺癌血漿miR-155表達(dá)量與患者年齡、性別、腫瘤大小等均無顯著相關(guān)性，而與腫瘤TNM分期呈顯著負(fù)相關(guān)(r=-0.323，P=0.01)。經(jīng)ROC分析，胰腺癌對正常對照組的AUC為0.943(95%CI0.902～0.985)，敏感性和特異性分別為87.1%和83.3%；胰腺癌對CP的AUC為0.762(95%CI0.678～0.846)，敏感性和特異性分別為64.5%和73.8%；胰腺癌對正常對照組+CP組的AUC為0.829(95%CI0.767～0.892)，敏感性和特異性分別為62.9%和84.5%。結(jié)論胰腺癌患者血漿miR-155表達(dá)量顯著升高，對胰腺癌的診斷可能有一定的應(yīng)用價值。

胰腺腫瘤； 微RNA； 診斷

血清CA19-9對胰腺癌的診斷敏感性為70%～80%[1]，而特異性不到50%[2]，這就需要發(fā)現(xiàn)新的胰腺癌腫瘤標(biāo)志物。眾多研究結(jié)果表明，microRNA(miRNA)與腫瘤的發(fā)病及預(yù)后相關(guān)，其中miR-155在胰腺癌組織中顯著高表達(dá)[3]。本研究檢測胰腺癌患者血漿miR-155表達(dá)量，評價其對胰腺癌的診斷價值。

資料和方法

一、臨床資料

收集2008年1月至2009年12月第二軍醫(yī)大學(xué)附屬長海醫(yī)院收治的62例胰腺癌患者和61例慢性胰腺炎(CP)患者的血漿標(biāo)本。所有胰腺癌患者均經(jīng)手術(shù)及病理證實。CP患者通過影像學(xué)診斷，并隨訪半年以上無腫瘤發(fā)生者。以36例年齡匹配、無任何腫瘤病史的健康體檢者作為正常對照組。

二、血漿miR-155檢測

采集2 ml外周靜脈血，置EDTA試管抗凝，4℃離心取上層血漿至1.5 ml Eppendorf管， 4℃ 12 000 g離心10 min后置-80℃保存。取200 μl血漿，加入5 μl作為內(nèi)參的50 pmol/L的線蟲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說明書抽提RNA。取2 μl RNA用TaqMan MicroRNA 逆轉(zhuǎn)錄試劑盒(Applied Biosystems公司)獲取cDNA，反應(yīng)條件：16℃ 30 min，42℃ 30 min，85℃ 5 min，4℃ 維持。取2 μl cDNA作為模板，用TaqMan Universal Master Mix Ⅱ試劑盒進(jìn)行實時 PCR，miR-155和miR-39探針為TaqMan公司提供。PCR條件：95℃ 1 min，95℃ 15 s，60℃ 30 s，50個循環(huán)。每份標(biāo)本設(shè)3個復(fù)孔。采用ABI 7500實時定量PCR儀進(jìn)行檢測，用SDS 2.1軟件計算標(biāo)本Ct值。miR-155表達(dá)量=-△△Ct=-[(Ct患者-Ct患者miR-39)-(Ct對照標(biāo)本-Ct對照miR-39)]。

三、統(tǒng)計學(xué)處理

結(jié) 果

一、一般情況

62例胰腺癌患者中，男42例，女20例，中位年齡62歲(36～79歲)。其中胰頭癌43例，胰體尾癌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，中位年齡48歲(19～76歲)。36例對照者中，男20例，女16例，中位年齡49歲(25～68歲)。

二、血漿miR-155表達(dá)量

胰腺癌、CP、對照組血漿miR-155表達(dá)量分別為5.41±3.14、2.59±2.49和0.77±1.17，胰腺癌組顯著高于CP組及對照組(P<0.01)。CP組血漿miRNA表達(dá)量雖高于對照組，但無統(tǒng)計學(xué)意義。

三、血漿miR-155水平與胰腺癌臨床病理參數(shù)的關(guān)系

血漿miR-155水平與胰腺癌患者年齡、性別、CA19-9水平、腫瘤最大直徑、局部淋巴結(jié)和遠(yuǎn)處轉(zhuǎn)移均無顯著相關(guān)性，但與T分期和臨床TNM分期呈顯著負(fù)相關(guān)(P<0.05，表1)。

表1 血漿miRNA表達(dá)量與胰腺癌臨床參數(shù)的關(guān)系

注：a：pearson相關(guān)分析,其他采用spearman秩相關(guān)分析

四、血漿miR-155表達(dá)量對胰腺癌的診斷價值

根據(jù)logistic回歸模型，血漿miR-155診斷胰腺癌的AUC及診斷敏感性、特異性、準(zhǔn)確性、陽性預(yù)測值、陰性預(yù)測值見表2。血漿miR-155表達(dá)量在鑒別胰腺癌與對照組時的敏感性和特異性較高。

表2 血漿miR-155對胰腺癌的診斷價值

討 論

miRNA是一類內(nèi)源性的非編碼調(diào)控單鏈小分子RNA，長度約18～25個核苷酸，通過對mRNA的調(diào)控，參與細(xì)胞增殖、分化、凋亡和代謝等過程的調(diào)節(jié)[4]，目前已可預(yù)測哺乳動物約30%基因表達(dá)的miRNA調(diào)控靶點[5]。通常情況下，miRNA通過沉默基因表達(dá)而調(diào)控細(xì)胞功能，其表達(dá)異常可導(dǎo)致腫瘤等疾病的發(fā)生[6-7]。已經(jīng)證實，miRNA在結(jié)腸癌、乳腺癌和肺癌等腫瘤中起負(fù)調(diào)控作用[8-9]，在血液系統(tǒng)淋巴瘤和白血病中miRNA異常表達(dá)[10-11]。

MiR-155在甲狀腺癌[12]、乳腺癌[2]、結(jié)腸癌[10]和胰腺導(dǎo)管腺癌[13-14]等腫瘤中高表達(dá)，并在腫瘤發(fā)生中起癌基因的作用[15]。研究表明，在胰腺癌早期腫瘤蛋白53誘導(dǎo)核蛋白1(TP53INP1)缺失，而miR-155參與了TP53INP1的轉(zhuǎn)錄后調(diào)控。胰腺癌細(xì)胞株轉(zhuǎn)染miR-155后伴有TP53INP1缺失[16]。本結(jié)果顯示，胰腺癌患者血漿miR-155表達(dá)量顯著高于CP患者和對照組，CP患者血漿miR-155表達(dá)量也顯著高于對照組，與正常胰腺到CP，再發(fā)展為胰腺癌的規(guī)律相符，提示miR-155具有胰腺癌的腫瘤特異性。血漿miR-155水平與胰腺癌大小、是否局部淋巴結(jié)和遠(yuǎn)處轉(zhuǎn)移等臨床參數(shù)之間無顯著相關(guān)性，而與腫瘤的T分期和臨床TNM分期呈負(fù)相關(guān)，這是否表示腫瘤浸潤胰腺周邊組織及血管者伴隨血漿miR-155的降低還需進(jìn)一步研究證實。通過logistic回歸及ROC曲線分析，血漿miR-155對胰腺癌診斷的敏感性和特異性較理想，有一定的臨床應(yīng)用價值。

[1] Slesak B, Harlozinska-Szmyrka A, Knast W, et al. Tissue polypeptide specific antigen (TPS), a marker for differentiation between pancreatic carcinoma and chronic pancreatitis. A comparative study with CA 19-9. Cancer,2000,89: 83-88.

[2] Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res,2005,65:7065-7070.

[3] 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.

[4] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell,2004,116: 281-297.

[5] Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet,2008,9:102-114.

[6] Slack FJ, Weidhaas JB. MicroRNA in cancer prognosis. N Engl J Med,2008,359:2720-2722.

[7] Visone R, Croce CM. MiRNAs and cancer. Am J Pathol,2009,174:1131-1138.

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

[9] 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.

[10] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA,2004,101:11755-11760.

[11] Lawrie CH, Soneji S, Marafioti T, et al. MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma. Int J Cancer,2007,121:1156-1161.

[12] Nikiforova MN, Tseng GC, Steward D, et al. MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility. J Clin Endocrinol Metab,2008,93:1600-1608.

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

[14] Szafranska AE, Davison TS, John J, et al. MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene,2007,26:4442-4452.

[15] Esquela-Kerscher A, Slack FJ. Oncomirs-microRNAs with a role in cancer. Nat Rev Cancer,2006,6:259-269.

[16] Gironella M, Seux M, Xie MJ, et al. Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development. Proc Natl Acad Sci USA,2007,104:16170-16175.

(本文編輯：呂芳萍)

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：高軍

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

國家科技支撐計劃資助項目(2006BAI02A12)

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

李兆申，Email：lizhaoshen@yahoo.com

2010-12-29)