張晶 趙晨燕 劉清華 余黨會(huì) 陳穎 史敏 倪燦榮 朱明華

·論著·

胰腺導(dǎo)管腺癌microRNA表達(dá)譜的研究

張晶 趙晨燕 劉清華 余黨會(huì) 陳穎 史敏 倪燦榮 朱明華

目的應(yīng)用microRNA(miRNA)高通量生物芯片篩選胰腺導(dǎo)管腺癌及癌旁組織差異表達(dá)的miRNA，分析其相關(guān)的靶基因。方法收集9例新鮮的胰腺導(dǎo)管腺癌和3例癌旁組織，運(yùn)用標(biāo)記713個(gè)miRNA的Agilent miRNA生物芯片篩選胰腺導(dǎo)管腺癌差異表達(dá)的miRNA，應(yīng)用熒光實(shí)時(shí)定量PCR方法驗(yàn)證表達(dá)上調(diào)的miRNA。采用TargetScan 5.1和miRandaV5分析軟件分析差異表達(dá)miRNAs的靶基因。結(jié)果miRNA芯片篩選出11個(gè)胰腺導(dǎo)管腺癌相關(guān)的差異表達(dá)的miRNA，其中miR-194*、miR-192*、miR-602、miR-194表達(dá)上調(diào)，miR-139-3p、miR-513a-5p、miR-630、miR-30c-1*、miR-887、miR-508-5p、miR-516a-5p表達(dá)下調(diào)。miR-192、miR-194及其同源體的表達(dá)在31例胰腺癌組織中得到驗(yàn)證。經(jīng)軟件分析，miR-192靶基因有ZEB2、CXCL-2、EEF1A1、ERCC3，miR-192*靶基因有DCC、SMAD4、FAS，miR-194靶基因有DACH1、IGSF11、PTPN2、RBBP4，miR-194*靶基因有CD40LG、CIDEB、FHL1。結(jié)論胰腺導(dǎo)管腺癌存在11個(gè)表達(dá)差異的miRNA，這些miRNA可能與胰腺導(dǎo)管腺癌的發(fā)生、發(fā)展有關(guān)。

胰腺腫瘤； 微RNAs； 微陣列分析； 基因表達(dá)譜

MicroRNA(miRNA)是近年來(lái)發(fā)現(xiàn)的一類(lèi)長(zhǎng)度為19～23 nt的、廣泛存在于動(dòng)植物體內(nèi)的、非編碼單鏈RNA。miRNA通過(guò)作用于其靶mRNA，降解靶基因或抑制其翻譯，發(fā)揮負(fù)調(diào)控作用。研究表明[1-6]，miRNA參與生命過(guò)程中一系列的重要進(jìn)程，包括早期發(fā)育、細(xì)胞增殖及分化、凋亡和免疫調(diào)節(jié)等，還具有癌基因和抑癌基因的作用。在不同類(lèi)型的腫瘤中有其特異的miRNA表達(dá)譜。本研究采用miRNA芯片技術(shù)篩選胰腺導(dǎo)管腺癌與癌旁正常胰腺組織差異表達(dá)的miRNA，并分析其相關(guān)靶基因。

材料與方法

一、組織標(biāo)本

收集2007年至2008年長(zhǎng)海醫(yī)院外科切除的9例胰腺癌組織標(biāo)本及3例配對(duì)的癌旁正常胰腺標(biāo)本送上海生物芯片有限公司行miRNA芯片檢測(cè)，其中男性4例，女性5例，年齡47～75歲。另取2009年3月至2010年5月手術(shù)切除的31例胰腺癌及配對(duì)的癌旁新鮮組織標(biāo)本，液氮冷凍后置-80℃保存，行實(shí)時(shí)定量PCR；其中男性15例，女性16例，年齡45～77歲。所有患者術(shù)前均未進(jìn)行抗腫瘤治療，均簽署知情同意書(shū)，并經(jīng)第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院倫理委員會(huì)批準(zhǔn)。所有標(biāo)本經(jīng)病理檢查均證實(shí)為胰腺導(dǎo)管腺癌。

二、miRNA芯片檢測(cè)

應(yīng)用miRNA抽提試劑盒(Ambion， AM1560)抽提新鮮組織的mRNA，并富集miRNA。應(yīng)用miRNA標(biāo)記和雜交試劑盒(Agilent 5190-0408)對(duì)miRNA進(jìn)行CY3熒光標(biāo)記。miRNA微陣列基因芯片和質(zhì)控探針由上海生物芯片有限公司提供(Agilnent公司產(chǎn)品)，其miRNA探針序列信息來(lái)自于SangermiRBase Release10.0版本數(shù)據(jù)庫(kù)(http://microrna. sanger. ac. uk/sequences/)，涵蓋了Sanger數(shù)據(jù)庫(kù)中713個(gè)miRNA探針。每張芯片都設(shè)計(jì)了與實(shí)驗(yàn)樣品無(wú)同源性的核酸序列作為質(zhì)控RNA。組織標(biāo)本預(yù)處理及探針雜交按Agilent芯片實(shí)驗(yàn)操作步驟進(jìn)行。設(shè)空白對(duì)照及采用與實(shí)驗(yàn)樣品無(wú)同源性的核酸序列探針的陰性對(duì)照組。芯片的每個(gè)探針雜交位點(diǎn)至少有3個(gè)重復(fù)。

采用Agilent掃描儀掃描采集雜交圖像，采用Feature Extraction軟件讀取圖像數(shù)據(jù)，最后采用Feature Extraction進(jìn)行處理分析，得到的原始數(shù)據(jù)通過(guò)VSN(variance stabilizing normalization)方法進(jìn)行歸一化。

三、熒光實(shí)時(shí)定量PCR

應(yīng)用miRNA抽提試劑盒抽提31例配對(duì)的胰腺導(dǎo)管腺癌和癌旁胰腺組織miRNAs，應(yīng)用TaqMan microRNA reverse transcription試劑盒將miRNAs逆轉(zhuǎn)錄成cDNA，應(yīng)用TaqMan microRNA assays檢測(cè)miRNAs的表達(dá)。PCR反應(yīng)條件：95℃ 30 s，95℃ 5 s，60℃ 34 s，40個(gè)循環(huán)。以U6 SnRNA作為內(nèi)參照，采用△CT方法計(jì)算miRNA的相對(duì)表達(dá)量。

四、差異miRNA靶基因及其相關(guān)疾病分析

采用生物信息學(xué)分析方法。應(yīng)用TargetScan5.1和miRandaV5分析軟件預(yù)測(cè)并初步分析差異表達(dá)miRNAs的靶基因，并分析與其相關(guān)的疾病。

五、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、胰腺導(dǎo)管腺癌組織中差異表達(dá)的miRNA

miRNA芯片雜交信號(hào)強(qiáng)度由綠-黑-紅色依次增加。胰腺導(dǎo)管腺癌組織中差異表達(dá)≥1.2倍的miRNA共12個(gè)，其中5個(gè)上調(diào)，為miR-194*、miR-192*、miR-602、miR-801、miR-194；7個(gè)下調(diào)，為miR-139-3p、miR-513a-5p、miR-630、miR-30c-1*、miR-887、miR-508-5p、miR-516a-5p(表1)。因miR-801被證實(shí)不是真正的miRNA，而是一個(gè)U11RNA的片段，故在聚類(lèi)分析中被剔除(圖1)。上調(diào)最高的miRNA為miR-194*，上調(diào)倍數(shù)為2.38倍。

表1 胰腺導(dǎo)管腺癌中差異表達(dá)的miRNAs

注：帶*號(hào)的miRNA與其相應(yīng)的miRNA來(lái)自同一前體

圖1 差異表達(dá)的miRNAs聚類(lèi)分類(lèi)圖

二、胰腺癌組織miR-192、miR-192*、miR-194和miR-194*表達(dá)的驗(yàn)證

31例胰腺導(dǎo)管腺癌和配對(duì)癌旁胰腺組織的miR-192、miR-192*、miR-194和miR-194*表達(dá)均顯著上調(diào)(P值均<0.05，表2)。

miRNA胰腺癌組織癌旁組織P值miR-192-0.66±4.08-5.49±7.140.001miR-192*-6.63±4.97-8.98±3.510.010miR-194-1.56±4.15-6.51±7.790.001miR-194*-9.35±5.51-11.95±2.860.022

三、差異表達(dá)miRNA的靶基因及其相關(guān)疾病

miR-192靶基因有ZEB2、CXCL-2、EEF1A1，miR-192*靶基因有DCC、SMAD4、FAS，miR-194靶基因有DACH1、IGSF11、PTPN2、RBBP4，miR-194*靶基因有CD40LG、CIDEB、FHL1。相關(guān)的疾病見(jiàn)表3。

表3 差異表達(dá)miRNA的靶基因及相關(guān)疾病

討 論

人類(lèi)約50%已知的miRNAs基因定位于與腫瘤相關(guān)的染色體區(qū)，提示其在腫瘤發(fā)生中具有重要作用[6]。在慢性淋巴細(xì)胞性白血病、兒童Burkitt淋巴瘤、彌漫性大B細(xì)胞淋巴瘤、肺癌和乳腺癌等惡性腫瘤中，miRNAs表達(dá)均發(fā)生顯著變化[7-14]，并可抑制或促進(jìn)腫瘤的發(fā)生和發(fā)展。2006年Volinia等[15]報(bào)道，胰腺癌中異常高表達(dá)的miRNA有55個(gè)，低表達(dá)的miRNA有2個(gè)。隨后，Lee、Bloomston及Szafanska等[16-18]檢測(cè)了胰腺癌、癌旁組織、慢性胰腺炎組織中miRNA表達(dá)譜，發(fā)現(xiàn)胰腺癌中異常高表達(dá)的miRNA有miR-155、miR-21、miR-221、miR-222、miR-196a等，異常低表達(dá)的有miR-181、miR-148、miR-216和miR-217等。Szafanska等[18]還發(fā)現(xiàn)miRNA表達(dá)譜可用于鑒別胰腺癌、慢性胰腺炎和正常胰腺。

本研究應(yīng)用Agilent miRNA芯片篩選出胰腺導(dǎo)管腺癌表達(dá)上調(diào)的miRNA 4個(gè)，下調(diào)的miRNA 7個(gè)，其中miR-192*和miR-194*上調(diào)大于1.8倍。通過(guò)胰腺癌組織標(biāo)本的檢測(cè)，證實(shí)了miRNA芯片的結(jié)果。

Mees等[19]在miRNA上游轉(zhuǎn)錄因子的研究中發(fā)現(xiàn)，高侵襲、高轉(zhuǎn)移的胰腺癌細(xì)胞株中miR-194的水平明顯高于侵襲力和浸潤(rùn)、轉(zhuǎn)移能力較差的胰腺癌細(xì)胞株。Meng等[20]發(fā)現(xiàn)miR-194在肝上皮細(xì)胞中高表達(dá)，它可作為肝上皮細(xì)胞的標(biāo)志物，并能抑制小鼠體內(nèi)肝癌細(xì)胞的轉(zhuǎn)移。miR-194也表達(dá)于胃腸道和腎臟，但肝臟枯否細(xì)胞和星狀細(xì)胞無(wú)高表達(dá)。

通過(guò)靶基因預(yù)測(cè)軟件檢索，miR-194的靶基因有DACH1。DACH1蛋白是黑腹果蠅dachshund(Dac)基因在脊椎動(dòng)物中表達(dá)的類(lèi)似物，是一種新型的抑癌基因，它通過(guò)阻斷腫瘤上皮細(xì)胞的DNA合成、抑制腫瘤基質(zhì)層中腫瘤集落的形成和腫瘤細(xì)胞的有絲分裂達(dá)到阻止腫瘤細(xì)胞增殖的目的。有研究[21]證明SIP1為miR-192的靶基因。Kato等[22-23]研究發(fā)現(xiàn)，miR-192在纖維化的腎臟高度特異性表達(dá)，促進(jìn)細(xì)胞外基質(zhì)(ECM)蛋白的蓄積；并通過(guò)雙熒光素檢測(cè)確定SIPl的3′UTR為miR-192調(diào)控靶點(diǎn)，證明miR-192通過(guò)mRNA降解途徑降低SIPl蛋白的表達(dá)。我們的結(jié)果并沒(méi)有顯示SIP1為miR-192的靶基因，這可能與生物信息學(xué)分析的方法不同、生物信息學(xué)分析與實(shí)驗(yàn)檢測(cè)存在一定的差異有關(guān)。

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MicroRNAprofilesinpancreaticductaladenocarcinoma

ZHANGJing,ZHAOChen-yan,LIUQing-hua,YUDang-hui,CHENYing,SHIMin,NICan-rong,ZHUMing-hua.

DepartmentofPathology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

ZHUMing-hua,Email:mhzhu2000@hotmail.com

ObjectiveTo study the differentially expressed microRNA (miRNA) between pancreatic ductal adenocarcinoma (PDAC) and para-cancerous tissues, and determine related target genes.MethodsNine fresh PDAC tumor tissues and 3 adjacent normal pancreatic tissues were collected, then Agilent miRNA microarray with 713 miRNA loci was used to identify the differentially expressed miRNA. Real-time PCR method was applied to verify the up-regulated miRNA. TargetScan 5.1 and miRandaV5 software were used to analyze the related target genes.ResultsmiRNA microarray identified 11 PDAC related miRNAs, among them, the expressions of miR-194*, miR-192*, miR- 602, miR-194 were up-regulated, while the expressions of miR-139-3p, miR-513a-5p, miR-630, miR-30c-1*, miR-887, miR-508-5p, miR-516a-5p were down-regulated. The expressions of miR-192, miR-194 and their homolog were verified in 31 PDAC tumor tissues. After software analysis, it was found that target genes of miR-192 were ZEB2, CXCL-2, EEF1A1, ERCC3, and target genes of miR-192* were DCC, SMAD4, FAS, and target genes of miR-194 included DACH1, IGSF11, PTPN2, RBBP4, while target genes of miR-194* included CD40LG, CIDEB, FHL1.ConclusionsEleven differentially expressed miRNAs are present in PDC, and they may be involved in the occurrence and development of PDC.

Pancreatic neoplasms; MicroRNA； Microarray analysis； Gene expression profiling

10.3760/cma.j.issn.1674-1935.2012.05.007

國(guó)家自然科學(xué)基金(30770996；81172310)

200433 上海，第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院病理科(張晶、劉清華、余黨會(huì)、陳穎、史敏、倪燦榮、朱明華)；復(fù)旦大學(xué)附屬婦產(chǎn)科醫(yī)院病理科(趙晨燕)

共同第一作者：趙晨燕

朱明華，Email:mhzhu2000@hotmail.com

2012-06-13)

(本文編輯：屠振興)