胰腺導管腺癌microRNA表達譜的研究

張晶 趙晨燕 劉清華 余黨會 陳穎 史敏 倪燦榮 朱明華

·論著·

胰腺導管腺癌microRNA表達譜的研究

張晶 趙晨燕 劉清華 余黨會 陳穎 史敏 倪燦榮 朱明華

目的應用microRNA(miRNA)高通量生物芯片篩選胰腺導管腺癌及癌旁組織差異表達的miRNA，分析其相關的靶基因。方法收集9例新鮮的胰腺導管腺癌和3例癌旁組織，運用標記713個miRNA的Agilent miRNA生物芯片篩選胰腺導管腺癌差異表達的miRNA，應用熒光實時定量PCR方法驗證表達上調的miRNA。采用TargetScan 5.1和miRandaV5分析軟件分析差異表達miRNAs的靶基因。結果miRNA芯片篩選出11個胰腺導管腺癌相關的差異表達的miRNA，其中miR-194*、miR-192*、miR-602、miR-194表達上調，miR-139-3p、miR-513a-5p、miR-630、miR-30c-1*、miR-887、miR-508-5p、miR-516a-5p表達下調。miR-192、miR-194及其同源體的表達在31例胰腺癌組織中得到驗證。經軟件分析，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。結論胰腺導管腺癌存在11個表達差異的miRNA，這些miRNA可能與胰腺導管腺癌的發生、發展有關。

胰腺腫瘤； 微RNAs； 微陣列分析； 基因表達譜

MicroRNA(miRNA)是近年來發現的一類長度為19～23 nt的、廣泛存在于動植物體內的、非編碼單鏈RNA。miRNA通過作用于其靶mRNA，降解靶基因或抑制其翻譯，發揮負調控作用。研究表明[1-6]，miRNA參與生命過程中一系列的重要進程，包括早期發育、細胞增殖及分化、凋亡和免疫調節等，還具有癌基因和抑癌基因的作用。在不同類型的腫瘤中有其特異的miRNA表達譜。本研究采用miRNA芯片技術篩選胰腺導管腺癌與癌旁正常胰腺組織差異表達的miRNA，并分析其相關靶基因。

材料與方法

一、組織標本

收集2007年至2008年長海醫院外科切除的9例胰腺癌組織標本及3例配對的癌旁正常胰腺標本送上海生物芯片有限公司行miRNA芯片檢測，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月手術切除的31例胰腺癌及配對的癌旁新鮮組織標本，液氮冷凍后置-80℃保存，行實時定量PCR；其中男性15例，女性16例，年齡45～77歲。所有患者術前均未進行抗腫瘤治療，均簽署知情同意書，并經第二軍醫大學長海醫院倫理委員會批準。所有標本經病理檢查均證實為胰腺導管腺癌。

二、miRNA芯片檢測

應用miRNA抽提試劑盒(Ambion， AM1560)抽提新鮮組織的mRNA，并富集miRNA。應用miRNA標記和雜交試劑盒(Agilent 5190-0408)對miRNA進行CY3熒光標記。miRNA微陣列基因芯片和質控探針由上海生物芯片有限公司提供(Agilnent公司產品)，其miRNA探針序列信息來自于SangermiRBase Release10.0版本數據庫(http://microrna. sanger. ac. uk/sequences/)，涵蓋了Sanger數據庫中713個miRNA探針。每張芯片都設計了與實驗樣品無同源性的核酸序列作為質控RNA。組織標本預處理及探針雜交按Agilent芯片實驗操作步驟進行。設空白對照及采用與實驗樣品無同源性的核酸序列探針的陰性對照組。芯片的每個探針雜交位點至少有3個重復。

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

三、熒光實時定量PCR

應用miRNA抽提試劑盒抽提31例配對的胰腺導管腺癌和癌旁胰腺組織miRNAs，應用TaqMan microRNA reverse transcription試劑盒將miRNAs逆轉錄成cDNA，應用TaqMan microRNA assays檢測miRNAs的表達。PCR反應條件：95℃ 30 s，95℃ 5 s，60℃ 34 s，40個循環。以U6 SnRNA作為內參照，采用△CT方法計算miRNA的相對表達量。

四、差異miRNA靶基因及其相關疾病分析

采用生物信息學分析方法。應用TargetScan5.1和miRandaV5分析軟件預測并初步分析差異表達miRNAs的靶基因，并分析與其相關的疾病。

五、統計學分析

結 果

一、胰腺導管腺癌組織中差異表達的miRNA

miRNA芯片雜交信號強度由綠-黑-紅色依次增加。胰腺導管腺癌組織中差異表達≥1.2倍的miRNA共12個，其中5個上調，為miR-194*、miR-192*、miR-602、miR-801、miR-194；7個下調，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被證實不是真正的miRNA，而是一個U11RNA的片段，故在聚類分析中被剔除(圖1)。上調最高的miRNA為miR-194*，上調倍數為2.38倍。

表1 胰腺導管腺癌中差異表達的miRNAs

注：帶*號的miRNA與其相應的miRNA來自同一前體

圖1 差異表達的miRNAs聚類分類圖

二、胰腺癌組織miR-192、miR-192*、miR-194和miR-194*表達的驗證

31例胰腺導管腺癌和配對癌旁胰腺組織的miR-192、miR-192*、miR-194和miR-194*表達均顯著上調(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miRNA的靶基因及其相關疾病

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。相關的疾病見表3。

表3 差異表達miRNA的靶基因及相關疾病

討 論

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

本研究應用Agilent miRNA芯片篩選出胰腺導管腺癌表達上調的miRNA 4個，下調的miRNA 7個，其中miR-192*和miR-194*上調大于1.8倍。通過胰腺癌組織標本的檢測，證實了miRNA芯片的結果。

Mees等[19]在miRNA上游轉錄因子的研究中發現，高侵襲、高轉移的胰腺癌細胞株中miR-194的水平明顯高于侵襲力和浸潤、轉移能力較差的胰腺癌細胞株。Meng等[20]發現miR-194在肝上皮細胞中高表達，它可作為肝上皮細胞的標志物，并能抑制小鼠體內肝癌細胞的轉移。miR-194也表達于胃腸道和腎臟，但肝臟枯否細胞和星狀細胞無高表達。

通過靶基因預測軟件檢索，miR-194的靶基因有DACH1。DACH1蛋白是黑腹果蠅dachshund(Dac)基因在脊椎動物中表達的類似物，是一種新型的抑癌基因，它通過阻斷腫瘤上皮細胞的DNA合成、抑制腫瘤基質層中腫瘤集落的形成和腫瘤細胞的有絲分裂達到阻止腫瘤細胞增殖的目的。有研究[21]證明SIP1為miR-192的靶基因。Kato等[22-23]研究發現，miR-192在纖維化的腎臟高度特異性表達，促進細胞外基質(ECM)蛋白的蓄積；并通過雙熒光素檢測確定SIPl的3′UTR為miR-192調控靶點，證明miR-192通過mRNA降解途徑降低SIPl蛋白的表達。我們的結果并沒有顯示SIP1為miR-192的靶基因，這可能與生物信息學分析的方法不同、生物信息學分析與實驗檢測存在一定的差異有關。

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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

國家自然科學基金(30770996；81172310)

200433 上海，第二軍醫大學長海醫院病理科(張晶、劉清華、余黨會、陳穎、史敏、倪燦榮、朱明華)；復旦大學附屬婦產科醫院病理科(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)