胰腺癌細(xì)胞株P(guān)ANC1異常甲基化miRNA的分析

彭泉 張立潔 蔡輝華 高文濤 趙成功 錢祝銀 苗毅

·論著·

胰腺癌細(xì)胞株P(guān)ANC1異常甲基化miRNA的分析

彭泉 張立潔 蔡輝華 高文濤 趙成功 錢祝銀 苗毅

目的分析胰腺癌細(xì)胞株P(guān)ANC1與正常胰腺組織表達(dá)有差異的啟動(dòng)子區(qū)甲基化miRNA，尋找與胰腺癌相關(guān)的高甲基化miRNA。方法抽提PANC1與正常胰腺組織基因組DNA，超聲斷裂。應(yīng)用抗5-甲基化嘧啶核苷抗體和免疫磁珠法獲取甲基化DNA片段。通過(guò)DNA甲基化芯片篩選出PANC1與正常胰腺組織表達(dá)差異的高甲基化miRNA，采用重亞硫酸鹽修飾的PCR(BSP)和TA克隆測(cè)序的方法進(jìn)行驗(yàn)證。提取胰腺癌細(xì)胞株BxPC3、CFPAC1、PANC1、SW1990基因組DNA，采用結(jié)合重亞硫酸鹽的限制性內(nèi)切酶法(combined bisulfite restriction analysis，COBRA)驗(yàn)證芯片篩選出的差異表達(dá)的甲基化miRNA。結(jié)果PANC1細(xì)胞與正常胰腺組織存在8個(gè)差異表達(dá)的甲基化miRNA，從中挑選出5個(gè)進(jìn)行BSP+TA克隆測(cè)序驗(yàn)證，其中miR-615、miR-663、miR-663b在PANC1細(xì)胞的甲基化率明顯高于正常組織(60.6%比7.6%,88.8%比22.2%,94.4%比13.0%)；miR-675的甲基化率與正常胰腺組織無(wú)明顯差異(76.0%比100%)；miR1826因測(cè)序結(jié)果誤差較大而舍去。經(jīng)COBRA驗(yàn)證，PANC1的上述4種miRNA均高甲基化；BxPC除miR-675外，其他3種均高甲基化；CFPAC1的miR-663、miR-663b高甲基化；SW1990的miR-615、miR-663高甲基化。結(jié)論胰腺癌細(xì)胞株與正常胰腺組織間存在差異表達(dá)的高甲基化miRNA，其中miR-663高甲基化與胰腺癌可能相關(guān)。

胰腺腫瘤； DNA甲基化； 微RNAs； TA克隆

DNA甲基化(DNA methylation)是最早被發(fā)現(xiàn)的表觀遺傳學(xué)改變途徑之一。位于啟動(dòng)子區(qū)的CpG島甲基化通常會(huì)導(dǎo)致基因表達(dá)沉默，是許多腫瘤發(fā)生中一個(gè)頻發(fā)的早期事件。微小RNA(miRNAs)是重要的轉(zhuǎn)錄后調(diào)節(jié)機(jī)制[1]，在腫瘤中表達(dá)異常，并且調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞增殖、凋亡等重要生物學(xué)過(guò)程。自Saito等[2]在2006年首次報(bào)道m(xù)iRNA-127受到DNA甲基化調(diào)控起抑癌基因作用以來(lái)，miRNA的啟動(dòng)子高甲基化導(dǎo)致其表達(dá)下調(diào)的報(bào)道增多[3-7]。本研究旨在尋找胰腺癌相關(guān)的異常甲基化的miRNA，以期對(duì)胰腺癌的診斷和治療發(fā)揮推動(dòng)作用。

材料與方法

一、材料

胰腺癌細(xì)胞株P(guān)ANC1、BxPC3、CFPAC1、SW1990均購(gòu)自上海細(xì)胞庫(kù)，常規(guī)培養(yǎng)、傳代，取對(duì)數(shù)生長(zhǎng)期細(xì)胞。3例正常胰腺組織取自意外死亡者，均獲得家屬同意，并簽署知情書(shū)。

二、DNA甲基化芯片分析

應(yīng)用DNA提取試劑盒(Omega公司)抽提胰腺癌細(xì)胞株P(guān)ANC1與正常胰腺組織基因組DNA，超聲打斷成400～500 bp的DNA片段，加熱變性為單鏈DNA。部分單鏈DNA樣品中加入抗5-甲基化胞嘧啶核苷抗體，使用免疫磁珠法分離甲基化的DNA片段。將甲基化DNA樣本及未處理的DNA片段樣本分別進(jìn)行擴(kuò)增，熒光標(biāo)記，標(biāo)記后將兩樣本混合、變性、與上?？党缮锕咎峁┑募谆疍NA微陣列芯片雜交，用高解析度芯片掃描儀檢測(cè)雜交信號(hào)，對(duì)結(jié)果進(jìn)行數(shù)據(jù)分析。

三、重亞硫酸鹽修飾的PCR(BSP)和TA克隆測(cè)序

對(duì)PANC1和正常胰腺組織的基因組DNA進(jìn)行重亞硫酸鹽處理，使未甲基化的胞嘧啶轉(zhuǎn)化成胸腺嘧啶，而甲基化的胞嘧啶不變。亞硫酸氫鹽修飾過(guò)程見(jiàn)文獻(xiàn)[8]。以重亞硫酸鹽處理后的DNA作為模板，采用Touchdown-PCR方法擴(kuò)增。引物由Invitrogen公司設(shè)計(jì)并合成。引物盡量設(shè)計(jì)位于miRNA發(fā)夾序列的上游500 bp之內(nèi)[9]，miRNA引物序列及與相鄰CpG位置、擴(kuò)增范圍見(jiàn)表1和圖1。擴(kuò)增產(chǎn)物經(jīng)電泳分離，回收、純化。純化的擴(kuò)增產(chǎn)物行TA克隆。挑選6個(gè)陽(yáng)性單克隆菌落的菌液行PCR，并送Invitrogen公司測(cè)序。

表1 miRNA引物序列及擴(kuò)增位置

注：+：表示在發(fā)夾序列上游，-：表示在下游

圖1UCSC數(shù)據(jù)庫(kù)上miRNA與相鄰CpG島位置及BSP擴(kuò)增范圍

四、結(jié)合重亞硫酸鹽的限制性內(nèi)切酶法(combined bisulfite restriction analysis，COBRA)

提取胰腺癌細(xì)胞株BXPC3、CFPAC1、PANC1、SW1990基因組DNA，行BSP，應(yīng)用內(nèi)切酶對(duì)PCR產(chǎn)物進(jìn)行酶切。酶切8 h后以2%瓊脂糖凝膠電泳分離PCR產(chǎn)物，伊文思蘭(EB)染色，成像。

結(jié) 果

一、DNA甲基化芯片分析結(jié)果

芯片分析公司提供序列上游2000 bp范圍內(nèi)覆蓋10個(gè)以上探針的miRNA共52個(gè)，其中PANC1細(xì)胞株與正常胰腺組織甲基化程度差異明顯的miRNA共8個(gè)，分別為miR-483、miR-574、miR-615、miR-663、miR-663b、miR-675、miR-1247、miR-1258、miR-1826(圖2)。

圖2在PANC1和正常胰腺組織差異表達(dá)的甲基化miRNA(綠色部分)

二、BSP+TA克隆測(cè)序結(jié)果

隨機(jī)挑選5個(gè)miRNA(miR-615、miR-663、miR-663b、miR-675、miR-1826)進(jìn)行BSP。PCR反應(yīng)：95℃ 5 min,94℃ 30 s、68℃ 30 s、72℃ 30 s，2個(gè)循環(huán)；95℃ 5 min，94℃ 30 s、66℃ 30 s、72℃ 30 s，3個(gè)循環(huán)；95℃ 5 min,94℃ 30 s、64℃ 30 s、72℃ 30 s，4個(gè)循環(huán)；95℃ 5 min,94℃ 30 s、62℃ 30 s、72℃ 30 s，35個(gè)循環(huán)，均獲得目的條帶(圖3)。TA克隆陽(yáng)性菌液測(cè)序結(jié)果見(jiàn)圖4。miR-615、miR-663、miR-663b在PANC1中的甲基化率明顯高于正常組織(60.6%比7.6%，88.8%比22.2%，94.4%比13.0%)；miR-675在PANC1和正常胰腺組織中的甲基化率無(wú)明顯差異(76.0%比100%)；miR-1826因測(cè)序結(jié)果誤差大而舍去。

圖3 5個(gè)miRNA的 BSP產(chǎn)物電泳圖

圖4miR-615、miR-663、miR-663b、miR-675經(jīng)TA克隆后測(cè)序結(jié)果 實(shí)心圓：甲基化的CpG島；空心圓：未發(fā)生甲基化的CpG島

三、COBRA驗(yàn)證結(jié)果

PANC1的上述4種miRNA均高甲基化；BxPC3除miR-675外，其他3種均高甲基化；CFPAC1的miR-665、miR-663b高甲基化；SW1990的miR-615、miR-663高甲基化(圖5)。

討 論

miRNA是21～25 nt長(zhǎng)的單鏈小分子RNA，廣泛存在于真核生物中。miRNA參與生命過(guò)程中一系列的重要進(jìn)程，包括早期胚胎發(fā)育、細(xì)胞分化、增殖和凋亡等，其中的一些miRNA可以調(diào)控正常細(xì)胞周期和終末分化，如發(fā)生突變可導(dǎo)致細(xì)胞增殖和凋亡異常。近年來(lái)專家們對(duì)與腫瘤相關(guān)的miRNA的研究取得不少新進(jìn)展。Chen等[10]發(fā)現(xiàn)，miR-17-192與miR-155在鼻咽癌中表達(dá)上調(diào)；miR-34、miR-143和miR-145的表達(dá)則顯著下調(diào)。這些表達(dá)下調(diào)的miRNA與多種生物學(xué)途徑相關(guān)，如TGF-Wnt途徑、VEGF信號(hào)轉(zhuǎn)導(dǎo)通路和IP3信號(hào)轉(zhuǎn)導(dǎo)通路等。此外，在包括胰腺癌在內(nèi)的多種腫瘤組織中亦發(fā)現(xiàn)多種miRNA的表達(dá)和功能異常[11-14]。Lu等[15]還證明miRNA的表達(dá)特征能用于劃分人類癌癥以及區(qū)別正常細(xì)胞和癌細(xì)胞，表明miRNA的活動(dòng)模式還能夠被用于診斷癌癥。2009年Rabinowits等[16]的研究已指出，miRNA可以用來(lái)對(duì)肺癌進(jìn)行篩選。

B:BxPC1；P:PANC1；C：CFPAC1；SW：SW1990

圖54個(gè)miRNA在胰腺癌細(xì)胞株中的甲基化狀況

目前，對(duì)miRNA發(fā)生異常表達(dá)的調(diào)控機(jī)制研究尚處于初級(jí)階段，但已有研究證實(shí)miRNA受多種因素調(diào)控，例如染色質(zhì)結(jié)構(gòu)異常、miRNA加工受損等。近幾年有多篇報(bào)道指出，啟動(dòng)子區(qū)的甲基化程度會(huì)影響miRNA的表達(dá)水平，表明DNA甲基化異常也是miRNA表達(dá)異常的重要機(jī)制之一[3-7]。

本實(shí)驗(yàn)通過(guò)甲基化芯片篩選出52個(gè)miRNA，選取其中5個(gè)進(jìn)行TA 克隆驗(yàn)證，發(fā)現(xiàn)miR-615、miR-663、miR-663b在胰腺癌細(xì)胞株P(guān)ANC1中存在高甲基化，并通過(guò)BxPC3、CFPAC1、SW1990胰腺癌細(xì)胞驗(yàn)證，只有miR-663在4株胰腺癌細(xì)胞中均為高甲基化，它可能是胰腺癌相關(guān)的特異性的高甲基化miRNA。當(dāng)然，這還有待于在大樣本的胰腺癌組織標(biāo)本中進(jìn)行驗(yàn)證。

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AbnormalmethylationofmiRNAinpancreaticcancercelllinePANC1

PENGQuan,ZHANGLi-jie,CAIHui-hua,GAOWen-tao,ZHAOCheng-gong,QIANZhu-yin,MIAOYi.
DepartmentofGeneralSurgery,The105thCentralHospitalofPeople′sLiberationArmy,Hefei230032,China

QIANZhu-yin,Email:qianzhusilver@163.com

ObjectiveTo investigate the methylation of the promoter region in miRNA in pancreatic cancer cell line PANC1 and normal pancreatic tissue, to discover the miRNA with hypermethylation associated with pancreatic cancer.MethodsThe genomic DNA of PANC1 and normal pancreatic tissue was extracted, and fractured by ultrasound. Methylation DNA fragments were obtained by 5-methyl of pyrimidine nucleoside antibodies and immunomagnetic beads. The hypermethylation miRNA differentially expressed between PANC1 and normal pancreatic tissue was selected by using methylation DNA chip. BSP (bisulfite genomic sequencing PCR) and TA clone sequencing was performed for further validation. The genomic DNA of pancreatic cancer cell lines BXPC3, CFPAC1, PANC1 and SW1990 was extracted. The COBRA (combined bisulfite restriction analysis) was used to validate differentially expressed hypermethylation miRNA.ResultsEight differentially expressed hypermethylation miRNAs were screened from the DNA methylation chips, then five of them were selected for sequencing. The methylation status of miRNA-615,-663,-663b was significantly higher in the PANC1 than in normal tissues (60.6%vs7.6%, 88.8%vs22.2%, 94.4%vs13.0%); the methylation status of miRNA-675 was not significantly different between PANC1 and normal pancreatic tissue (76.0%vs100%). Due to large error in sequencing, miRNA1826 was excluded. The results of COBRA confirmed all the 4 miRNAs were highly methylated in PANC1; except for miRNA-675, other 3 miRNAs were highly methylated in BxPC,miRNA-663, miRNA-663b were highly methylated in CFPAC1, while miRNA-615, miRNA-663 were highly methylated in SW1990.ConclusionsHypermethylation miRNAs were differentially expressed between pancreatic cancer cell lines and normal pancreatic tissue, among them, highly methylated miRNA-663 was possibly associated with pancreatic cancer.

Pancreatic neoplasms; DNA methylation; miRNA; TA cloning

10.3760/cma.j.issn.1674-1935.2012.01.004

江蘇省自然科學(xué)基金(BK2006241)；人事部留學(xué)回國(guó)人員基因(303070460IA10)

230032 安徽合肥，解放軍第105醫(yī)院普外科(彭泉、趙成功)，腫瘤科(張立潔)；南京醫(yī)科大學(xué)第一附屬醫(yī)院普外科(蔡輝華、高文濤、錢祝銀、苗毅)

錢祝銀，Email：qianzhusilver@163.com

2011-04-06)

(本文編輯：屠振興)