呂順莉 高軍 杜奕奇 黃浩杰 王小瑋 金晶 龔燕芳 張玲 李兆申

·論著·

胰腺癌細(xì)胞株原鈣黏附蛋白8基因的甲基化狀態(tài)

呂順莉 高軍 杜奕奇 黃浩杰 王小瑋 金晶 龔燕芳 張玲 李兆申

目的分析原鈣黏附蛋白8(protocadherin 8，PCDH8)基因在胰腺癌細(xì)胞株的甲基化狀態(tài)。方法抽提6株胰腺癌細(xì)胞株P(guān)ANC1、ASPC1、BxPC3、CFPAC、PaTu8988、SW1990和2例正常胰腺組織的總RNA，以甲基化特異性PCR(MSP)法檢測(cè)PCDH8甲基化情況。應(yīng)用DNA甲基化轉(zhuǎn)移酶(DNMT)抑制劑5-氮雜-2′-脫氧胞苷(5-Aza-dC)處理6株胰腺癌細(xì)胞株，采用實(shí)時(shí)定量PCR法檢測(cè)處理前后細(xì)胞的PCDH8 mRNA表達(dá)。結(jié)果2例正常胰腺組織PCDH8基因未發(fā)生甲基化， PANC1、BxPC3、CFPAC胰腺癌細(xì)胞株 PCDH8基因部分甲基化，而PaTu8988、ASPC1、SW1990細(xì)胞完全甲基化。PCDH8 mRNA在PANC1、SW1990、PaTu8988胰腺癌細(xì)胞株中有表達(dá)，表達(dá)的相對(duì)值(RQ)分別為1.576±0.648、0.013±0.008、0.002±0.001；BxPC3、CFPAC、ASPC1細(xì)胞株無PCDH8 mRNA表達(dá)。5-Aza-dC處理后，胰腺癌細(xì)胞株P(guān)ANC1、ASPC1、BxPC3、CFPAC、PaTu8988、SW1990均有PCDH8 mRNA表達(dá)，表達(dá)量較處理前明顯升高，相對(duì)表達(dá)量分別為7.463±2.628、10.696±1.539、7.852±2.762、421.815±1.493、118.595±4.089、6.690±1.884。結(jié)論P(yáng)CDH8基因啟動(dòng)子高甲基化是導(dǎo)致該基因在胰腺癌細(xì)胞株表達(dá)下降的主要原因之一。

胰腺腫瘤； 甲基化； 聚合酶鏈反應(yīng)； 原鈣黏附蛋白8

DNA甲基化是在DNA甲基化轉(zhuǎn)移酶等作用下，給CpG二核苷酸胞嘧啶的第5位碳原子加上一個(gè)甲基基團(tuán)，使非甲基化狀態(tài)的CpG位點(diǎn)變?yōu)榧谆癄顟B(tài)的CpG位點(diǎn)，阻礙了轉(zhuǎn)錄因子復(fù)合體與DNA的結(jié)合及延伸，導(dǎo)致基因轉(zhuǎn)錄受抑及表達(dá)沉默。DNA的異常甲基化在腫瘤的發(fā)生發(fā)展過程中起著重要作用，抑癌基因啟動(dòng)子CpG島高甲基化可導(dǎo)致基因表達(dá)抑制，功能喪失[1-3]。原鈣黏附蛋白8(protocadherin 8，PCDH8)屬于原鈣黏附蛋白家族，其在胰腺癌發(fā)生、發(fā)展中的作用尚未見報(bào)道。為此，我們檢測(cè)胰腺癌細(xì)胞株P(guān)CDH8的表達(dá)及其甲基化狀態(tài)。

材料和方法

一、實(shí)驗(yàn)材料

ASPC1、PANC1胰腺癌細(xì)胞株購(gòu)于中國(guó)科學(xué)院細(xì)胞庫(kù)；BxPC3、CFPAC、SW1990胰腺癌細(xì)胞株購(gòu)于美國(guó)ATCC公司；PaTu8988細(xì)胞株為德國(guó)Marburg Phillips大學(xué)Elsasser博士惠贈(zèng)。2例正常胰腺組織取自上海長(zhǎng)海醫(yī)院胰腺外科因胰腺囊腫行手術(shù)治療的患者，均獲得患者的知情同意。

二、甲基化特異性PCR

以酚氯仿法抽提2例正常胰腺組織和6株胰腺癌細(xì)胞株的DNA，分光光度計(jì)(NanoDrop ND-1000，美國(guó))測(cè)定DNA純度和濃度。參照亞硫酸鹽處理試劑盒說明書(QIAgen)對(duì)抽提的DNA進(jìn)行亞硫酸鹽處理。通過methyl-primer express1.0軟件設(shè)計(jì)引物序列。甲基化引物(M)序列已申請(qǐng)專利(專利號(hào)為201010125668.0)，產(chǎn)物大小為125 bp；非甲基化引物(U)上游5′-GAGGTGGTGTAGTTTTTGTAAGAGAT-3′，下游5′-ACACTCTTTACAAACCCTATACAAAA-3′，產(chǎn)物大小為125 bp，均由上海英駿生物有限公司合成。采用甲基化特異性PCR(MSP)法檢測(cè)甲基化狀態(tài)。PCR反應(yīng)條件：95℃ 3 min；95℃ 30 s，61.3℃(M引物)或57.3℃(U引物) 30 s，72℃ 30 s，共35循環(huán)；72℃ 10 min。PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳，復(fù)日FR-980生物電泳圖像分析系統(tǒng)攝影。

三、實(shí)時(shí)定量PCR

所有胰腺癌細(xì)胞株常規(guī)培養(yǎng)，待細(xì)胞生長(zhǎng)至60%時(shí)更換含5-氮雜-2′-脫氧胞苷(5-Aza-dC)的培養(yǎng)液繼續(xù)培養(yǎng)48 h。采用Trizol試劑(Invitrogen)提取總RNA。分光光度計(jì)法測(cè)定RNA純度和濃度。取5 μg總RNA在20 μl反應(yīng)體系逆轉(zhuǎn)錄合成cDNA。取1 μl cDNA行實(shí)時(shí)定量PCR反應(yīng)。PCDH8上游引物5′-ATGAGTCCTGTGAGGCGTTG-3′，下游引物5′-GCTTGTGTCACCCGATACTTT-3′，產(chǎn)物大小為186 bp；內(nèi)參GAPDH上游引物5′-GCACCGTCAAGGCTGAGAAC-3′，下游引物5′-ATGGTGGTGAAGACGCCAGT-3′，產(chǎn)物大小為142 bp。均由上海英駿生物有限公司合成。PCR反應(yīng)條件：95℃ 10 s，95℃ 5 s，60℃ 34 s， 40循環(huán)。利用Real-Time PCR儀自帶軟件獲得Ct值，以2例正常胰腺組織作為對(duì)照。△△Ct=△Ct待測(cè)樣品-△Ct正常胰腺?！鰿t待測(cè)樣品=Ct待測(cè)樣品-Ct內(nèi)參，△Ct正常胰腺=Ct正常胰腺-Ct內(nèi)參，表達(dá)量RQ=2-△△C。實(shí)驗(yàn)重復(fù)3次，取均值。

結(jié) 果

一、胰腺癌細(xì)胞株P(guān)CDH8基因的甲基化狀態(tài)

2例正常胰腺組織PCDH8未發(fā)生甲基化， PANC1、BxPC3、CFPAC細(xì)胞 PCDH8基因部分甲基化，PaTu8988、ASPC1、SW1990細(xì)胞PCDH8基因完全甲基化(圖1)。

二、5-Aza-dC處理后胰腺癌細(xì)胞株P(guān)CDH8 mRNA的表達(dá)變化

5-Aza-dC 處理前PANC1、SW1990、PaTu8988細(xì)胞有PCDH8 mRNA表達(dá)，表達(dá)量分別為1.576±0.648、0.013±0.008、0.002±0.001；BxPC3、CFPAC、ASPC1細(xì)胞株無PCDH8 mRNA表達(dá)。5-Aza-dC處理后，胰腺癌細(xì)胞株P(guān)ANC1、ASPC1、BxPC3、CFPAC、PaTu8988、SW1990均有PCDH8 mRNA表達(dá)，表達(dá)量分別為7.463±2.628、10.696±1.539、7.852±2.762、421.815±1.493、118.595±4.089、6.690±1.884(圖2)，均較處理前明顯升高。

圖1MSP法檢測(cè)正常胰腺組織(N1,N2)和胰腺癌細(xì)胞株中PCDH8基因的甲基化狀態(tài)

討 論

原鈣黏附蛋白為膜結(jié)合蛋白，屬鈣黏蛋白大家族中具有特殊功能的新一類分支家族。該家族除具有黏附功能外，其胞內(nèi)區(qū)參與細(xì)胞骨架形成和胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)，調(diào)控細(xì)胞的生長(zhǎng)和分化[4-5]。依據(jù)在基因組的分布和種系發(fā)生學(xué)等情況主要分為成簇和非成簇原鈣黏附蛋白，其中非成簇原鈣黏附蛋白又包含Delta型和其他類型家族，Delta型家族又分Delta1和Delta2兩亞型，兩者區(qū)別在于胞內(nèi)區(qū)包含的保守基序。研究發(fā)現(xiàn)，多數(shù)原鈣黏附蛋白家族均顯示為抑癌基因的特性[6-14]：在腫瘤組織中高甲基化、雜合性缺失、突變和低表達(dá)，相鄰正常組織低甲基化和高表達(dá)，轉(zhuǎn)染野生型可導(dǎo)致細(xì)胞生長(zhǎng)抑制、遷移抑制等抑制腫瘤惡性表型的功能[6, 10-12]。

圖25-Aza-dC處理前后胰腺癌細(xì)胞株P(guān)CDH8 mRNA的表達(dá)量(LogRQ)

本實(shí)驗(yàn)結(jié)果顯示，PCDH8基因在6株胰腺癌細(xì)胞株均存在不同程度的甲基化，其中PANC1、BxPC3、CFPAC細(xì)胞部分甲基化，而PaTu8988、ASPC1、SW1990細(xì)胞完全甲基化；2例正常胰腺組織PCDH8未發(fā)生甲基化。PANC1、SW1990、PaTu8988細(xì)胞有PCDH8 mRNA表達(dá)，而BxPC3、CFPAC、ASPC1細(xì)胞株無PCDH8 mRNA表達(dá)。胰腺癌細(xì)胞株P(guān)CDH8甲基化狀況和mRNA表達(dá)的結(jié)果不一致，提示PCDH8基因在胰腺癌中低表達(dá)的原因不只是高甲基化導(dǎo)致，還存在其他原因共同參與，可能包括雜合性缺失、突變等原因。

目前已有針對(duì)DNA甲基化轉(zhuǎn)移酶(DNMT)的抑制劑，5-Aza-dC是其中去甲基化作用最強(qiáng)的一種，屬于非特異性的去甲基化藥物[15]，本實(shí)驗(yàn)應(yīng)用該抑制劑對(duì)6株胰腺癌細(xì)胞株進(jìn)行干預(yù)，檢測(cè)處理前后mRNA表達(dá)水平，結(jié)果顯示處理后PCDH8的mRNA表達(dá)量增加，證實(shí)了PCDH8基因的高甲基化是導(dǎo)致低表達(dá)的一個(gè)重要因素，但不是唯一的因素。

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2009-10-23)

(本文編輯：呂芳萍)

MethylationofPCDH8inpancreaticcarcinomacelllines

LVShun-li,GAOJun,DUYi-qi,HUANGHao-jie,WANGXiao-wei,JINJing,GONGYan-fang,ZHANGLing,LIZhao-shen.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

Correspondingauthor:LIZhao-shen,Email:zhsli@81890.net

ObjectiveTo investigate the methylation status of PCDH8 gene in pancreatic carcinoma.MethodsMethylation of PCDH8 gene in 2 samples of normal pancreatic tissues and 6 pancreatic carcinoma cell lines (PANC1, ASPC1, BxPC3, CFPAC, PaTu8988 and SW1990) was detected by the methylation-specific PCR (MSP) method. The expression of PCDH8 mRNA was detected with 5-Aza-2-deoxycytidine (5-Aza-dC) treatment, a kind of DNA methyltransferase (DNMT) inhibitor in 6 pancreatic carcinoma cell lines by real-time-PCR.ResultsThe methylation of PCDH8 gene was not detected in normal tissues, while it was partially methylated in PANC1, BxPC3, CFPAC and it was totally methylated in PaTu8988, ASPC1, SW1990. PCDH8 mRNA was expressed in PANC1, SW1990, PaTu8988 and the relative quantities of mRNA expression (RQ) were 1.576±0.648, 0.013±0.008, 0.002±0.001; PCDH8 mRNA was not expressed in BxPC3, CFPAC, ASPC1. After 5-Aza-dC treatment, PCDH8 mRNA was expressed in PANC1, ASPC1, BxPC3, CFPAC, PaTu8988, SW1990 and the relative quantities of mRNA expression all significantly increased, and they were 7.463±2.628, 10.696±1.539, 7.852±2.762, 421.815±1.493, 118.595±4.089, 6.690±1.884.ConclusionsThe methylation of PCDH8 gene may be the major mechanism of down-regulated expression of PCDH8 gene in pancreatic carcinoma.

Pancreatic neoplasms; Methylation; Polymerase chain reaction; Protocadherin 8

10.3760/cma.j.issn.1674-1935.2010.03.014

200433 上海，第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院消化內(nèi)科

李兆申，Email:zhsli@81890.net