mTOR信号通路在胰腺癌发病中的作用

江华 沈晓莹 刘雁冰 张小燕 熊伍军 郜恒骏

·论著·

mTOR信号通路在胰腺癌发病中的作用

江华 沈晓莹 刘雁冰 张小燕 熊伍军 郜恒骏

目的检测mTOR信号通路在胰腺癌组织中的表达，探讨其在胰腺癌发病中的作用。方法选择经手术、病理证实的6例胰腺癌及相应癌旁组织，抽提RNA，应用Agilent人全基因表达谱芯片进行检测，生物信息学分析mTOR信号通路在胰腺癌组织中的表达。结果总共筛选到1276个差异基因，其中癌组织上调的有691个，下调的有585个。KEGG通路的Enrichment和基因计数两项指标得分最高的是mTOR信号通路中的hsa04150，其Enrichment为4.5622519，基因计数为9，基因计数百分率为1.15%，EASE ScoreP值为6.23E-04，最有生物学意义，其中ULK2、PIK3R3、PDPK1、EIF4EBP1、PGF、VEGFB、ULK3、RICTOR与PIK3R5等9个关键基因具有显著性差异(P值均<0.05)。结论胰腺癌发病与mTOR信号通路激活密切相关。

胰腺肿瘤； mTOR； 信号通路； 基因表达谱

近年来，尽管胰腺癌分子发病机制的研究已经取得了较大的进展，但早期诊治胰腺癌依然很困难。因此，广泛、深入研究胰腺癌发病的分子机制，寻求早期诊断的特异性标记物和特异性治疗靶点是有效防治胰腺癌、改善患者预后的关键。最近发现，mTOR信号通路在人某些恶性肿瘤异常激活[1-2]。本研究探讨mTOR信号通路在胰腺癌中的状况。

材料与方法

一、总RNA抽提及标记

取6例性别、年龄等均无明显差异的胰腺癌及相应癌旁组织新鲜标本，采用Trizol(Invitrogen, Carlsbad, USA)抽提组织总RNA，使用间接法进行样品标记。具体步骤：以 1 μg总RNA为起始量，T7-oligo(dT)为引物反转录合成cDNA一链、二链。柱纯化后使用Agilent Low RNA Input Linear Amplification Kit(Agilent, Pal alto, USA)进行体外转录放大，在此过程中以修饰的UTP[aaUTP,5-(3-aminoallyl)-UTP]替换UTP。掺入的aaUTP可以与Cy3 NHS ester起反应，形成荧光标记产物。荧光的掺入率用Nanodrop ND-1000进行检测。

二、芯片杂交及数据分析

使用Agilent Oligonucleotide Microarray in Situ Hybridization Plus Kit 配制杂交混合物。750 ng的荧光标记物在60℃进行片段化后加在Human Whole-Genome 60-mer oligo-chips 仪(G4112F, Agilent Technologies)上进行杂交，杂交温度为60℃，转速10 r/min，时间17 h。杂交结束后，根据Agilent提供的方法使用Agilent Gene Expression Wash Buffer Kit洗涤。然后用Agilent扫描仪和提供的软件Feature Extraction获得原始信号值。对原始信号进行标准化处理，即中位数调整对齐，过滤低表达量的探针。筛选指标为“gIsFound”是否为0和1，筛选方法为严格选择所有样本中“gIsFound”等于1的探针；合并重复探针。然后利用成对T检验分析，对每个探针平行计算P值，根据P≤0.05来筛选癌和癌旁组织的差异探针，将筛选到的探针对应的GeneBank Accession number转换成Entrez IDs。

三、KEGG体积分析

KEGG通路数据库是应用最为广泛和注释信息较全面的数据库。具有显著性通路分类的判断标准为EASE ScoreP值≤0.05，且显著性通路分类按照P值从低到高进行排序，同时还根据实际差异基因在各个通路分类中的相对比率，得出通路分类的Enrichment，该值越高说明差异基因占该分类的比重越大，也说明该通路分类参与表达差异的贡献越高。Enrichment和基因计数两项指标得分最高的通路分类最有生物学意义。

结 果

一、样品RNA的鉴定

所有样本的总RNA电泳条带清晰，28S与18S rRNA条带亮度接近2∶1，紫外分光光度计检测A260/A280介于1.8～2.1，符合表达谱芯片实验的要求。

二、基因芯片杂交

基因芯片符合质控标准，实验体系稳定，荧光信号强度强、均一(图1)。12张芯片均有27 574个表达信号的探针，占总41 000个探针数的67.3%。

图1 基因芯片杂交荧光信号图

三、基因芯片数据分析

共筛选到1276个差异基因。其中，癌组织上调691个，下调585个。KEGG通路的Enrichment和基因计数两项指标得分最高的是mTOR信号通路中的hsa04150，其Enrichment为4.5622519，基因计数为9，基因计数百分率为1.15%，P=6.23E-04，其中，ULK2、PIK3R3、PDPK1、EIF4EBP1、PGF、VEGFB、ULK3、RICTOR、PIK3R5等9个关键基因具有显著性差异(P值均<0.05，表1)。

讨 论

TOR基因是于1991年在酵母作为雷帕霉素(rapamycin)靶蛋白时被发现的，与酵母TOR结构和功能相应的哺乳动物TOR称mTOR(mammalian target of rapamycin)，是一种非典型的丝氨酸/苏氨酸蛋白激酶，分子质量为289 000。人体的某些疾病，包括某些遗传性疾病和糖尿病的发生被认为与mTOR信号通路异常有关[3]，某些恶性肿瘤亦存在mTOR信号通路的异常激活[1-2]。该信号通路可作为肿瘤治疗的靶点[4-6]。

ULK2参与调节最基本的生物学过程[7]，在细胞自我吞噬的激活中起着关键的作用，ULK2激酶蛋白的失活可以抑制自我吞噬过程[8]。Soroceanu等[9]报道，IGF2-PIK3R3信号轴参与促进人神经胶质母细胞瘤的发生。Zhang等[10]报道，PIK3R3可作为上皮性卵巢癌治疗的潜在靶点。但PIK3R5与肿瘤相关性尚未见报道。EIF4EBP1、人胎盘型PGF[11]、VEGFB[12]与多种肿瘤相关，前者具有肿瘤抑制功能，可用作胃肠道肿瘤预后评估[13]。本结果显示，胰腺癌组织ULK2、PIK3R3、PIK3R5、EIF4EBP1、PGF、VEGFB表达下调，而ULK3表达上调，它们在胰腺癌发生中的作用值得进一步探讨。

表1 胰腺癌相关的mTOR信号通路关键基因

PDPK1在胃癌、结肠癌、肺癌、口腔鳞状细胞癌中表达异常[14]。本结果与其一致。Chakraborty等[15]发现了一种与mTOR蛋白质相互作用的新蛋白——人mTOR的雷帕霉素不敏感组分(RICTOR)，这两种蛋白质结合形成的复合物能够使Akt蛋白质的氨基酸磷酸化，从而激活Akt蛋白的活性。本结果亦显示RICTOR在胰腺癌显著上调，有望成为胰腺癌重要的诊断标记物与治疗靶点。

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2010-04-06)

(本文编辑：吕芳萍)

RoleofmTORsignalingpathwayinthepathogenesisofpancreaticcancer

JIANGHua,SHENXiao-ying,LIUYan-bing,ZHANGXiao-yan,XIONGWu-jun,GAOHeng-jun.

DepartmentofGastroenterology,EastHospital,TongjiUniversity,Shanghai200120,China

JIANGHua,Email:huajiang666@yahoo.com.cn;GAOHeng-jun,Email:hengjun_gao@shbiochip.com

ObjectivesTo investigate the expression of mTOR signaling pathway in pancreatic cancer and export its signification.Methods6 samples of pancreatic cancer and its paracancerous tissues specimens confirmed by surgery and pathologic examination were selected. RNA was extracted and expression profiles experiment was performed by using Agilent human whole genomic oligonucleotide microarrays. The expression of mTOR signaling pathway in pancreatic cancer was analyzed by bioinformatics.ResultsTotally 1276 differential gene were selected, and 691 were up-regulated in cancer tissue, while 585 were down-regulated. The highest score of KEGG pathway′s Enrichment and gene count was hsa04150 in mTOR signaling pathway, with its Enrichment of 4.5622519 and gene count of 9, and the percentage of gene count was 1.15%, the EASE ScorePvalue was 6.23E-04, which had the most biological significance. Among those, there was significantly difference of expression of nine key genes including ULK2, PIK3R3, PDPK1, EIF4EBP1, PGF, VEGFB, ULK3, RICTOR and PIK3R5(P<0.05).ConclusionsThe pathogenesis of pancreatic cancer is related to the activation of the mTOR signaling pathway.

Pancreatic neoplasms; mTOR; Signaling pathway; Gene expression profiling

10.3760/cma.j.issn.1674-1935.2011.01.010

上海市浦东新区科委科技创新资金项目(PKJ2007-Y10)

200120 上海，同济大学附属东方医院消化内科(江华、刘雁冰、熊伍军)；同济大学附属同济医院消化疾病研究所(郜恒骏)；生物芯片上海国家工程中心(沈晓莹、张小燕、郜恒骏)

江华，Email：huajiang666@yahoo.com.cn，郜恒骏，hengjun_gao@shbiochip.com