摘要
结直肠癌是严重危害我国人民健康的常见病，其发病率近年上升趋势明显。尽管结直肠癌发生发展的分子机制取得一定进展，仍有许多已知或未知的基因有待于进一步探索。随着人类基因组计划、单体型计划和癌症基因组解剖计划等大规模测序项目的完成，公共数据库积累了大量的基因组序列信息。表达序列标签(expressed sequence tag，EST)是cDNA单次测序结果，是寻找基因的重要方法。挖掘公共EST数据库(database of EST，dbEST)，将为我们的研究带来新思路。
本文从dbEST获取结直肠正常、炎症性肠病、腺瘤和腺癌等相关EST，利用dbEST和UniGene数据库的序列注释特征，开发生物信息学软件GetUni，成功实现了从EST到UniGene的聚类，构建结直肠正常(N)、炎症性肠病(IBD)、腺瘤(A)和腺癌(T)四个电子文库，各文库分别包含4375、3451、875和200608个UniGene，4108、2230、606和18891个非冗余UniGene，及4108、2201、592和14879个基因。美国癌症研究所(National Cancer Institute，NCI)结直肠正常及癌组织cDNA文库Xprofiler交叉验证GetUni的转化效率。电子文库间的直接比对以及基于Gene Ontology的生物信息学分析发现：除bA9F11.1(Hs．329040)外，T文库包括了N、IBD和A等3个文库的全部基因。T文库每个基因平均含1.27个转录子，显著高于其它三个文库(p＜0.01)。生物信息学分析发现50条信号通路在文库间富集程度差异有统计学显著性(p＜0.01)，核糖体蛋白基因与糖酵解／糖异生通路基因在文库A和IBD相对富集，整合素信号通路基因在N和IBD相对富集，七次跨膜受体(rhodopsin family)家族则在T文库相对富集。Q-PCR检测结果发现，RPS2、RPS12、RPS27a、RPL7a、RPL5和RPL10等6个核糖体蛋白基因分别有5例、6例、3例、5例、2例和3例腺瘤表达相对正常表达增高，21例、18例、17例、14例、21例和13例结直肠癌表达相对增高。但各基因在正常、腺瘤和腺癌等不同组织之间的表达水平差异未发现有显著统计学意义(p＞0.05)。系统聚类把腺瘤、结直肠癌分成核糖体蛋白基因富集程度较高的A组和相对较低的B组，各有25例(腺瘤7例)和23例(腺瘤1例)结直肠肿瘤，提示腺瘤核糖体蛋白基因富集程度显著高于结直肠癌(87.5％，7／8例腺瘤vs 45％，18／40例结直肠癌)，差异有统计学意义(p=0.020)。
在结直肠组织电子表达谱生物信息学分析的基础上，选取95个基因构建整合TaqMan定量PCR和微流体技术的低密度芯片(Low density array，LDA)，进行高通量定量PCR研究。这些基因大多与结直肠正常粘膜上皮分化关系密切，包括Wnt、TGFβ／BMP4、Hedgehog和Notch等四条信号通路、Polycomb Group(PcG)基因家族及其它细胞分化发育相关基因。96个基因(含GAPDH)中，仅DAAM1未见有效扩增信号。26例结直肠癌配对组织(包括27个癌、7个腺瘤及配对正常组织)LDA检测总体阳性率为97.2％(11520／11844)。同一组织不同LDAGAPDH检测Ct值之差最大为0.952，最小0.001，平均Ct值之差在0.279±0.254。Spearman相关分析和Logistic回归分析发现两次实验检测结果高度相关(p＜0.0001，r=0.911，r~2=0.829)。94个基因在不同实验之间的ΔCt值差异，94个基因两次实验间的ΔCt值差平均为0.36±0.33。Spearman相关分析和Logistic回归分析发现各基因两次实验间ΔCt差平均值与基因平均ΔCt值呈高度线性正相关(p＜0.0001，r=0.743，r~2=0.551)。
配对正常和腺癌组织之间LDA鉴定了79个差异表达基因(p＜0.05)，癌组织表达上调4个，下调75个，2倍以上34个。其中，Wnt信号通路基因NKD1和SOX9在结直肠癌组织表达显著上调，分别较正常上调15.15倍和1.62倍。APC、DAAM2、Tcf4等基因表达显著下调，分别较正常下调2.71倍、3.81倍和2.18倍。与正常组织相比，TGFβ／BMP4信号通路基因TGFβ1、SMAD4、L3MBTL等在结直肠癌组织分别下调2.41倍、2.51倍和1.39倍，HH信号通路基因IHH、DISP1、DISP2等在结直肠癌组织分别下调2.03倍、1.79倍和13.96倍，Notch信号通路基因NOTCH2、MAML1、MAL2、MAML3、HES1、HES2等基因在结直肠癌分别下调2.22倍、1.91倍、2.39倍、1.96倍、1.76倍、1.76倍，PcG基因家族EZH2在结直肠癌组织上调1.64倍，EPC1、EPC2、PCGF1、PCGF2、PCGF3、PCGF4、PCGF5、PCGF6等分别下调2.28倍、1.96倍、1.93倍、2.02倍、1.63倍、1.49倍、2.28倍和1.58倍。7例正常、腺瘤及腺癌配对组织LDA检测发现59个差异表达基因(p＜0.05)。在结直肠腺瘤，上述各基因的表达趋势基本上与腺癌一致。
SYBR Q-PCR检测了19个基因在22例正常、腺癌配对结直肠组织的表达，发现SOX9、EPC1、EPC2，CECR1、KLF9、METRNL、NKD1、NUMB、SPRED2、DISP2等10个基因在结直肠癌组织的表达改变有统计学显著意义(p＜0.05)。全部19个基因SYBR Q-PCR与LDA的表达趋势(2~(-ΔΔCt)值)一致。19个基因的中位表达变化倍数相关分析发现，两种方法检测结果高度相关(p=0.003，r=0.79，r~2=0.637)。
最后，我们对结直肠癌表达上调基因SOX9进行了比较全面的临床病理研究。20例结直肠癌及6个细胞系DNA测序未能发现SOX9基因编码区存在突变。21例结直肠正常、腺癌及5个伴发腺瘤配对组织Western-blot检测发现，SOX9、β-Catenin分别在16例和15例腺癌表达上调，各有5个和2个腺瘤表达上调。结直肠癌SOX9、β-catenin蛋白表达上调有显著统计学意义(Mean±SD：SOX9正常0.3293±0.3863，腺癌0.907±0.6413，p＜0.01；β-catenin正常0.3397±0.2921，腺癌0.6024±0.498，p＜0.05)。免疫组织化学染色发现，正常粘膜SOX9+细胞主要位于隐窝基底部，正常粘膜下部阳性细胞数(9.6±13.1％)显著高于上部(3.1±5.9％)(p＜0.001)，与Ki67染色的分布特征吻合。在结直肠腺瘤，SOX9+细胞沿隐窝弥漫分布，但腺瘤底部阳性细胞数要高于上部(50.7％±25.1％vs 32.9％±30.0％，p＜0.001)。结直肠腺瘤SOX9+细胞(39.8％±30.9％)要显著高于瘤旁残留黏膜(24.9％±18.2％)。结直肠癌SOX9总体表达率为36.7％±30.3％，显著高于正常粘膜和瘤旁残留粘膜(p＜0.001)，但与腺瘤表达差异无统计学意义(p＞0.05)。结直肠腺瘤SOX9阳性率为74.5％(70／94)，结直肠癌阳性率为60.1％(113／188)，腺瘤与癌的阳性率显著高于正常粘膜(10／110，9.09％)(p＜0.001)。如把“-”和“+”定义为SOX9低表达，“++”和“+++”合并为高表达，我们发现SOX9高表达分别见于50例(53.2％)结直肠腺瘤和64例(34.0％)结直肠癌，正常粘膜未见有SOX9高表达。结直肠腺瘤和癌组织SOX9高表达率显著高于正常粘膜(p＜0.001)。SOX9高表达在黏液腺癌／印戒细胞癌相对非黏液／印戒细胞癌少见(p＜0.05，x~2检验)。SOX9高表达的结直肠癌五年生存率为39.5％(17／43)，显著低于SOX9低表达的结直肠癌69.5％(66／95)(p＜0.01)。多因素COX分析发现。SOX9高表达是结直肠癌预后不良的独立指标(p＜0.05，RR=1.381，95％CI：1.051-1.815)。
通过上述研究，我们得出以下结论：
①结直肠癌在分子水平呈高度异质性，变异性剪接在结直肠癌发生过程中有重要作用，核糖体蛋白基因富集是结直肠腺瘤与炎症性肠病等癌前病变的重要分子特征。因此，结直肠电子文库的构建有助于揭示结直肠癌发生、发展过程中的总体分子特征，对于加深我们对结直肠癌发生机制的理解有重要意义。
②结直肠上皮细胞分化关系密切的Wnt信号通路激活及TGFβ／BMP、HH、Notch等信号通路抑制及PcG家族表达改变在结直肠癌的发生过程中有重要作用，且这些信号通路之间存在复杂的相互作用网络。
③Wnt信号通路下游基因SOX9与结直肠上皮细胞分化有关，其表达在结直肠腺瘤及腺癌表达增高，SOX9高表达是结直肠癌预后不良的独立评判指标。
④LDA是一种灵敏、可靠的高通量定量PCR检测方法，dbEST生物信息学分析结合LDA检测将为恶性肿瘤生物标志物的筛选提供新思路、新方法。
Colorectal cancer is one of the commonest cancers in China. The incidence of colorectal cancer is increasing rapidly in the last two decades. The molecular mechanisms underlying colorectal carcinogenesis have achieved great advance recently, but many novel genes which are associated with cancer initiation and progression remain to be explored. Astronomic data have been accumulating in the public database with the accomplishment of large-scale sequencing projects, such as human genome project, finished human genome project, haplotype project (HapMap) and cancer genome anatomy project (CGAP), etc. Expressed sequence tag (EST), a single-passed sequence of compliment DNA (cDNA), is one of the most important ways to find novel biomarkers for cancer. Mining database of EST (dbEST) from the Genobank will shed new light on cancer study.
In this manuscript, we downloaded ESTs relevant to colorectal normal mucosa, inflammatory bowel disease, adenoma and cancer from dbEST. We developed a bioinformatics software package-GetUni by utilizing the sequence annotation features in the dbEST and UniGene. We then successfully clustered all these ESTs into UniGene, thus constructed 4 electronic gene expression libraries, normal mucosa (N), inflammatory bowel disease (IBD), adenoma (A) and cancer (T). There are 4,375, 3,451, 875 and 200,608 UniGenes, 4,108,2,230, 606 and 18,891 non-redundant UniGenes, or 4,108,2,230, 592 and 14,879 genes in library N, IBD, A and T, respectively. The cDNA Xprofiler analysis of colorectal normal mucosa and cancer in the National Cancer Institute (NCI) has cross-validated the efficiency of our GetUni software package. Subsequently, overall features for these libraries were analyzed by GOTM (GOTree Machine). Genes in library N, IBD and A were all found in library T only except bA9F11.1 (Hs.329040), which was only present in library A. Each gene in library T had an average of 1.27 transcripts, significantly higher than that in other libraries (p<0.01, x~2 test). Differences among these libraries in gene enrichment were statistically significant in 50 signaling pathways as revealed by WebGestalt (p<0.01), such as enrichment of ribosome protein genes or genes of KEGG Glycolysis/Gluconeogenesis pathway in library A and IBD, Integrin pathway genes in IBD and N and 7 transmembrane receptor (rhodopsin family) genes in cancers. Quantitative PCR found elevated expression of RPS2, RPS12, RPS27a, RPL7a, RPL5 and RPL10 in 5,6, 3, 5, 2 and 3 of 8 adenomas, or 21, 18, 17, 14, 21 and 13 of 40 colorectal cancers, respectively. However, the expression of these ribosomal protein genes among normal mucosa, adenoma and cancer was not statistically significant (p>0.05). Hierarchial analysis showed 2 distinct groups of colorectal adenomas and cancers. There are 7 adenomas and 35 cancers in the group with high ribosomal protein gene enrichment and 1 adenoma and 23 cancers in the group with low ribosomal protein gene enrichment. The enrichment of ribosomal protein genes was significantly more common in colorectal adenomas (7/8, 87.5%) than that in cancers (18/40,45%) (p=0.020).
Next, we selected 95 genes from electronic gene expression profile in colorectal cancer to construct a low-density array (LDA), a high throughput PCR approach based on the combination of TaqMan quantitative PCR and microfluidic principle. All these candidate genes were focused on cell differentiation and development including 4 signaling pathway (Wnt, TGFβ/BMP4, Hedgehog and Notch), Polycomb Group (PcG) family and others. GAPDH was used as the internal control for normalization. We only failed to detect DAAM1 among all 96 genes by using LDA approach. Totally, 97.2% sample gene tests [11,520/11,844 (384×96)] were successfully amplified. To evaluate the reproductivity of LDA, we compared the threshold cycles (Ct) of GAPDH between different experiments. We found that the range of Ct variation between different times for a specific sample is 0.001～0.952, with a mean±SD of 0.279±0.254. Spearman correlation and Logistic regression analysis showed a significant correlation between different tests in all 60 samples (p<0.0001, r=0.911,r~2=0.829). We next analyzed the ΔCt variations between tests in the 94 target genes. The average ΔCt variation was 0.36±0.33. The ΔCt variation was significantly correlated with the mean of ΔCt (p<0.0002, r=0.743,r~2=0.551).
By using LDA, we identified 79 differentially expressed genes between colorectal cancers and normal mucosa (p<0.05), of which, 4 were upregulated and 75 downregulated as compared with those in the normal mucosa. Thirty-four genes had a change-fold more than 2. In Wnt pathway, NKD1 and SOX9 were significantly upregulated with medium change-folds of 15.15 and 1.62, respectively, and APC, DAAM2 and Tcf4 downregulated 2.71, 2.82 and 2.18 folds in colorectal cancers as compared with those in normal mucosa, respectively (p<0.05). In comparison with those in normal tissue, genes in TGFβ7BMP4 pathway, Hedgehog pathway and Notch pathway, were significantly downregulated in colorectal cancers with medium change-folds of TGFβ1 2.41, SMAD4 2.51, L3MBTL 1.39, IHH 2.03, DISP1 1.79, DISP2 13.96, NOTCH2 2.22, MAML1 1.91, MAML2 2.39, MAML3 1.96, HES1 1.76 and HES2 1.78, respectively (p<0.05). In PcG gene family, only EZH2 showed higher expression level in colorectal cancers with a medium fold of 1.64 and others had lower expression with medium change-folds of EPC1 2.28, EPC2 1.96, PCGF1 1.93, PCGF2 2.02, PCGF3 1.63, PCGF4 1.49, PCGF5 2.28 and PCGF6 1.58. In 7 normal, adenoma and cancer individual-matched cases, 59 differentially expressed genes were found (p<0.05). Most of these genes showed consistent expression alterations in adenomas as in cancers. We applied SYBR Green Q-PCR to detect 19 differentially expressed genes, which were identified by LDA, in 22 normal and cancer individual-matched colorectal patients. A total of 10 genes, SOX9, EPC1, EPC2, CECR1, KLF9, METRNL, NKD1, NUMB, SPRED2 and DISP2, were significantly differentially expressed in colorectal cancers (p<0.05). As expected, all of the 19 genes had the same directional changes (2~(-ΔΔCt)) between SYBR Green approach and LDA in colorectal cancers. The medium change-folds of these genes were significantly correlated between these two approaches (p=0.003, r=0.79, r2=0.637).
Finally, we carried out a large clinicopathological survey on one of the upregulated genes in colorectal caner-SOX9, a novel downstream transcriptor in the Wnt pathway. There were no significant mutations in the coding region of SOX9 in 20 colorectal cancers and 5 colonic cancer cell lines. Western-blot showed SOX9 and β-Catenin upregulation in 16 or 15 of 21 colorectal cancers and 5 or 2 of 5 adenomas, respectively. SOX9 and β-Catenin were significantly upregulated in cancers as compared with those in normal
mucosa (Mean±SD: SOX9 0.3293±0.3863 in normal mucosa, 907±0.6413 in cancer,
p<0.01;β-Catenin 0.3397±0.2921 in normal mucosa, 0.6024±0.498 in cancer, p<0.05). SOX9+ cells were invariably located in the lower part of normal colonic mucosa, showing a characteristic pattern of Ki67 staining. There were more SOX9+ cells in the lower zone (9.6±13.1%) of colonic mucosa than those in the upper zone (3.1 ±5.9%) (p<0.001). In colorectal adenomas, SOX9+ cells could be seen along the whole dysplastic crypt. However, the lower zone contained more SOX9+ cells than that in the upper zone (50.7%±25.1% vs 32.9%±30.0%, p<0.001). There were more SOX9+ cells in colorectal adenomas (39.8%±30.9%) than that in the peri-adenomatous normal mucosa (24.9%±18.2%) (p<0.01). The rate of SOX9+ cells in cancer is 36.7%±30.3%, significantly higher than that in normal mucosa and peri-carcinomatous normal residues, but not in adenomas (p>0.05). The SOX9+ incidence of both adenomas (74.5%, 70/94) and cancers (60.1%, 113/188) was higher than that in the normal mucosa (10/110,9.09%) (p<0.001), if a working protocol of a
simple additive scoring system for immunostaining assessment was applied. As we defined "-" and "+" as "low-expression" and "++" and "+++" as "overexpression", we found that SOX9 overexpression is significantly more common in colorectal adenomas (53.2%, 50/94) and cancers (34.0%, 64/188) than that in the normal mucosa, where none showed SOX9 overexpression. SOX9 overexpression was less common in mucin-producing cancer (signet-ring cell cancer and mutinous adenocarcinoma) than that in non-mucin-producing cancer (p<0.05). The 5-year overall survival was significantly lower in colorectal cancer patients with SOX9 overexpression (39.5%, 17/43) than those with low-expression (69.5%, 66/95) (p<0.01). Survival analysis and COX proportional haphazard model indicated that SOX9 overexpression is an independent adverse prognosticator in colorectal cancers (p<0.05,RR=1.381, 95% CI:1.051-1.815).
In conclusion, we demonstrated that colorectal caner is heterogenous at the molecular level. Alternative splicing is common in cancers implying its potential role in carcinogenesis. Enrichment of ribosomal protein genes is an important molecular feature for two most important colorectal precursor lesions, adenoma and inflammatory bowel disease. Thus, the electronic gene expression library will help us to explore the underlying general molecular features in the process of colorectal cancer initiation and progression. We also suggested that dysregulation of colonocyte differentiation associated pathways and gene families, such as Wnt activation, inhibition of TGFβ/BMP4, Hedgehog and Notch, and aberrant expression of PcG family genes, had potential roles in the colorectal carcinogenesis. Wnt pathway might play a central role in the intricate network of these pathways. Particularly, we found that SOX9, a novel transcriptor in Wnt pathway, was associated with intestinal epithelial differentiation. SOX9 was overexpressed in colorectal adenomas and cancers and its overexpression was an adverse prognosticator for colorectal cancer, thus it might serve as a potential gene therapeutic target in the future. Finally, we demonstrated that LDA was a sensitive and reliable high throughput quantitative PCR method. A combined application of dbEST data mining and LDA was a novel methodology for cancer biomarkers detection.
引文
1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin. 2005;55:10-30.
2. Dennis JL, Vass JK, Wit EC, et at. Identification from public data of molecular markers of adenocarcinoma characteristic of the site of origin. Cancer Res. 2002,62: 5999-6005.
3. Adams MD, Kelley JM, Gocayne JD, et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science. 1991; 252:1651-6.
4. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW. Serial analysis of gene expression. Science. 1995;270:484-7.
5. Buckhaults P, Rago C, St Croix B, et al. Secreted and cell surface genes expressed in benign and malignant colorectal tumors. Cancer Res. 2001; 61:6996-7001.
6. Notterman DA, Alon U, Sierk AJ, Levine AJ. Transcriptional Gene Expression Profiles of Colorectal Adenoma, Adenocarcinoma, and Normal Tissue Examined by Oligonucleotide Arrays. Cancer Res. 2001;61:3124-3130.
7. Grade M, Hormann P, Becker S, et al. Gene expression profiling reveals a massive, aneuploidy-dependent transcriptional deregulation and distinct differences between lymph node-negative and lymph node-positive colon carcinomas. Cancer Res. 2007;67:41-56.
8. Fujita Y, Nakanishi T, Hiramatsu M, et al. Proteomics-based approach identifying autoantibody against peroxiredoxin Ⅵ as a novel serum marker in esophageal squamous cell carcinoma. Clin Cancer Res. 2006; 12:6415-20.
9. Chen EI, Hewei J, Felding-Habermann B, Yates JR 3rd. Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT). Mol Cell Proteomics. 2006;5:53-6.
10. Kuska B. Cancer genome anatomy project set for take-off. J Natl Cancer Inst. 1996;88: 1801-3.
11. Strausberg RL, Feingold EA, Klausner RD, Collins FS. The mammalian gene collection. Science. 1999:286:455-7.
12. Ota T, Suzuki Y, Nishikawa T, et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet. 2004;36:40-5.
13. Luo MJ, Lai MD. Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH. World J Gastroenterol. 2001; 7:726-31.
14. Zhang Y, Lai M, Lv B, et al. Overexpression of Reg Ⅳ in colorectal adenoma. Cancer Lett. 2003;200:69-76.
15. Shao L, Huang Q, Luo M, Lai M. Detection of the differentially expressed gene IGF-binding protein-related protein-1 and analysis of its relationship to fasting glucose in Chinese colorectal cancer patients. Endocr Relat Cancer. 2004;11:141-8.
16. Xing X, Lai M, Gartner W, et al. Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin. Proteomics. 2006;6:2916-2923.
17. Weston AD, Hood L. Systems biology, proteomics, and the future of health care: toward predictive, preventative, and personalized medicine. J Proteome Res. 2004;3:179-96.
18. Lal A, Lash AE, Altschul SF, et al. A public database for gene expression in human cancers. Cancer Res. 1999;59:5403-7.
19. Barrett T, Troup DB, Wilhite SE, et al. NCBI GEO: mining tens of millions of expression profiles-database and tools update. Nucleic Acids Res. 2007;35:D760-5.
20. Aung PP, Oue N, Mitani Y, et al. Systematic search for gastric cancer-specific genes based on SAGE data: melanoma inhibitory activity and matrix metalloproteinase-10 are novel prognostic factors in patients with gastric cancer. Oncogene. 2006;25:2546-57.
21. Brazma A, Hingamp P, Quackenbush J, et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001 ;29:365-71.
22. Scott M, Hyland PL, McGregor G, et al. Multimodality expression profiling shows SEPT9 to be overexpressed in a wide range of human tumours. Oncogene, 2005;24:4688～4700.
23. Couzin J. Genomics. Microarray data reproduced, but some concerns remain. Science. 2006;313:1559.
24. Schmitt AO, Specht T, Beckmann G, et al. Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues. Nucleic Acids Res. 1999;27:4251-60.
25. Qiu P, Benbow L, Liu S, et al. Analysis of a human brain transcriptome map. BMC Genomics. 2002;3:10.
26. Hood L, Heath JR, Phelps ME, Lin B. Systems biology and new technologies enable predictive and preventative medicine. Science. 2004;306:640-3.
27. Scheurle D, De Young MP, Binninger DM, et al. Cancer gene discovery using digital differential display. Cancer Res. 2000;60:4037-43.
28. De Young MP, Damania H, Scheurle D, et al. Bioinformatics-based discovery of a novel factor with apparent specificity to colon cancer. In Vivo. 2002;16:239-48.
29. Jiang Z, Hu J, Li X, et al. Expression analyses of 27 DNA repair genes in astrocytoma by TaqMan low-density array. Neurosci Lett. 2006;409: 112-7.
30. Steinbach D, Schramm A, Eggert A, et al. Identification of a set of seven genes for the monitoring of minimal residual disease in pediatric acute myeioid leukemia. Clin Cancer Res. 2006; 12:2434-41.
31. Langmann T, Mauerer R, Schmitz G. Human ATP-binding cassette transporter TaqMan low-density array: analysis of macrophage differentiation and foam cell formation. Ciin Chem. 2006;52:310-3.
32. Steg A, Wang W, Blanquicett C, et al. Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma. J Mol Diagn. 2006;8:76-83.
33. Gallagher WM, Bergin OE, Rafferty M, et al. Multiple markers for melanoma progression regulated by DNA methylation: insights from transcriptomic studies. Carcinogenesis. 2005;26:1856-67.
34. Abruzzo LV, Lee KY, Fuller A, et al. Validation of oligonucleotide microarray data using microfluidic low-density arrays: a new statistical method to normalize real-time RT-PCR data. Biotechniques. 2005;38:785-92.
35. Gyorffy B, Surowiak P. Kiesslich O, et al. Gene expression profiling of 30 cancer cell lines predicts resistance towards 11 anticancer drugs at clinically achieved concentrations, Int J Cancer. 2006; 118:1699-712
36. Dahl A, Sultan M, Jung A, et al. Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips. Biomed Microdevices. 2007 Jan 3; [Epub ahead of print]
37. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000; 100:57-70.
38. Domon-Dell C, Wang Q, Kim S, et al. Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells. Gut. 2002;50:525-9.
39. Daly K, Shirazi-Beechey SP. Microarray analysis of butyrate regulated genes in colonic epithelial cells. DNA Cell Biol. 2006;25:49-62.
40. Mariadason JM, Corner GA, Augenlicht LH. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res. 2000;60:4561-72.
41. Tan S, Seow TK, Liang RC, et al. Proteome analysis of butyrate-treated human colon cancer cells (HT-29). Int J Cancer. 2002;98:523-31.
42. Radtke F, Clevers H. Self-renewal and cancer of the gut: two sides of a coin. Science. 2005;307:1904-09.
43. Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275:1787-90.
44. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell. 2000; 103:311-20.
45. Blache P, van de Wetering M, Duluc I, et al. SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes. J Cell Biol 2004; 166:37-47.
46. Yano F, Kugimiya F, Ohba S, et al. The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner. Biochem Biophys Res Commun 2005;333:1300-8.
47. Akiyama H, Lyons JP, Mori-Akiyama Y, et al. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev 2004; 18:1072-87.
48. Jay P, Berta P, Blache P. Expression of the carcinoembryonic antigen gene is inhibited by SOX9 in human colon carcinoma cells. Cancer Res 2005;65:2193-8.
49. Drivdahl R, Haugk K.H, Sprenger CC, et al. Suppression of growth and tumorigenicity in the prostate tumor cell line M12 by overexpression of the transcription factor SOX9. Oncogene 2004;23:4584-93.
50. Kato N, Fukase M, Motoyama T. Expression of a transcription factor, SOX9, in Sertoli-stromal cell tumors of the ovary. Int J Gynecol Pathol 2004;23:180-1.
51. Wehrli BM, Huang W, De Crombrugghe B, et al. Sox9, a master regulator of chondrogenesis, distinguishes mesenchymal chondrosarcoma from other small blue round cell tumors. Hum Pathol 2003;34:263-9.
52. Vidal VP, Chaboissier MC, Lutzkendorf S, et al. Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment. Curr Biol. 2005;15:1340-51.
53. Xia Y, Papalopulu N, Vogt PK, Li J. The oncogenic potential of the high mobility group box protein Sox3. Cancer Res 2000;60:6303-6.
54. Weigle B, Ebner R, Temme A, et al. Highly specific overexpression of the transcription factor SOX11 in human malignant gliomas. Oncol Rep 2005;13:139-44.
55. Cook AL, Smith AG, Smit DJ, et al. Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro. Exp Cell Res 2005;308:222-35.
56. Perl AK, Kist R, Shan Z, et al. Normal lung development and function after Sox9 inactivation in the respiratory epithelium. Genesis 2005;41:23-32.
57. Valk-Lingbeek ME, Bruggeman SW, van Lohuizen M. Stem cells and cancer; the polycomb connection. Cell. 2004; 118:409-18.
58.Gil J, Bernard D, Peters G Role of Polycomb Group Proteins in Stem Cell Self-Renewal and Cancer. DNA Cell Biol.2005;24:117-25.
59. Lee TI, Jenner RG, Boyer LA, et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell. 2006;125:301-13.
60. Tateishi K, Ohta M, Kanai F, et al. Dysregulated expression of stem cell factor Bmil in precancerous lesions of the gastrointestinal tract. Clin Cancer Res. 2006; 12:6960-6.
61.van Leenders GJ, Dukers D, Hessels D, et al. Polycomb-Group Oncogenes EZH2, BMI1, and RING1 Are Overexpressed in Prostate Cancer With Adverse Pathologic and Clinical Features. Eur Urol. 2006; [Epub ahead of print]
62. Bachmann IM, Halvorsen OJ, Collett K, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol. 2006;24:268-73.
63. Mimori K, Ogawa K, Okamoto M, et al. Clinical significance of enhancer of zeste homoiog 2 expression in colorectal cancer cases. Eur J Surg Oncol. 2005;31:376-80.
64. Ding L, Kleer CG. Enhancer of zeste 2 as a marker of preneoplastic progression in the breast. Cancer Res. 2006;66:9352-5.
65. Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer. 2006;6:846-56.
66. Zhao W, Hisamuddin IM, Nandan MO, et al. Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene. 2004;23:395-402.
67. Reeves HL, Narla G, Ogunbiyi O, et al. Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer. Gastroenterology. 2004; 126:1090-103.
68. Widschwendter M, Fiegl H, Egle D, et al. Epigenetic stem cell signature in cancer. Nat Genet. 2007;39:157-8.
66. Zhuo D, Zhao WD, Wright FA, et al. Assembly, annotation, and integration of UNIGENE clusters into the human genome draft. Genome Res. 2001, 11:904-918.
67. The Gene Ontology Consortium. Gene ontology: tool for the unification of biology. Nat. Genet. 2000, 25:25-29.
68. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990, 61:759-767.
69. Hanash S. Integrated global profiling of cancer. Nat Rev Cancer. 2004, 4:638-644.
70．吕炳建，崔晶，徐静，等。结肠腺瘤-正常(A-N)文库的生物信息学分析。遗传，2006；28：385-92。
71. Hamilton SR, Altonin LA. Pathology and Genetics: tumours of the Digestive System. WHO classifications Third Series, RIAS, Ryon, France 2001
72. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004, 431:931-945.
73. Johnson JM, Castle J, Garrett-Engele P, et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science. 2003;302:2141-4.
74. Hui L, Zhang X, Wu X, et al. Identification of alternatively spliced mRNA variants related to cancers by genome-wide ESTs alignment. Oncogene. 2004;23(17):3013-23.
75. Ku TH, Hsu FR. Mining colon cancer specific alternative splicing in EST database. AMIA Annu Syrup Proc. 2005;: 1012.
76. Aretz S, Uhlhaas S, Sun Y, et al. Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene. Hum Murat. 2004;24:370-80.
77. Marchler-Bauer A, Anderson JB, Cherukuri PF, et al. CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res. 2005, 33 :D192-196.
78. Ruggero D, Pandolfi PP. Does the ribosome translate cancer? Nat Rev Cancer. 2003, 3:179-92.
79. Kasai H, Nadano D, Hidaka E, et al. Differential expression of ribosomal proteins in human normal and neoplastic colorectum. J Histochem Cytochem. 2003, 51:567-74.
80. Pogue-Geile K, Geiser JR, Shu M, et al. Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein. Mol Cell Biol. 1991, 11:3842-9.
81. Wong JM, Mafune K, Yow H, et al. Ubiquitin-ribosomal protein S27a gene overexpressed in human colorectal carcinoma is an early growth response gene. Cancer Res. 1993, 53:1916-20.
82. Bee A, Ke Y, Forootan S, et al. Ribosomal protein L19 is a prognostic marker for human prostate cancer. Clin Cancer Res. 2006,12:2061-5.
83. Cheng Q, Lau WM, Chew SH, et al. Identification of molecular markers for the early detection of human squamous cell carcinoma of the uterine cervix. Br J Cancer. 2002, 86:274-81.
84. Kim JH, You KR, Kim IH, et al. Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma. Hepatology. 2004, 39:129-38.
85. Huang XP, Zhao CX, Li QJ, et al. Alteration of RPL14 in squamous cell carcinomas and preneoplastic lesions of the esophagus. Gene. 2006, 366:161-8.
86. 20. Zhou YH, Hess KR, Liu L, et al. Modeling prognosis for patients with malignant astrocytic gliomas: quantifying the expression of multiple genetic markers and clinical variables. Neuro-oncol. 2005, 7:485-94.
87. Du J, Shi Y, Pan Y, et al. Regulation of multidrug resistance by ribosomal protein 16 in gastric cancer cells. Cancer Biol Ther. 2005, 4:242-7.
88. Feagan BG, Greenberg GR, Wild G, et al. Treatment of ulcerative colitis with a humanized antibody to the alpha4beta7 integrin. N Engl J Meal. 2005, 352:2499-507.
89. Enns A, Korb T, Schluter K, et al. Alphavbeta5-integrins mediate early steps of metastasis formation. Eur J Cancer. 2005, 41:1065-72.
90. Sullivan DC, Huminiecki L, Moore JW, et al. EndoPDI, a novel protein-disulfide isomemse-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor. J Biol Chem. 2003;278:47079-88.
91. Coppock DL, Kopman C, Scandalis S, et al. Preferential gene expression in quiescent human lung fibroblasts. Cell Growth Differ. 1993, 4:483-93.
92. Mork H, al-Taie OH, Bahr K, et al. Inverse mRNA expression of the selenocysteine-containing proteins GI-GPx and SeP in colorectal adenomas compared with adjacent normal mucosa. Nutr Cancer. 2000;37:108-16.
93. Bhattacharya M., Babwah AV, Ferguson SSG. Small GTP-binding protein-coupled receptors. Biochem Soc Trans. 2004, 32, 1040-50.
94. Malarstig A, Tenno T, Jossan S, et al. A quantitative real-time PCR method for tissue factor mRNA. Thromb Res. 2003;112:175-83.
95. Katoh M. WNT/PCP signaling pathway and human cancer (review). Oncol Rep. 2005;14:1583-8.
96. Segditsas S, Tomlinson I. Colorectal cancer and genetic alterations in the Wnt pathway. Oncogene. 2006;25:7531-7.
97. Koch A, Waha A, Hartmann W, et al. Elevated expression of Wnt antagonists is a common event in hepatoblastomas. Clin Cancer Res. 2005; 11:4295-304.
98. Lee S, Hwang KS, Lee HJ, et al. Aberrant CpG island hypermethylation of multiple genes in colorectal neoplasia. Lab Invest. 2004;84:884-93.
99. Gonzalez-Sancho JM, Aguilera O, Garcia JM, et al. The Wnt antagonist DICKKOPF-1 gene is a downstream target of beta-catenin/TCF and is downregulated in human colon cancer. Oncogene. 2005;24:1098-103.
100. Howe JR, Roth S, Ringold JC, et al. Mutations in the SMAD4/DPC4 gene in juvenile polyposis. Science. 1998;280:1086-8.
101. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type Ⅱ TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 1995;268:1336-8.
102. Kim JS, Crooks H, Dracheva T, et al. Oncogenic beta-catenin is required for bone morphogenetic protein 4 expression in human cancer cells. Cancer Res. 2002;62:2744-8.
103. Attisano L, Labbe E. TGFbeta and Wnt pathway cross-talk. Cancer Metastasis Rev. 2004;23:53-61.
104. Hellemans J, Preobrazhenska O, Willaert A, et al. Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and meiorheostosis. Nat Genet. 2004;36:1213-8.
105. van den Brink GR, Bleuming SA, Hardwick JCH, et al. Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation. Nature Genet. 2004;36, 277-82.
106. Madison BB, Braunstein K, Kuizon E, et al. Epithelial hedgehog signals pattern the intestinal crypt-villus axis. Development. 2005;132:279-89.
107. Taipale J, Beachy PA. The Hedgehog and Wnt signalling pathways in cancer. Nature. 2001;411:349-54.
108. Mizuno T, Tokuoka S, Kishikawa M, et al. Molecular basis of basal cell carcinogenesis in the atomic-bomb survivor population: p53 and PTCH gene alterations. Carcinogenesis. 2006;27:2286-94.
109. Burke R, Nellen D, Bellotto M, et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell. 1999;99:803-15.
110. Murone M, Luoh SM, Stone D, et al. Gli regulation by the opposing activities of fused and suppressor of fused. Nat Cell Biol. 2000;2:310-2.
111. Chert MH, Gao N, Kawakami T, Chuang PT. Mice deficient in the fused homolog do not exhibit phenotypes indicative of perturbed hedgehog signaling during embryonic development. Mol Cell Biol. 2005;25:7042-53.
112. Oniscu A, James RM, Morris RG, et al. Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia. J Pathol. 2004 Aug;203(4):909-17.
113. Wilson A, Radtke F. Multiple functions of Notch signaling in self-renewing organs and cancer. FEBS Lett. 200;580:2860-8.
114. Duan L, Yao J, Wu X, Fan M. Growth suppression induced by Notchl activation involves Wnt-beta-catenin down-regulation in human tongue carcinoma cells. Biol Cell. 2006;98:479-90.
115. Politi K, Feirt N, Kitajewski J. Notch in mammary gland development and breast cancer. Semin Cancer Biol. 2004;14:341-7.
116. Bracken AP, Pasini D, Capra M, et al. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 2003;22:5323-35.
117. Vire E, Brenner C, Deplus R, et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature. 2006;439:871-4.
118. Schlesinger Y, Straussman R, Keshet I, et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet. 2006; [Epub ahead of print]
119. Breuer RH, Snijders PJ, Sutedja GT, et al. Expression of the p16(INK4a) gene product, methylation of the p16(INK4a) promoter region and expression of the polycomb-group gene BMI-1 in squamous cell lung carcinoma and premalignant endobronchial lesions. Lung Cancer. 2005;48:299-306.
120. Dukers DF, van Galen JC, Giroth C, et al. Unique polycomb gene expression pattern in Hodgkin's lymphoma and Hodgkin's lymphoma-derived cell lines. Am J Pathol. 2004;164:873-81.
121. Reinisch C, Kandutsch S, Uthman A, Pammer J. BMI-1: a protein expressed in stem cells, specialized cells and tumors of the gastrointestinal tract. Histol Histopathol. 2006; 21:1143-9.
122. Kim JH, Yoon SY, Kim CN, et al. The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced p16INK4a/p 14ARF proteins. Cancer Lett. 2004;203:217-24.
123. Suarez-Merino B, Hubank M, Revesz T, et al. Microarray analysis of pediatric ependymoma identifies a cluster of 112 candidate genes including four transcripts at 22q12.1-q13.3. Neuro-oncol. 2005;7:20-31.
124. Kanno M, Hasegawa M, Ishida A, et al. reel-18, a Polycomb group-related mammalian gene, encodes a transcriptional negative regulator with tumor suppressive activity. EMBO J. 1995;14:5672-8.
125. Colter DC, Piera-Velazquez S, Hawkins DF, et al. Regulation of the human Sox9 promoter by the CCAAT-binding factor. Matrix Biol. 2005;24:185-97.
126. Ueno H, Murphy J, Jass JR, et al. Tumour 'budding' as an index to estimate the potential of aggressiveness in rectal cancer. Histopathology 2002;40:127-32.
127．徐芳英，董健康，朱益民，等。结直肠癌独立预后因素：TNM分期、肿瘤芽、神经周围侵犯、瘤旁淋巴细胞浸润和尿糖。中华流行病学杂志，2005：26：366-9。
128. Leake R, Barnes D, Pinder S, et al. Immunohistoehemical detection of steroid receptors in breast cancer: a working protocol. UK Receptor Group, UK NEQAS, The Scottish Breast Cancer Pathology Group, and The Receptor and Biomarker Study Group of the EORTC. J Clin Pathol 2000;53:634-5.
129. Konishi K, Yamochi T, Makino R, et al. Molecular differences between sporadic serrated and conventional colorectal adenomas. Clin Cancer Res. 2004;10:3082-90.
130. Yokota N, Mainpdze TG, Taylor MD, et al. Identification of differentially expressed and developmentally regulated genes in medulloblastoma using suppression subtraction hybridization. Oncogene. 2004;23:3444-53.
131. Liu P, Ramachandran S, Ali Scyed M, et al. Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells. Cancer Res. 2006;66:4011-9.
132. Aaboe M, Birkenkamp-Demtroder K, Wiuf C, et al. SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization. Cancer Res. 2006;66:3434-42.
133. Katoh M. Expression of human SOX7 in normal tissues and tumors. Int J Mol Med. 2002;9:363-8.
134. Foster JW, Dominguez-Steglich MA, Guioli S, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 1994;372:525-30.
135. Wang H, McKnight NC, Zhang T, et al. SOX9 is expressed in normal prostate basal cells and regulates androgen receptor expression in prostate cancer cells. Cancer Res. 2007;67:528-36.
136. Miller SJ, Rangwala F, Williams J, et al. Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues. Cancer Res. 2006;66:2584-91.
137. Preston SL, Wong WM, Chan AO, et al. Bottom-up histogenesis of eolorectal adenomas: origin in the monoeryptal adenoma and initial expansion by crypt fission. Cancer Res. 2003;63:3819-25.
138. Wong SC, Chan AT, Lo ES, Lo YM. Nuclear beta-catenin expression is rare and its potential association with short survival in colorectal signet-ring cell carcinoma. Appl Immunohistechem Mol Morphol2005;13:248-51.
139. Allegra CJ, Paik S, Colangelo LH, et al. Investigation of the prognostic and predictive value of thymidylate synthase, p53, and Ki67 in patients with locally advanced colon cancer. J Clin Oncol 2002;20:1735-43.
140. Garrity MM, Burgart LJ, Mahoney MR, et al. Prognostic value of proliferation, apoptosis, defective DNA mismatch repair, and p53 overexpression in patients with resected Dukes' B2 or C colon cancer: a North Central Cancer Treatment Group Study. J Clin Oncol 2004;22:1572-82.
1. Hall PA, Russell SH. The pathobioiogy of the septin gene family. J Pathol 2004, 204: 489～505.
2. Hartwell LH. Genetic control of the division cycle in yeast. (Ⅳ). Genes controlling bud emergence and cytokinesis. Exp Cell Res, 1972,69:265～276.
3. Macara IG, Baldarelli R, Field CM, et al. Mammalian septins nomenclature. Mol Biol Cell, 2002,3: 4111～4113.
4. Hall PA, Jung K, Hillan KJ, Russell SE. Expression profiling the human septin gene family. J Pathol 2005, 206: 269～278.
5. Martinez C, Sanjuan MA, Dent JA, et al. Huaman septin-septin interaction as a prerequisite for targeting septin complexes in the cytosol. Biochem J, 2004,382:783-791.
6. Kinoshita M, Noda M. Roles of septin in the mammalian cytokinesis machinery. Cell Struct Funct, 2001, 26:667-670.
7. Surka MC, Tsang CW, Trimble WS. The mammalian septin MSF localizes with microtubules and is required for completion of cytokinesis. Mol Biol Cell, 2002,13:3532-3545.
8. Spiliotis ET, Kinoshita M, Nelson WJ. A mitotic septin scaffold required for Mammalian chromosome congression and segregation. Science, 2005, 307:1781-1785.
9. Sheffield PJ, Oliver CJ, Kremer BE, et al. Borg/septin interactions and the assembly of mammalian septin heterodimers, trimers, and filaments J Biol Chem, 2003,278:3483-3488.
10.Kinoshita M, Field CM, Coughlin ML, et al. Self- and actin-templated assembly of Mammalian septins. DevCel,12002,3:791-802.
11. Schmidt K, Nichols BJ. Functional interdependence between septin and actin cytoskeleton. BMC Cell Biol, 2004,5:43.
12. Kremer BE, Haystead T, Macara IG Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4. Mol Biol Cell, 2005,16:4648-4659
13. Caviston JP, Longtine M, Pringle JR, Bi E. The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast. Mol Biol Cell, 2003,14:4051-4066.
14. Nagata K, Inagaki M. Cytoskeletal modification of Rho guanine nucleotide exchange factor activity: identification of a Rho guanine nucleotide exchange factor as a binding partner for Sept9b, a mammalian septin. Oncogene, 2005,24:65-76.
15. Ito H, Iwamoto I, Morishita R, et al. Possible role of Rho/Rhotekin signaling in mammalian septin organization. Oncogene, 2005 Jun 27; [Epub ahead of print]
16. Joberty G, Perlungher RR, Sheffield PJ, et al. Borg proteins control septin organization and are negatively regulated by Cdc42. Nat Cell Biol, 2001,3:861-866.
17. Kartmann B, Roth D. Novel roles for mammalian septins: from vesicle trafficking to oncogenesis. J Cell Sci, 2001, 114:839-844.
18. Hsu SC, Hazuka CD, Roth R, et al. Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron, 1998,20:1111-1122.
19. Dent J, Kato K, Peng XR, et al. A prototypic platelet septin and its participation in secretion. Proc Natl Acad Sci U S A,2002,99:3064-3069.
20. Beites CL, Xie H, Bowser R, Trimble WS. The septin CDCrel-1 binds syntaxin and inhibits exocytosis. Nat Neurosci, 1999,2:434-439.
21. Gottfried Y, Rotem A, Lotan R, et al. The mitochondrial ARTS protein promotes apoptosis through targeting XIAP. EMBO J, 2004,23:1627-1635.
22. Choi P, Snyder H, Petrucelli L, et al. SEPT5_v2 is a parkin-binding protein. Brain Res Mol Brain Res, 2003, 117:179-189.
23. Ihara M, Tomimoto H, Kitayama H, et al. Association of the cytoskeletal GTP-binding protein Sept4/H5 with cytoplasmic inclusions found in Parkinson's disease and other synucleinopathies. J Biol Chem, 2003,278:24095-240102.
24. Takehashi M, Alioto T, Stedeford T, et al. Septin 3 gene polymorphism in Alzheimer's disease. Gene Expr, 2004,11:263-270.
25. Cheon MS, Fountoulakis M, Dierssen M, et al. Expression profiles of proteins in fetal brain with Down syndrome. J Neural Transm Suppl, 2001 ,(61 ):311 -319.
26. Slater DJ, Hilgenfeld E, Rappaport EF, et al. MLL-SEPTIN6 fusion recurs in novel translocation of chromosomes 3, X, and 11 in infant acute myelomonocytic leukaemia and in t(X;l 1) in infant acute myeloid leukaemia, and MLL genomic breakpoint in complex MLL-SEPTIN6 rearrangement is a DNA topoisomerase II cleavage site. Oncogene, 2002,21:4706-47014.
27. Larisch S. The ARTS connection: role of ARTS in apoptosis and cancer. Cell Cycle, 2004, 3:1021-1023.
28. Kalikin LM, Sims HL, Petty EM. Genomic and expression analyses of alternatively spliced transcripts of the MLL septin-like fusion gene (MSF) that map to a 17q25 region of loss in breast and ovarian tumors. Genomics, 2000,63:165-172.
29. Montagna C, Lyu MS, Hunter K, et al. The Septin 9 (MSF) gene is amplified and overexpressed in mouse mammary gland adenocarcinomas and human breast cancer cell lines. Cancer Res, 2003,63:2179～2187.
30. Chacko AD, Hyland PL, McDade SS, et al. SEPT9_v4 expression induces morphological change, increased motility and disturbed polarity. J Pathol, 2005,206:458～465.
31. Scott M, Hyland PL, McGregor G, et al. Multimodality expression profiling shows SEPT9 to be overexpressed in a wide range of human tumours. Oncogene, 2005,24:4688～4700.
32. Kim DS, Hubbard SL, Peraud A. Analysis of mammalian septin expression in human malignant brain tumors. Neoplasia, 2004,6:168～178.
33. Tanaka M, Kijima H, Itoh J, et al. Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo. Cancer Gene Ther, 2002,9:483～488.
34. Tanaka M, Tanaka T, Matsuzaki S, et al. Rapid and quantitative detection of human septin family Bradeion as a practical diagnostic method of colorectal and urologic cancers. Med Sci Monit, 2003,9:MT61～68.
35. Ying-Tao Z, Yi-Ping G, Lu-Sheng S, Yi-Li W. Proteomic analysis of differentially expressed proteins between metastatic and non-metastatic human colorectal carcinoma cell lines. Eur J Gastroenterol Hepatol, 2005,17:725～732.
36．陆东东，张锡燃．肝癌组织中septin1、septin2、septin6基因表达及其功能的初步研究．肿瘤研究与临床．2002，14：366～368．
37. Sui L, Zhang W, Liu Q, et al. Cloning and functional characterization of human septin 10, a novel member of septin family cloned from dendritic cells. Biochem Biophys Res Commun, 2003,304:393～398.
38. Ihara M, Kinoshita A, Yamada S, et al. Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa. Dev Cell, 2005,8:343～352.
1. Lindor NM, Rabe K, Petersen GM, et al. Lower cancer incidence in Amsterdam-I criteria familics without mismatch repair deficiency: familial colorectal cancer type X. JAMA. 2005;293:1979-85.
2. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst. 1997;89:1758-62.
3. Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer inst. 2004;96:261-8.
4. Pinol V, Castells A, Andreu M, et al. Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer. JAMA. 2005;293:1986-94.
5. Wullenweber HP, Sutter C, Autschbach F, et al. Evaluation of Bethesda guidelines in relation to microsatellite instability. Dis Colon Rectum. 2001;44:1281-9.
6．来茂德，黄智达，黄琼，等。PER+结直肠癌组织转化生长因子βⅡ型受体的突变。中华病理学杂志，2004：33：6-10。
7．吕炳建，来茂德，程蕾，等。Bat26-DHPLC法检测胃癌微卫星不稳定性。遗传，2004：26：574-78。
8. Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 2005;352:1851-60.
9．来茂德。WHO新的结直肠肿瘤分类的特点。中华病理学杂志，2003：32：170-2。
10. Deng G, Bell I, Crawley S, et al. BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res. 2004;10:191-5.
11. Domingo E, Laiho P, Oilikainen M, et al. BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet. 2004;41:664-8.
12. Hendriks Y, Franken P, Dierssen JW, et al. Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors. Am J Pathol. 2003; 162:469-77.
13．黄智达，来茂德。大肠腺瘤发生的分子机制。临床与实验病理学杂志，1999：15：235-8。
14. Kaplan KB, Burds AA, Swedlow JR, et al. A role for the Adenomatous Polyposis Coli protein in chromosome segregation. Nat Cell Biol. 2001 ;3:429-32.
15. Kawasaki Y, Sato R, Akiyama T. Mutated APC and Asef are involved in the migration of colorectal tumour cells. Nat Cell Biol. 2003;5:211-5.
16. Béroud C, Soussi T. APC gene: database of germline and somatic mutations in human tumors and cell lines. Nucleic Acids Research, 1996, 24:121-4.
17. Laken SJ, Petersen GM, Gruber SB, et al. Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC. Nat Genet. 1997; 17:79-83.
18. Bertario L, Russo A, Sala P, et al. Multiple approach to the exploration of genotype-phenotype correlations in familial adenomatous polyposis. J Clin Oncol. 2003;21:1698-707.
19. Wallis YL, Morton DG, McKeown CM, et al. Molecular analysis of the APC gene in 205 families: extended genotype-phenotype correlations in FAP and evidence for the role of APC amino acid changes in colorectal cancer predisposition. J Med Genet. 1999;36:14-20.
20. Bisgaard ML, Buiow S. Familial adenomatous polyposis (FAP): genotype correlation to FAP phenotype with osteomas and sebaceous cysts. Am J Med Genet A. 2006;140:200-4.
21. Lynch HT, Smyrk T, MeGinn T, et al. Attenuated familial adenomatous polyposis (AFAP). A phenotypically and genotypically distinctive variant of FAP. Cancer. 1995 15;76:2427-33.
22. Su LK, Barnes CJ, Yao W, et al. Inactivation of germline mutant APC alleles by attenuated somatic mutations: a molecular genetic mechanism for attenuated familial adenomatous polyposis. Am J Hum Genet. 2000 ;67:582-90.
23. Heppner Goss K, Trzepacz C, Tuohy TM, et al. Attenuated APC alleles produce functional protein from internal translation initiation. Proc Natl Acad Sci USA. 2002;99:8161-6.
24. Baglioni S, Mclean G, Gensini F, et al. A kindred with MYH-associated polyposis and pilomatrieomas. Am J Med Genet A. 2005;134:212-4.
25. Sampson JR, Jones S, Dolwani S, et al. MutYH (MYH) and colorectal cancer. Biochem Soc Trans. 2005;33:679-83.
26. Sieber OM, Lipton L, Crabtree M, et al. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med. 2003;348:791-9.
27. Sampson JR, Doiwani S, Jones S, et al. Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH. Lancet. 2003 ;362:39-41
28. Croitoru ME, Cleary SP, Di Nicola N, et al. Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk. J Natl Cancer Inst. 2004;96:1631-4.
29. Nugent KP, Talbot IC, Hodgson SV, Phillips RK. Solitary juvenile polyps: not a marker for subsequent malignancy. Gastroenterology 1993; 105: 698-700.
30. Howe JR, Mitros FA, Summers RW. The risk of gastrointestinal carcinoma in familial juvenile polyposis. Ann. Stag. Oncol. 1998; 5: 751-6.
31. Giardiello FM, Hamilton SR, Kern SE, et al. Colorectal neoplasia in juvenile polyposis or juvenile polyps. Arch Dis Child. 1991;66:971-5.
32. Howe JR, Roth S, Ringold JC et al. Mutations in the SMAD4/DPC4 gene in juvenile polyposis. Science 1998;280:1086-8.
33. Chow E, Macrae F. A review of juvenile polyposis syndrome. Journal of Gastroenterology and Hepatology 2005;20; 1634-1640.
34. Kinzler KW, Vogelstein B. Landscaping the cancer terrain. Science 1998;280:1036-7.
35. Woodford-Richens KL, Rowan AJ, Pouisom R, et al. Comprehensive analysis of SMAD4 mutations and protein expression in juvenile polyposis: evidence for a distinct genetic pathway and polyp morphology in SMAD4 mutation carders. Am. J. Pathol 2001; 159:1293-300.
36. Howe JR, Balr JL, Sayed MG, et al. Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis. Nat. Genet. 2001 ;28:184-7.
37. Howe JR, Ahmed AF, Ringold J, et al. The prevalence of MADH4 and BMPR1A mutations in juvenile polyposis and absence of BMPR2, BMPR1B, and ACVR1 mutations. J. Med. Genet.2004; 41:484-91.
38. Friedl W, Uhlhaas S, Schulmann K, et al. Juvenile polyposis:massive gastric polyposis is more common in MADH4 mutation carriers than in BMPR1A mutationcarriers. Hum. Genet. 2002;111:108-11.
39. Handra-Luca A, Condroyer C, de Moncuit C, et al. Vessels' morphology in SMAD4 and BMPR1A-related juvenile polyposis. Am J Med Genet A. 2005;138:113-7.
40. Waite KA, Eng C. Protean PTEN: form and function. Am J Hum Genet. 2002;70:829-44.
41. Sweet K, Willis J, Zhou XP, et al. Molecular Classification of Patients With Unexplained Hamartomatous and Hyperplastic Polyposis. JAMA. 2005;294:2465-73.
42. Marsh DJ, Kum JB, Lunetta KL, et al. PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum Mol Genet 1999;8:1461-72.
43. Hanssen AMN, Fryns JP. Cowden syndrome. J Med Genet 1995; 32:117-9.
44. StarinkTM, van der Veen JPW, Arwert F de Waal LP, et al. The Cowden syndrome. A clinical and genetic study in 21 patients. Clin Genet 1986;29:222-33.
45. Kato M, Mizuki A, Hayashi T, et al. Cowden's disease diagnosed through mucocutaneous lesions and gastrointestinal polyposis with recurrent hematochezia, unrevealed by initial diagnosis. Intern Med. 2000;39:559-63.
46. Brugarolas J, Kaelin WG Jr. Dysregulation of HIF and VEGF is a unifying feature of the familial hamartoma syndromes. Cancer Cell. 20041;6:7-10.
47. Gorlin RJ, Cohen MM Jr, Condon LM, et al. Bannayan-Riley-Ruvalcaba syndrome. Am J Med Genet 1992;44:307-14.
48. Giardiello FM, Welsh SB, Hamilton SR, et al Increased risk of cancer in the Peutz-Jeghers syndrome. N Engl J Med. 1987;316:1511-4.
49. Lim W, Hearle N, Shah B, et al. Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome. Br J Cancer. 2003;89:308-13.
50. Boardman LA, Thibodeau SN, Schaid DJ, et al. Increased risk for cancer in patients with the Peutz-Jeghers syndrome. Ann Intern Med. 1998;128:896-9.
51. Hemminki A, Markie D, Tomlinson I, et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature. 1998;391:184-7.
52. Schumacher V, Vogel T, Leube B, et al. STK 11 genotyping and cancer risk in Peutz-Jeghers syndrome. J Med Genet. 2005;42:428-35.
53. Wang Q, Lasset C, Desseigne F, et al. Neurofibromatosis and early onset of cancers in hMLH1-deficient children. Cancer Res. 1999;59:294-7.
54. Whitelaw SC, Murday VA, Tomlinson IP, et al. Clinical and molecular features of the hereditary mixed polyposis syndrome. Gastroenterology. 1997;112:327-34.
55. Chow E, Lipton L, Lynch E, et al. Hyperplastic Polyposis Syndrome: Phenotypic Presentations and the Role of MBD4 and MYH. Gastroenterology. 2006; 131:30-9.
56. Ferrandez A, Samowitz W, DiSario JA, et al. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol. 2004 ;99:2012-8.
57. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum. 2004;47:2101-4.
58. Rashid A, Houlihan PS, Booker S, et al. Phenotypic and molecular characteristics of hypcrplastic polyposis. Gastroenterology. 2000; 119:323-32.