胃癌相关新基因的克隆及生物信息学分析
|
摘要
目的:从胃癌高频率杂合性缺失区域8p21着手,克隆染色体8p21-22区域EST(DA931869)所代表的胃癌相关新基因,初步分析预测该基因的结构及编码的蛋白质分子与功能,为进一步鉴定胃癌发病相关新基因奠定坚实的工作基础。
方法:收集42例胃癌手术切除新鲜标本,切取胃癌组织为实验组,以远离癌组织(≥5cm)胃黏膜组织作为正常对照组,所有样本均经病理学确诊,分别提取胃癌及正常胃黏膜组织总RNA,运用RT-PCR方法验证EST的表达水平;将EST作为起始序列,通过BLAST分析dbEST数据库,进行电子克隆(in silico cloning),以DNASTAR软件进行序列拼接与延伸,获得该基因的全长cDNA序列;采用生物信息软件,预测该cDNA序列的开放阅读框,染色体定位,及其所编码的蛋白质同源性分析,二级结构及其功能预测等;运用染色体步移(Chromosome walking)技术实验验证EST所代表的未知基因全长序列。
结果:RT-PCR验证结果:EST在42例胃癌组织中阳性表达率为40.48%(17/42),在正常胃黏膜组织中阳性表达率为83.33%(35/42),两者差异有统计学意义(p<0.05)。
电子克隆及基因结构预测结果:将EST通过BLAST进行dbEST数据库检索,寻找同源序列进行拼接延伸,第一次延伸往3’端方向延长87bp,随后往5’端方向依次延长443bp、343bp、421bp和473bp,经五次延伸拼接之后,此cDNA序列达最大长度为2326bp。将该cDNA序列与人类基因组草图大规模测序数据库中的序列NT-167187.1进行比较分析,将此cDNA精确定位于8p21,在染色体上跨度770Kbp(24,080-24,850Kbp),由3个外显子和2个内含子组成。ORF FINDER在线软件预测最大开放阅读框长度为354bp,编码117个氨基酸,起始密码子预测分值为0.78。在线软件预测分析结果显示,于序列367位碱基发现TATA盒信号:TGTATAAAAA;加尾信号位于第1932位碱基:GAAATAAAAT;在序列202-587位存在一CpG岛,G+C%为59%。运用DNAMAN软件进行限制性酶切分析到60个酶切位点。将该cDNA序列与nr数据库BLAST比对,结果显示,EST(DA931869)所代表的未知基因序列与神经丝中间多肽基因序列覆盖率为55%。
蛋白质结构及功能预测分析:利用DNASTAR软件的Protean程序对编码蛋白进行蛋白质理化特性分析,该蛋白相对分子质量为13016.04 Daltons,等电点为11.70,为一碱性蛋白质。蛋白质二级结构预测显示,α螺旋分别为1-9,13-18,40-49,β折叠为111-117,其余为β转角及无规则卷曲。蛋白质疏水性预测分析显示该蛋白为一亲水性蛋白质。利用SMART数据库进行蛋白质结构域分析,结果显示28-37和71-91位氨基酸残基存在低复杂性区域,32-43位氨基酸发现一膜占位识别连接结构域。在此编码蛋白中未发现跨膜片段及信号肽片段。同源性蛋白质分析结果显示,此编码蛋白与蛋白质转运蛋白Sec16B及视交叉上核昼夜节律振动蛋白SCOP相似。
染色体步移结果:逐步设计特异性引物进行RT-PCR扩增,T-A克隆后测序,经两次步移,共获得此cDNA3’末端序列641bp,经BLAST比对基因组序列,结果一致。
结论:
1.采用电子克隆,得到EST(DA931869)所代表基因序列长度为2326bp,由3个外显子和2个内含子组成,ORF长度为354bp,精确定位于染色体8p21,含有加尾信号。
2.该基因编码一个由117位氨基酸组成的蛋白质分子,含有一个MORN结构域(为32-43位氨基酸),为亲水性蛋白质。
3.染色体步移,获得EST(DA931869)所代表的未知基因3’末端cDNA序列641bp。
Objective: To screen high frequency Loss of Heterozygosity sites associated with gastric carcinoma exist on chromosome 8p21-22, and clone the novel gene which replaced of EST(DA931869), primaly analyze the structure and function of the unknown gene, which provided basis for further identify novel genes associated with the development of gastric carcinoma.
Methods: 42 cases of gastric cancer surgery samples were collected as cancer groups, corresponding normal gastric mucosa away from the cancer point as the normal control groups. All normal and cancer samples were pathologically diagnosed. The total RNA of samples was extracted out, with reverse transcription polymerase chain reaction, to screen the expression of EST (DA931869). Then in silico clone the EST which expressed differently between gastric carcinoma and normal gastric mucosa through the tool of BLAST soft ware,joint and extend the cDNA with the software of DNASTAR. It was predicted the ORF, Chromosome Location for the cDNA, and predicted protein homology, protein’s secondary structure and functions for the protein which coding by the cDNA. The full lenth of unknown gene which replaced by EST (DA931869) was identified by chromosome walking.
Results: The RT-PCR results showed that EST (DA931869) expressed significantly different between gastric carcinoma and normal gastric mucosa by RT-PCR approach (p<0.05). The positive expression rate of EST was 83.33%(35/42)in normal gastric mucosa, and 40.48%(17/42)in gastric carcinoma, reduced or absent expressed compared with in normal.
In silico clonging results: The EST was blasted with dbESTdatabase, extended the homology. Firstly we extended 87bp to3’end, and then extended respectively 443bp, 343bp, 421bp and 473bp to5’end. The full lenth of cDNA was 2326bp through extending five times. It’s located on chromosome 8p21, accrossed 770Kbp(24,080-24,850Kbp),the genome sequence (NT-167187.1)included the cDNA. The cDNA was blasted with nr database, the result showed that the identity of the unknown gene and neurofilament, medium polypeptide were 55%.
The lenth of ORF which consisted of 117aa was 354bp which predicted By online software ORF FINDER.The predicted values of starting codon was 0.78. Bioinformatics tools found that the TATA box at 367bp, the poly(A) signal at 1932bp, and existed a CpG island within 202-587bp, G+C% was 59%.
Protein mocular weight was 13016.04 Daltons, PI was 11.70, which analyzed by DNASTAR software. The protein might be basic protein. The secondary structure of protein was predicted thatαhelix1-9,13-18,40-49,βsheet111-117, respectively. Structure domain predicted results showed that 32-43aa was MORN domain. The protein was blasted with protein database of NCBI, the result showed that the protein was similar with Sec16B and SCOP.
The results of Chromosome Walking showed that, we have got the 641bp to 3’end of the cDNA through walking two times. Blasting with genome sequences, the results of sequencing were correctly.
Conclusion:
1. We have obtained the full lenth of cDNA sequence 2326bp represented by the EST(DA931869) through in silico cloning, which consisted of 3 extrons and 2 introns. the lenth of ORF was 354bp. It’s precisely located at chromosome 8p21, contains PolyA tails.
2. Protein which consisted of 117aa contains MORN domain(32-43aa). The protein was Hydrophilia protein.
3. we have got the 641bp to 3’end of the cDNA which represented by the EST(DA931869) through Chromosome Walking techniques.
方法:收集42例胃癌手术切除新鲜标本,切取胃癌组织为实验组,以远离癌组织(≥5cm)胃黏膜组织作为正常对照组,所有样本均经病理学确诊,分别提取胃癌及正常胃黏膜组织总RNA,运用RT-PCR方法验证EST的表达水平;将EST作为起始序列,通过BLAST分析dbEST数据库,进行电子克隆(in silico cloning),以DNASTAR软件进行序列拼接与延伸,获得该基因的全长cDNA序列;采用生物信息软件,预测该cDNA序列的开放阅读框,染色体定位,及其所编码的蛋白质同源性分析,二级结构及其功能预测等;运用染色体步移(Chromosome walking)技术实验验证EST所代表的未知基因全长序列。
结果:RT-PCR验证结果:EST在42例胃癌组织中阳性表达率为40.48%(17/42),在正常胃黏膜组织中阳性表达率为83.33%(35/42),两者差异有统计学意义(p<0.05)。
电子克隆及基因结构预测结果:将EST通过BLAST进行dbEST数据库检索,寻找同源序列进行拼接延伸,第一次延伸往3’端方向延长87bp,随后往5’端方向依次延长443bp、343bp、421bp和473bp,经五次延伸拼接之后,此cDNA序列达最大长度为2326bp。将该cDNA序列与人类基因组草图大规模测序数据库中的序列NT-167187.1进行比较分析,将此cDNA精确定位于8p21,在染色体上跨度770Kbp(24,080-24,850Kbp),由3个外显子和2个内含子组成。ORF FINDER在线软件预测最大开放阅读框长度为354bp,编码117个氨基酸,起始密码子预测分值为0.78。在线软件预测分析结果显示,于序列367位碱基发现TATA盒信号:TGTATAAAAA;加尾信号位于第1932位碱基:GAAATAAAAT;在序列202-587位存在一CpG岛,G+C%为59%。运用DNAMAN软件进行限制性酶切分析到60个酶切位点。将该cDNA序列与nr数据库BLAST比对,结果显示,EST(DA931869)所代表的未知基因序列与神经丝中间多肽基因序列覆盖率为55%。
蛋白质结构及功能预测分析:利用DNASTAR软件的Protean程序对编码蛋白进行蛋白质理化特性分析,该蛋白相对分子质量为13016.04 Daltons,等电点为11.70,为一碱性蛋白质。蛋白质二级结构预测显示,α螺旋分别为1-9,13-18,40-49,β折叠为111-117,其余为β转角及无规则卷曲。蛋白质疏水性预测分析显示该蛋白为一亲水性蛋白质。利用SMART数据库进行蛋白质结构域分析,结果显示28-37和71-91位氨基酸残基存在低复杂性区域,32-43位氨基酸发现一膜占位识别连接结构域。在此编码蛋白中未发现跨膜片段及信号肽片段。同源性蛋白质分析结果显示,此编码蛋白与蛋白质转运蛋白Sec16B及视交叉上核昼夜节律振动蛋白SCOP相似。
染色体步移结果:逐步设计特异性引物进行RT-PCR扩增,T-A克隆后测序,经两次步移,共获得此cDNA3’末端序列641bp,经BLAST比对基因组序列,结果一致。
结论:
1.采用电子克隆,得到EST(DA931869)所代表基因序列长度为2326bp,由3个外显子和2个内含子组成,ORF长度为354bp,精确定位于染色体8p21,含有加尾信号。
2.该基因编码一个由117位氨基酸组成的蛋白质分子,含有一个MORN结构域(为32-43位氨基酸),为亲水性蛋白质。
3.染色体步移,获得EST(DA931869)所代表的未知基因3’末端cDNA序列641bp。
Objective: To screen high frequency Loss of Heterozygosity sites associated with gastric carcinoma exist on chromosome 8p21-22, and clone the novel gene which replaced of EST(DA931869), primaly analyze the structure and function of the unknown gene, which provided basis for further identify novel genes associated with the development of gastric carcinoma.
Methods: 42 cases of gastric cancer surgery samples were collected as cancer groups, corresponding normal gastric mucosa away from the cancer point as the normal control groups. All normal and cancer samples were pathologically diagnosed. The total RNA of samples was extracted out, with reverse transcription polymerase chain reaction, to screen the expression of EST (DA931869). Then in silico clone the EST which expressed differently between gastric carcinoma and normal gastric mucosa through the tool of BLAST soft ware,joint and extend the cDNA with the software of DNASTAR. It was predicted the ORF, Chromosome Location for the cDNA, and predicted protein homology, protein’s secondary structure and functions for the protein which coding by the cDNA. The full lenth of unknown gene which replaced by EST (DA931869) was identified by chromosome walking.
Results: The RT-PCR results showed that EST (DA931869) expressed significantly different between gastric carcinoma and normal gastric mucosa by RT-PCR approach (p<0.05). The positive expression rate of EST was 83.33%(35/42)in normal gastric mucosa, and 40.48%(17/42)in gastric carcinoma, reduced or absent expressed compared with in normal.
In silico clonging results: The EST was blasted with dbESTdatabase, extended the homology. Firstly we extended 87bp to3’end, and then extended respectively 443bp, 343bp, 421bp and 473bp to5’end. The full lenth of cDNA was 2326bp through extending five times. It’s located on chromosome 8p21, accrossed 770Kbp(24,080-24,850Kbp),the genome sequence (NT-167187.1)included the cDNA. The cDNA was blasted with nr database, the result showed that the identity of the unknown gene and neurofilament, medium polypeptide were 55%.
The lenth of ORF which consisted of 117aa was 354bp which predicted By online software ORF FINDER.The predicted values of starting codon was 0.78. Bioinformatics tools found that the TATA box at 367bp, the poly(A) signal at 1932bp, and existed a CpG island within 202-587bp, G+C% was 59%.
Protein mocular weight was 13016.04 Daltons, PI was 11.70, which analyzed by DNASTAR software. The protein might be basic protein. The secondary structure of protein was predicted thatαhelix1-9,13-18,40-49,βsheet111-117, respectively. Structure domain predicted results showed that 32-43aa was MORN domain. The protein was blasted with protein database of NCBI, the result showed that the protein was similar with Sec16B and SCOP.
The results of Chromosome Walking showed that, we have got the 641bp to 3’end of the cDNA through walking two times. Blasting with genome sequences, the results of sequencing were correctly.
Conclusion:
1. We have obtained the full lenth of cDNA sequence 2326bp represented by the EST(DA931869) through in silico cloning, which consisted of 3 extrons and 2 introns. the lenth of ORF was 354bp. It’s precisely located at chromosome 8p21, contains PolyA tails.
2. Protein which consisted of 117aa contains MORN domain(32-43aa). The protein was Hydrophilia protein.
3. we have got the 641bp to 3’end of the cDNA which represented by the EST(DA931869) through Chromosome Walking techniques.
引文
[1]徐飚,王建明.胃癌流行病学研究[J].中华肿瘤防治杂志, 2006, 13(1):1-7.
[2] Ryoji Kaizaki, Masakazu Yashiro, Osamu Shinto, et al. Expression of ERas Oncogene in Gastric Carcinoma[J]. Anticancer Res, 2009, 29: 2189 - 2193.
[3] Pinto de Sousa J, David L, Almeida R, et al. c-erb B-2 expression is associated with tumor location and venous invasion and influences survival of patients with gastric carcinoma[J]. Int J Surg Pathol, 2002, 10(4):247-256.
[4] Kuraoka K, Matsumura S, Hamai Y, et al. A single nucleotide polymorphism in the transmembrane domain coding region of HER-2 is associated with development phenotype of gastric cancer[J]. Int J Cancer, 2003, 107(4):593-596.
[5] Chan AW, Chan MW, Lee TL, et al. Promoter hypermethylation of Death- associated protein-kinase gene associated with advance stage gastric cancer[J]. Oncol Rep. 2005, 13(5):937-941.
[6] Roa JC, Araya JC, Villaseca MA, et al. Microsatellite instability and loss of heterozygosity in neoplastic and preneoplastic gastric lesions[J]. Rev Med Chil. 2003, 131(11):1227-36.
[7] Hara T, Ooi A, Kobayashi M, et al. Amplification of c-myc, K-sam, and c-met in gastric cancers detection by fluorescence in situ hybridization[J]. Lab Invest, 1998, 78(9):1143.
[8] Pinto de Sousa J, Silva F, David L, et al. Clinicopathological significance and survival influence of p53 protien expression in gastric carcinoma[J] . Histopathology, 2004, 44(4):323-331.
[9] Migliavacca M, Ottini L, Bazan V, et al. TP53 in gastric cancer mutation in the 13 loop and LSH motify DNA-binding domains of TP53 predict poor outcome[J]. J Cell Physiol, 2004, 200(3):476-485.
[10] Wen-Bo Qin, Shi-Ling Zhang, Yu-Ming Kang, et al.Expression of tumor suppressor gene p16, mutant p53 and c-erbB-2 protein in patients with gastric carcinoma[J]. FASEB J, 2008, 2: 1226.39.
[11] Tahara E. Genetic alterations in human gastrointestinal cancers. The application to molecular diagnosis[J]. Cancer, 1995, 75(6s):1410.
[12] L Wang, F Zhang, PP Wu, et al. Disordered beta-catenin expression and E-cadherin/CDH1 promoter methylation in gastric carcinoma[J]. World J Gastroenterol, 2006; 12(26): 4228-31.
[13] JC Cai, D Liu, HP Zhang, et al. Frequent promoter hypermethylation of several tumor suppressor genes in gastric carcinoma and foveolar epithelium[J]. Zhonghua Zhong Liu Za Zhi, 2007, 29(7): 510-3.
[14] Drebber U, Baldus SE, Nolden B, et al.The overexpression of c-met as a prognostic indicator for gastric carcinoma compared to p53 and p21 nuclear accumulation[J]. Oncol Rep, 2008, 19(6): 1477-83.
[15] Emoto K, Sawada H, Yamada Y, et al. Annexin II overexpression is correlated with poor prognosis in human gastric carcinoma[J]. Anticancer Res, 2001, 21 ( 2B ) : 1339-45.
[16] Vukobrat-Bijedi? Z, Radovi? S, Husi?-Selimovi? A, et al. Tumor suppresser gene p53 expression in premalignant lesions and gastric carcinoma - prognostic value[J]. Bosn J Basic Med Sci, 2007, 7(1):7-10.
[17]张成岗,贺福初.生物信息学在新基因全长cDNA序列分析及功能预测中的应用[J].生物化学与生物物理进展,2003,30(1):159-163.
[18] Wiemann S, Weil B, Wellenreuther R, et al. Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs[J]. Genome Research, 2001, 11(3):422.
[19] Claverie JM. Computational methods for the identification of differential and coordinated gene expression[J]. Hum Mol Genet, 1999, 8(10): 1821- 1832.
[20] Julien JP, C?téF, Beaudet L, et al. Sequence and structure of the mouse gene coding for the largest neurofilament subunit[J].Gene.,1988, 68(2):307-314.
[21] Abe A, Numakura C, Saito K, et al. Neurofilament light chain polypeptide gene mutations in Charcot-Marie-Tooth disease: nonsense mutation probably causes a recessive phenotype[J]. J. Hum. Genet. 2009, 54: 94-97.
[22] Hagihara A, Miyamoto K, Furuta J, et al.Identification of 27 5' CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers[J]. Oncogene, 2004, 23(53):8705-8710.
[23] Catto JW, Abbod MF, Linkens DA, et al. Neurofuzzy modeling to determine recurrence risk following radical cystectomy for nonmetastatic urothelial carcinoma of the bladder[J].Clin Cancer Res, 2009, 15(9):3150-3155.
[24] Quyun C, Ye Z, Lin SC, et al. Recent Patents and Advances in Genomic Biomarker Discovery for Colorectal Cancers[J].DNA Gene Seq, 2010 Apr 29. [Epub ahead of print]
[25] schultz j,milpetz f, et al. SMART, a simple modular architecture research tool: identification of signalling domains[J]. proc natl. acad. sci. 1998, 95: 5857 -5864
[26] Takeshima H, Komazaki S, Nishi M, et al.Junctophilins: a novel family of junctional membrane complex proteins[J]. Mol Cell. 2000; 6: 11-22 .
[27] Nishi M, Mizushima A, Nakagawara Ki, et al.Characterization of human junctophilin subtype genes[J]. Biochem Biophys Res Commun. 2000; 273: 920-7
[28] Dibyendu Bhattacharyya, Benjamin S Glick. Two Mammalian Sec16 Homologues Have Nonredundant Functions in Endoplasmic Reticulum (ER) Export and Transitional ER Organization[J]. Molecular Biology of the Cell. 2007, 18(3): 839–849.
[29] Shimizu K, Okada M, Takano A, et al. SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus[J].FEBS Lett. 1999, 458 (3):363-369.
[30] Shimizu K, Okada M, Nagai K,et al. Suprachiasmatic nucleus circadian oscillatory protein, a novel binding partner of K-Ras in the membrane rafts, negatively regulates MAPK pathway[J]. J Biol Chem. 2003, 278(17):14920-5.
[31] Liu, B, et al. Progress of the PCR amplification techniques for chromosome walking[J]. Yi Chuan, 2006. 28(5): 587-95.
[32] Walker JA, Kilroy GE, Xing J, et al.Human DNA quantitation using Alu element-based polymerase chain reaction[J].Anal Biochem. 2003, 315(1):122-8.
[33] Huang H, Wang G, Zhao Y, et al.Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR [J]. Appl Microbiol Biotechnol. 2010 Apr 27. [Epub ahead of print]
[34] Bae JH, Sohn JH. Template-blocking PCR: an advanced PCR technique for genome walking[J].Anal Biochem. 2010 , 398(1):112-6.
[35] Gibaud A, Vogt N, Hadj-Hamou NS, et al.Extrachromosomal amplification mechanisms in a glioma with amplified sequences from multiple chromosome loci[J].Hum Mol Genet. 2010 , 19(7):1276-85.
[1] Jemal A, Siegel R,Ward E, et a1.Cancer statistics, 2006 [J]. CACancer J Clin, 2006, 56 (2) : 1062130.
[2] Houghton J,Wang TC. Helicobacter pylori and gastric cancer: a new paradigm for inflammation2associated ep ithelial cancers [J].Gastroenterology, 2005, 128 (6) : 156721578.
[3] Correa P. Helicobacter pylori and gastric carcinogenesis[J]. Am J Surg Pathol, 1995, 19 ( Supp l 1) : 37241.
[4] Sagawa T, Takayama T, Oku T, et a1.Argon plasma coagulation for successful treatment of early gastric cancer with intramucosal invasion[J] . Gut , 2003 , 52 (3) : 334 - 339.
[5]房殿春,王东旭,罗元辉,等. PCR检测胃癌MCC、DCC基因和YNZ22位点串联重复序列的杂合性丢失[J] .中华消化内镜杂志, 1997 , 14 (2) : 203 - 206.
[6] Unger Z, Molnar B, Pronai L,et a1.Mutant p53 expression and apoptotic activity of Helicobacter pylori positive and negative gastritis in correlation with the presence of intestinal metaplasia [J] . Eur J Gas troenterol Hepatol , 2003 , 15 (4) : 389 - 393.
[7] Kim B, Byun SJ, Kim YA, et a1.Cell cycle regulators, APC/beta-catenin, NF-kappaB and Epstein-Barr virus in gastric carcinomas [J].Pathology. 2010 ,42(1):58-65.
[8] Ksiaa F, Ziadi S, Amara K, et a1.Biological significance of promoter hypermethylation of tumor-related genes in patients with gastric carcinoma [J].Clin Chim Acta. 2009, 27;404(2):128-33.
[9] Wang D, Fang D, Luo Y, et a1.Study of loss of heterozygosity at DCC and APC/MCC genetic loci of gastric cancer [J].Chin Med Sci J. 1999 ,14(2):107-11.
[10] Hibi K, Sakata M, Sakuraba K, et a1. Methylation of the DCC gene is lost in advanced gastric cancer [J].Anticancer Res. 2010 ,30(1):107-9.
[11] Zhang R,Mayhoed T, Lipari P, et a1.Fluorescence polarization assay and inhibitor design for mdm2/p53 interaction[J]. Anal Biochem, 2004,331:138- 1461
[12]孙利平,李岩,张宁,等.Mdm2基因扩增和蛋白表达与胃癌相关性的研究[J].世界华人消化杂志, 2003, l1: 1800–1801.
[13]张健慧,李琰,王端等.中国人食管癌及肺癌发病风险与p53基因多态性[J].中国肿瘤杂志, 2003, 25 (4) : 365– 3671.
[14] Zhang Y, Shi Y, Li X, et a1. Inhibition of the p53-MDM2 interaction by adenovirus delivery of ribosomal protein L23 stabilizes p53 and induces cell cycle arrest and apoptosis in gastric cancer[J]. J Gene Med. 2010,12(2):147-56.
[15] Mira A,MCGuirkM, Hockenberry TN, et a1.Human surviving is negatively regulated by wide type p53 and participates in p53 depend entapoptofie partway[J]. Oncogene, 2002, 21: 26131
[16] Zhu Y, Qiu M, Zhou J, et a1.Analysis of the P53 transcriptional activity in gastric cancer[J].Zhonghua Yi Xue Yi Chuan Xue Za Zhi.2010 , 27(1):60-5.
[17] Dimrl GP, Itahan ak, Acosta M, et a11Regulation of a senescence check point response by the E2F - l, transcription factor and pl4 tumor suppresson[J]. Mol Cell Boil, 2000, 20(1):273– 2851.
[18] Tomorawa S, Tsuno NH, Sunami E, et a1.Cyclooxygenase - 2 over expression correlates with tumour recurrence, especially haematogenous metastasis of colorectal cancer[J]. Br J Cancer, 2000, 83:324-3281
[19] Subbaramaiah K, Ahorki N, ChungWJ, et a1. Inhibition of cyclooxygerrnse - 2 gene expression by p53[J].J Biol Chem, 1999, 274 (16):10911– 10915.
[20] BouretM, EllislM,Nishirakim, et a1.Adenorrusmediated wide type p53 gene transfer down regulates vaseular endothelial growth factor expression and inhibits angiogenesis in human colon caneer[J]. Cancer Res, 1998, 58 (1):2288– 2292.
[21]马波,赵吉生,丁大勇.p53、VEGF的表达与胃癌生物学行为相关性的研究[J].中国实验诊断学, 2005, 9 (6):920– 9221.
[22] GottesmanMM, Pastan L .Biochemistry of muhidm resistance mediated by the muhidrug transporter[J]. Annu RevBiochem, 1993, 62: 3851-3852.
[23] Kamb A, Gruis N A, Wearer-Feldans J,et al.A cell cycle regulator potentially involved in genesis of many tumor types[J]. Science,1994,264(5157):436-440.
[24] Blok P , Craanen ME , Offerhaus G, et al . Molecular alteration in early gastric carcinomas [J] . AmJ Clin Pathol , 1999,111:241-246.
[25] Murakami N, Koufuji K, Shirouzu K. Influence of hepatocyte growth factor secreted from fibroblasts on the growth and invasion of scirrhous gastric cancer [J] . Int Surg, 2001,86 (3):151 - 157.
[26] Tian H, Zhang N, Suo WH, et al.Gastrin suppresses the interdependent expression of p16 and anion exchanger 1 favouring growth inhibition of gastric cancer cells[J].Int J Cancer. 2009 , 17(3):321-325.
[27] Pinto de.Sousa J,Silva F,David L,et al. Clinicopathological significance and survival influence of p53 protein expression in gastric carcinoma[J] . Histopathology, 2004, 44(4):323.
[28] He XS, Rong YH, Su Q,et al. Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance[J].World J Gastroenterol, 2005,11(15):2218.
[29] Mino A,Onoda N,Yashiro M,et al. Frequent p16 CpG island hypermethylation in primary remnant gastric cancer suggesting an independent carcinogenic pathway[J]. Oncol Rep, 2006,15(3):615.
[30] Lee YY, Kang SH, Seo JY, et al. Alterations of p16 INK4A and p15 INK4B genes in gastric carcinomas [J]. Cancer, 1997, 80(10):1889-1896.
[31] Song SH, Jong HS, Choi HH, et al. Methylation of specific CpG sites in the promoter region could significantly down2regulate p16 INK4a expression in gastric adenocarcinoma [J]. Int J Cancer, 2000, 87 (2):236-240.
[32] Sirak I, Petera J, Hatlova J, et al. Expression of p53, p21 and p16 does not correlate with response to preoperative chemoradiation in gastric carcinoma[J]. Hepatogastroenterology. 2009 ,56(93):1213-8.
[33] Shim YH, Kang GH, Ro JY. Correlation of p16 hypermethylation with p16 protein loss in sporadic gastric carcinomas [J]. Lab In.vest, 2000, 80(5) : 689-695.
[34] Park GS,Joo YE,Kim HS,et al. Expression of PTEN and its correlation with angiogenesis in gastric carcinoma [J]. Korean J Gastroenterol, 2005, 46(3):196.
[35] Semba S, Satake S, Matsushita M, et al .Phosphatase activity of nuclear PTEN is required for CDX2-mediated intestinal differentiation of gastric carcinoma[J]. Cancer Lett. 2009 ,8;274(1):143-50.
[36] Zheng HC, Li YL, Sun JM, et al. Growth, invasion, metastasis, differentiation, angiogenesis and apoptosis of gastric cancer regulated by expression of PTEN encoding products[J].World J Gastroenterol,2003, 9(8):1662.
[37] Stec-Michalska K, Peczek L, Michalski B, et al.Helicobacter pylori infection and family history of gastric cancer decrease expression of FHIT tumor suppressor gene in gastric mucosa of dyspeptic patients. Helicobacter[J]. 2009,14(5):126-34.
[38] Zheng H, Takahashi H, Murai Y, et al.Low expression of FHIT and PTEN correlates with malignancy of gastric carcinomas: tissue-array findings[J].Appl Immunohistochem Mol Morphol. 2007 ,15(4):432-40.
[39] Zhao P, Liu W, Lu YL. Clinicopathological significance of FHIT protein expression in gastric adenocarcinoma patients[J]. World J Gastroenterol, 2005,11(36):5735.
[40] Kouraklis G, Katsoulis IE, Theocharis S, et al.Does the expression of cyclin E, pRb, and p21 correlate with prognosis in gastric adenocarcinoma? [J].Dig Dis Sci. 2009,54(5):1015-20.
[41] Moundhri MS, Nirmala V, Al- Hadabi I,et al. The prognostic significance of p53 p27 kip1 p21 waf1 HER- 2/neu and Ki67 proteins expression in gastric cancer: a clinicopathological and immunohistochemical study of 121 Arab patients[J]. J Surg Oncol,2005,91(4):243.
[42] Dammann R,Li C, Yoon JH, et al. Epigenetic inactivation of a Ras association domain family protein from the lung tumor suppressor locus 3p21.3[J]. Nat Genet, 2000, 25 (3):315～319.
[43] Khokhlatchev A, Rabizadeh S, Xavier R, et al. Identification of a novel Ras - regulated proapoptotic pathway[J]. Curr Biol, 2002,(4) : 253～265.
[44] Dammann R, Takahashi T, Pfeifer GP, et al. The CpG Island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas[ J ]. Oncogene, 2001, 20 (27) :3563～3567.
[45] Shivakumar L,Minna J, Sakamaki T, et al. The RASSFIA tumor suppressor blocks cell cycle p rogression and inhibits cyclinD1 accumulation[ J ]. Mol Cell Biol, 2002, 22 (12) : 4309-4318.
[46] Guo W, Dong Z, Chen Z, et al.Aberrant CpG island hypermethylation of RASSF1A in gastric cardia adenocarcinoma[J].Cancer Invest. 2009,27(4):459-65.
[47] Byun DS, Lee MG, Chae KS, et al. Frequent ep igenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma[J]. Cancer Rer, 2001, 61 ( 19 ) : 7034-7038.
[48] Wang YC, Yu ZH, Liu C, et al. Detection of RASSF1A promoter hypermethylation in serum from gastric and colorectal adenocarcinoma patients[J].World J Gastroenterol. 2008,21;14(19):3074-80.
[49] Kang GH, Lee S, KIm JS, et al. Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis [J]. Lab Invest, 2003, 83 (5): 635～641.
[50] Toyooka S, Carbone M , Toyooka KO, et al. Progressivea berrant methylation of the RASSFIA gene insimian virus40 infected human mesothelial cells [J]. Oncogene, 2002, 21(27):4340-4343.
[51] Kang GH, Lee S, Kim WH, et al. Epstein - barr virus– positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype -positive gastric carcinoma[J]. Am J Pathol, 2002, 160 (3):787-794.
[2] Ryoji Kaizaki, Masakazu Yashiro, Osamu Shinto, et al. Expression of ERas Oncogene in Gastric Carcinoma[J]. Anticancer Res, 2009, 29: 2189 - 2193.
[3] Pinto de Sousa J, David L, Almeida R, et al. c-erb B-2 expression is associated with tumor location and venous invasion and influences survival of patients with gastric carcinoma[J]. Int J Surg Pathol, 2002, 10(4):247-256.
[4] Kuraoka K, Matsumura S, Hamai Y, et al. A single nucleotide polymorphism in the transmembrane domain coding region of HER-2 is associated with development phenotype of gastric cancer[J]. Int J Cancer, 2003, 107(4):593-596.
[5] Chan AW, Chan MW, Lee TL, et al. Promoter hypermethylation of Death- associated protein-kinase gene associated with advance stage gastric cancer[J]. Oncol Rep. 2005, 13(5):937-941.
[6] Roa JC, Araya JC, Villaseca MA, et al. Microsatellite instability and loss of heterozygosity in neoplastic and preneoplastic gastric lesions[J]. Rev Med Chil. 2003, 131(11):1227-36.
[7] Hara T, Ooi A, Kobayashi M, et al. Amplification of c-myc, K-sam, and c-met in gastric cancers detection by fluorescence in situ hybridization[J]. Lab Invest, 1998, 78(9):1143.
[8] Pinto de Sousa J, Silva F, David L, et al. Clinicopathological significance and survival influence of p53 protien expression in gastric carcinoma[J] . Histopathology, 2004, 44(4):323-331.
[9] Migliavacca M, Ottini L, Bazan V, et al. TP53 in gastric cancer mutation in the 13 loop and LSH motify DNA-binding domains of TP53 predict poor outcome[J]. J Cell Physiol, 2004, 200(3):476-485.
[10] Wen-Bo Qin, Shi-Ling Zhang, Yu-Ming Kang, et al.Expression of tumor suppressor gene p16, mutant p53 and c-erbB-2 protein in patients with gastric carcinoma[J]. FASEB J, 2008, 2: 1226.39.
[11] Tahara E. Genetic alterations in human gastrointestinal cancers. The application to molecular diagnosis[J]. Cancer, 1995, 75(6s):1410.
[12] L Wang, F Zhang, PP Wu, et al. Disordered beta-catenin expression and E-cadherin/CDH1 promoter methylation in gastric carcinoma[J]. World J Gastroenterol, 2006; 12(26): 4228-31.
[13] JC Cai, D Liu, HP Zhang, et al. Frequent promoter hypermethylation of several tumor suppressor genes in gastric carcinoma and foveolar epithelium[J]. Zhonghua Zhong Liu Za Zhi, 2007, 29(7): 510-3.
[14] Drebber U, Baldus SE, Nolden B, et al.The overexpression of c-met as a prognostic indicator for gastric carcinoma compared to p53 and p21 nuclear accumulation[J]. Oncol Rep, 2008, 19(6): 1477-83.
[15] Emoto K, Sawada H, Yamada Y, et al. Annexin II overexpression is correlated with poor prognosis in human gastric carcinoma[J]. Anticancer Res, 2001, 21 ( 2B ) : 1339-45.
[16] Vukobrat-Bijedi? Z, Radovi? S, Husi?-Selimovi? A, et al. Tumor suppresser gene p53 expression in premalignant lesions and gastric carcinoma - prognostic value[J]. Bosn J Basic Med Sci, 2007, 7(1):7-10.
[17]张成岗,贺福初.生物信息学在新基因全长cDNA序列分析及功能预测中的应用[J].生物化学与生物物理进展,2003,30(1):159-163.
[18] Wiemann S, Weil B, Wellenreuther R, et al. Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs[J]. Genome Research, 2001, 11(3):422.
[19] Claverie JM. Computational methods for the identification of differential and coordinated gene expression[J]. Hum Mol Genet, 1999, 8(10): 1821- 1832.
[20] Julien JP, C?téF, Beaudet L, et al. Sequence and structure of the mouse gene coding for the largest neurofilament subunit[J].Gene.,1988, 68(2):307-314.
[21] Abe A, Numakura C, Saito K, et al. Neurofilament light chain polypeptide gene mutations in Charcot-Marie-Tooth disease: nonsense mutation probably causes a recessive phenotype[J]. J. Hum. Genet. 2009, 54: 94-97.
[22] Hagihara A, Miyamoto K, Furuta J, et al.Identification of 27 5' CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers[J]. Oncogene, 2004, 23(53):8705-8710.
[23] Catto JW, Abbod MF, Linkens DA, et al. Neurofuzzy modeling to determine recurrence risk following radical cystectomy for nonmetastatic urothelial carcinoma of the bladder[J].Clin Cancer Res, 2009, 15(9):3150-3155.
[24] Quyun C, Ye Z, Lin SC, et al. Recent Patents and Advances in Genomic Biomarker Discovery for Colorectal Cancers[J].DNA Gene Seq, 2010 Apr 29. [Epub ahead of print]
[25] schultz j,milpetz f, et al. SMART, a simple modular architecture research tool: identification of signalling domains[J]. proc natl. acad. sci. 1998, 95: 5857 -5864
[26] Takeshima H, Komazaki S, Nishi M, et al.Junctophilins: a novel family of junctional membrane complex proteins[J]. Mol Cell. 2000; 6: 11-22 .
[27] Nishi M, Mizushima A, Nakagawara Ki, et al.Characterization of human junctophilin subtype genes[J]. Biochem Biophys Res Commun. 2000; 273: 920-7
[28] Dibyendu Bhattacharyya, Benjamin S Glick. Two Mammalian Sec16 Homologues Have Nonredundant Functions in Endoplasmic Reticulum (ER) Export and Transitional ER Organization[J]. Molecular Biology of the Cell. 2007, 18(3): 839–849.
[29] Shimizu K, Okada M, Takano A, et al. SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus[J].FEBS Lett. 1999, 458 (3):363-369.
[30] Shimizu K, Okada M, Nagai K,et al. Suprachiasmatic nucleus circadian oscillatory protein, a novel binding partner of K-Ras in the membrane rafts, negatively regulates MAPK pathway[J]. J Biol Chem. 2003, 278(17):14920-5.
[31] Liu, B, et al. Progress of the PCR amplification techniques for chromosome walking[J]. Yi Chuan, 2006. 28(5): 587-95.
[32] Walker JA, Kilroy GE, Xing J, et al.Human DNA quantitation using Alu element-based polymerase chain reaction[J].Anal Biochem. 2003, 315(1):122-8.
[33] Huang H, Wang G, Zhao Y, et al.Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR [J]. Appl Microbiol Biotechnol. 2010 Apr 27. [Epub ahead of print]
[34] Bae JH, Sohn JH. Template-blocking PCR: an advanced PCR technique for genome walking[J].Anal Biochem. 2010 , 398(1):112-6.
[35] Gibaud A, Vogt N, Hadj-Hamou NS, et al.Extrachromosomal amplification mechanisms in a glioma with amplified sequences from multiple chromosome loci[J].Hum Mol Genet. 2010 , 19(7):1276-85.
[1] Jemal A, Siegel R,Ward E, et a1.Cancer statistics, 2006 [J]. CACancer J Clin, 2006, 56 (2) : 1062130.
[2] Houghton J,Wang TC. Helicobacter pylori and gastric cancer: a new paradigm for inflammation2associated ep ithelial cancers [J].Gastroenterology, 2005, 128 (6) : 156721578.
[3] Correa P. Helicobacter pylori and gastric carcinogenesis[J]. Am J Surg Pathol, 1995, 19 ( Supp l 1) : 37241.
[4] Sagawa T, Takayama T, Oku T, et a1.Argon plasma coagulation for successful treatment of early gastric cancer with intramucosal invasion[J] . Gut , 2003 , 52 (3) : 334 - 339.
[5]房殿春,王东旭,罗元辉,等. PCR检测胃癌MCC、DCC基因和YNZ22位点串联重复序列的杂合性丢失[J] .中华消化内镜杂志, 1997 , 14 (2) : 203 - 206.
[6] Unger Z, Molnar B, Pronai L,et a1.Mutant p53 expression and apoptotic activity of Helicobacter pylori positive and negative gastritis in correlation with the presence of intestinal metaplasia [J] . Eur J Gas troenterol Hepatol , 2003 , 15 (4) : 389 - 393.
[7] Kim B, Byun SJ, Kim YA, et a1.Cell cycle regulators, APC/beta-catenin, NF-kappaB and Epstein-Barr virus in gastric carcinomas [J].Pathology. 2010 ,42(1):58-65.
[8] Ksiaa F, Ziadi S, Amara K, et a1.Biological significance of promoter hypermethylation of tumor-related genes in patients with gastric carcinoma [J].Clin Chim Acta. 2009, 27;404(2):128-33.
[9] Wang D, Fang D, Luo Y, et a1.Study of loss of heterozygosity at DCC and APC/MCC genetic loci of gastric cancer [J].Chin Med Sci J. 1999 ,14(2):107-11.
[10] Hibi K, Sakata M, Sakuraba K, et a1. Methylation of the DCC gene is lost in advanced gastric cancer [J].Anticancer Res. 2010 ,30(1):107-9.
[11] Zhang R,Mayhoed T, Lipari P, et a1.Fluorescence polarization assay and inhibitor design for mdm2/p53 interaction[J]. Anal Biochem, 2004,331:138- 1461
[12]孙利平,李岩,张宁,等.Mdm2基因扩增和蛋白表达与胃癌相关性的研究[J].世界华人消化杂志, 2003, l1: 1800–1801.
[13]张健慧,李琰,王端等.中国人食管癌及肺癌发病风险与p53基因多态性[J].中国肿瘤杂志, 2003, 25 (4) : 365– 3671.
[14] Zhang Y, Shi Y, Li X, et a1. Inhibition of the p53-MDM2 interaction by adenovirus delivery of ribosomal protein L23 stabilizes p53 and induces cell cycle arrest and apoptosis in gastric cancer[J]. J Gene Med. 2010,12(2):147-56.
[15] Mira A,MCGuirkM, Hockenberry TN, et a1.Human surviving is negatively regulated by wide type p53 and participates in p53 depend entapoptofie partway[J]. Oncogene, 2002, 21: 26131
[16] Zhu Y, Qiu M, Zhou J, et a1.Analysis of the P53 transcriptional activity in gastric cancer[J].Zhonghua Yi Xue Yi Chuan Xue Za Zhi.2010 , 27(1):60-5.
[17] Dimrl GP, Itahan ak, Acosta M, et a11Regulation of a senescence check point response by the E2F - l, transcription factor and pl4 tumor suppresson[J]. Mol Cell Boil, 2000, 20(1):273– 2851.
[18] Tomorawa S, Tsuno NH, Sunami E, et a1.Cyclooxygenase - 2 over expression correlates with tumour recurrence, especially haematogenous metastasis of colorectal cancer[J]. Br J Cancer, 2000, 83:324-3281
[19] Subbaramaiah K, Ahorki N, ChungWJ, et a1. Inhibition of cyclooxygerrnse - 2 gene expression by p53[J].J Biol Chem, 1999, 274 (16):10911– 10915.
[20] BouretM, EllislM,Nishirakim, et a1.Adenorrusmediated wide type p53 gene transfer down regulates vaseular endothelial growth factor expression and inhibits angiogenesis in human colon caneer[J]. Cancer Res, 1998, 58 (1):2288– 2292.
[21]马波,赵吉生,丁大勇.p53、VEGF的表达与胃癌生物学行为相关性的研究[J].中国实验诊断学, 2005, 9 (6):920– 9221.
[22] GottesmanMM, Pastan L .Biochemistry of muhidm resistance mediated by the muhidrug transporter[J]. Annu RevBiochem, 1993, 62: 3851-3852.
[23] Kamb A, Gruis N A, Wearer-Feldans J,et al.A cell cycle regulator potentially involved in genesis of many tumor types[J]. Science,1994,264(5157):436-440.
[24] Blok P , Craanen ME , Offerhaus G, et al . Molecular alteration in early gastric carcinomas [J] . AmJ Clin Pathol , 1999,111:241-246.
[25] Murakami N, Koufuji K, Shirouzu K. Influence of hepatocyte growth factor secreted from fibroblasts on the growth and invasion of scirrhous gastric cancer [J] . Int Surg, 2001,86 (3):151 - 157.
[26] Tian H, Zhang N, Suo WH, et al.Gastrin suppresses the interdependent expression of p16 and anion exchanger 1 favouring growth inhibition of gastric cancer cells[J].Int J Cancer. 2009 , 17(3):321-325.
[27] Pinto de.Sousa J,Silva F,David L,et al. Clinicopathological significance and survival influence of p53 protein expression in gastric carcinoma[J] . Histopathology, 2004, 44(4):323.
[28] He XS, Rong YH, Su Q,et al. Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance[J].World J Gastroenterol, 2005,11(15):2218.
[29] Mino A,Onoda N,Yashiro M,et al. Frequent p16 CpG island hypermethylation in primary remnant gastric cancer suggesting an independent carcinogenic pathway[J]. Oncol Rep, 2006,15(3):615.
[30] Lee YY, Kang SH, Seo JY, et al. Alterations of p16 INK4A and p15 INK4B genes in gastric carcinomas [J]. Cancer, 1997, 80(10):1889-1896.
[31] Song SH, Jong HS, Choi HH, et al. Methylation of specific CpG sites in the promoter region could significantly down2regulate p16 INK4a expression in gastric adenocarcinoma [J]. Int J Cancer, 2000, 87 (2):236-240.
[32] Sirak I, Petera J, Hatlova J, et al. Expression of p53, p21 and p16 does not correlate with response to preoperative chemoradiation in gastric carcinoma[J]. Hepatogastroenterology. 2009 ,56(93):1213-8.
[33] Shim YH, Kang GH, Ro JY. Correlation of p16 hypermethylation with p16 protein loss in sporadic gastric carcinomas [J]. Lab In.vest, 2000, 80(5) : 689-695.
[34] Park GS,Joo YE,Kim HS,et al. Expression of PTEN and its correlation with angiogenesis in gastric carcinoma [J]. Korean J Gastroenterol, 2005, 46(3):196.
[35] Semba S, Satake S, Matsushita M, et al .Phosphatase activity of nuclear PTEN is required for CDX2-mediated intestinal differentiation of gastric carcinoma[J]. Cancer Lett. 2009 ,8;274(1):143-50.
[36] Zheng HC, Li YL, Sun JM, et al. Growth, invasion, metastasis, differentiation, angiogenesis and apoptosis of gastric cancer regulated by expression of PTEN encoding products[J].World J Gastroenterol,2003, 9(8):1662.
[37] Stec-Michalska K, Peczek L, Michalski B, et al.Helicobacter pylori infection and family history of gastric cancer decrease expression of FHIT tumor suppressor gene in gastric mucosa of dyspeptic patients. Helicobacter[J]. 2009,14(5):126-34.
[38] Zheng H, Takahashi H, Murai Y, et al.Low expression of FHIT and PTEN correlates with malignancy of gastric carcinomas: tissue-array findings[J].Appl Immunohistochem Mol Morphol. 2007 ,15(4):432-40.
[39] Zhao P, Liu W, Lu YL. Clinicopathological significance of FHIT protein expression in gastric adenocarcinoma patients[J]. World J Gastroenterol, 2005,11(36):5735.
[40] Kouraklis G, Katsoulis IE, Theocharis S, et al.Does the expression of cyclin E, pRb, and p21 correlate with prognosis in gastric adenocarcinoma? [J].Dig Dis Sci. 2009,54(5):1015-20.
[41] Moundhri MS, Nirmala V, Al- Hadabi I,et al. The prognostic significance of p53 p27 kip1 p21 waf1 HER- 2/neu and Ki67 proteins expression in gastric cancer: a clinicopathological and immunohistochemical study of 121 Arab patients[J]. J Surg Oncol,2005,91(4):243.
[42] Dammann R,Li C, Yoon JH, et al. Epigenetic inactivation of a Ras association domain family protein from the lung tumor suppressor locus 3p21.3[J]. Nat Genet, 2000, 25 (3):315～319.
[43] Khokhlatchev A, Rabizadeh S, Xavier R, et al. Identification of a novel Ras - regulated proapoptotic pathway[J]. Curr Biol, 2002,(4) : 253～265.
[44] Dammann R, Takahashi T, Pfeifer GP, et al. The CpG Island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas[ J ]. Oncogene, 2001, 20 (27) :3563～3567.
[45] Shivakumar L,Minna J, Sakamaki T, et al. The RASSFIA tumor suppressor blocks cell cycle p rogression and inhibits cyclinD1 accumulation[ J ]. Mol Cell Biol, 2002, 22 (12) : 4309-4318.
[46] Guo W, Dong Z, Chen Z, et al.Aberrant CpG island hypermethylation of RASSF1A in gastric cardia adenocarcinoma[J].Cancer Invest. 2009,27(4):459-65.
[47] Byun DS, Lee MG, Chae KS, et al. Frequent ep igenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma[J]. Cancer Rer, 2001, 61 ( 19 ) : 7034-7038.
[48] Wang YC, Yu ZH, Liu C, et al. Detection of RASSF1A promoter hypermethylation in serum from gastric and colorectal adenocarcinoma patients[J].World J Gastroenterol. 2008,21;14(19):3074-80.
[49] Kang GH, Lee S, KIm JS, et al. Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis [J]. Lab Invest, 2003, 83 (5): 635～641.
[50] Toyooka S, Carbone M , Toyooka KO, et al. Progressivea berrant methylation of the RASSFIA gene insimian virus40 infected human mesothelial cells [J]. Oncogene, 2002, 21(27):4340-4343.
[51] Kang GH, Lee S, Kim WH, et al. Epstein - barr virus– positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype -positive gastric carcinoma[J]. Am J Pathol, 2002, 160 (3):787-794.