Full-Length cDNA Cloning of FXYD6 Gene Related to Cholangiocarcinoma with Bioinformatic Localization of Its Functional Domain_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WAN Tao ¹ , ZHOU Ningxin ² , XU Donggang ³ , WU Jiajin ³ , FAN Wenhong ³ , HUANG Zhiqiang ¹

1.Institute of Hepatobiliary Surgery of General Hospital of PLA, Beijing 100853, China;;
2.Institute of Hepatobiliary and Gastrointestinal Diseases of The Second Artillery General Hospital of PLA, Beijing 100088, China;;
3.Institute of Basic Medicine, Academy of Military Medical Sciences of PLA， Beijing 100850, China;

Corresponding author：

Keywords：

Cholangiocarcinoma; Ion channel; FXYD6 gene; Cloning; Localization of functional domain

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To obtain the full-length gene and functional domains of FXYD6 gene which is a cholangiocarcinoma related gene.
Methods A new strategy with the integration of bioinformatics and molecular biology was used. Bioinformatical methods were used to analyze the full-length sequence, and to predict the functional domains of its protein. And the full-length sequence of FXYD6 was isolated by polymerase chain reaction from fetal hepatic, brain and spleen cDNA libraries, and then cloned in pGEM-T vector for sequence analyzing. Goldkey Sequence Analyzing Software was used to analyze the sequence of candidate domain without signal peptide.
Results The full-length sequence of FXYD6 was isolated by Touch-down PCR from fetal hepatic and brain cDNA library, but was not from spleen cDNA library. The open reading frame Finder software was used in the National Center for Biotechnology Information website to find the most probable encoding regions of FXYD6 gene. And the +1 phase was selected as the template sequence, from 67 bp to 354 bp, to predict the functional domains by Goldkey Sequence Analyzing Software. The signal peptide was located from 1 amino acid (aa) to 17 aa， and the main domain was composed from 18 aa to 34 aa. The region between 35 aa and 57 aa was the transmembrane region. The FHYD peptide chain was highly conserved amino acids.
Conclusion The study of full-length cDNA cloning of FXYD6 gene and its functional domains provides the basis for understanding the relationship between the structure and function of FXYD6. More work shall be performed on FXYD6 protein and its influence on the mechanism of cholangiocarcinoma.

Citation： WAN Tao ,ZHOU Ningxin,XU Donggang,WU Jiajin,FAN Wenhong,HUANG Zhiqiang. Full-Length cDNA Cloning of FXYD6 Gene Related to Cholangiocarcinoma with Bioinformatic Localization of Its Functional Domain. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(7): 707-712. doi: Copy

1.	Patel T. Worldwide trends in mortality from biliary tract malignancies [J]. BMC Cancer, 2002; 2: 1014.
2.	邹声泉. 胆管癌外科治疗的现状与展望 [J]. 中国普外基础与临床杂志, 2008; 15(2): 7780.
3.	Hsing AW, Gao YT, Devesa SS, et al. Rising incidence of biliary tract cancers in Shanghai, China [J]. Int J Cancer, 1998; 75(3): 368370.
4.	周宁新, 黄志强, 刘永雄, 等. 肝外胆道癌全国调查1 098例分析 [J]. 中华外科杂志, 1990; 28(9): 516521.
5.	Grainge MJ, West J, SolaymaniDodaran M, et al. The antecedents of biliary cancer: a primary care casecontrol study in the United Kingdom [J]. Br J Cancer, 2009; 100(1): 178180.
6.	万涛, 倪彬, 刘建伟, 等. 人胆管癌相关基因CCRL2/FXYD6大片段克隆与鉴定 [J]. 中华实验外科杂志, 2003; 20(5): 393395.
7.	肖梅, 周宁新, 高丽杰, 等. 半定量检测胆管癌组织中人fxyd6基因的表达 [J]. 中国普外基础与临床杂志, 2008; 15(2): 8387.
8.	http: // www.ncbi.nlm.nic.gov/genbank.
9.	Sweadner KJ, Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression [J]. Genomics, 2000; 68(1): 4156.
10.	周宁新, 黄志强, 张文智, 等. 402例肝门部胆管癌临床分型、手术方式与远期疗效的综合分析 [J]. 中华外科杂志, 2006; 44(23): 15991603.
11.	Hsing AW, Sakoda LC, Rashid A, et al. Variants in inflammation genes and the risk of biliary tract cancers and stones: a populationbased study in China [J]. Cancer Res, 2008; 68 (15): 64426452.
12.	Bishop JM. Molecular themes in oncogenesis [J]. Cell, 1991; 64(2): 235248.
13.	Moolgavkar SH, Luebeck EG. Multistage carcinogenesis and the incidence of human cancer [J]. Genes Chromosomes Cancer, 2003; 38(4): 302306.
14.	Vogelstein B, Kinzler KW. The multistep nature of cancer [J]. Trends Genet, 1993; 9(4): 138141.
15.	Levi S, UrbanoIspizua A, Gill R, et al. Multiple Kras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerse chain reaction technique [J]. Cancer Res, 1991; 51(13): 34973502.
16.	Yoshida S, Todoroki T, Ichikawa Y, et al. Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers [J]. Cancer Res, 1995; 55(13): 27562760.
17.	Lee CS. Ras p21 protein immunoreactivity and its relationship to p53 expression and prognosis in gallbladder and extrahepatic biliary carcinoma [J]. Eur J Surg Oncol, 1997; 23(3): 233237.
18.	Hahn SA, Bartsch D, Schroers A, et al. Mutations of the DPC4/Smad4 gene in biliary tract carcinoma [J]. Cancer Res, 1998; 58(6): 11241126.
19.	Tannapfel A, Engeland K, Weinans L, et al. Expression of p73, a novel protein related to the p53 tumour suppressor p53, and apoptosis in cholangiocellular carcinoma of the liver [J]. Br J Cancer, 1999; 80(7): 10691074.
20.	Ahrendt SA, Eisenberger CF, Yip L, et al. Chromosome 9p21 loss and p16 inactivation in primary sclerosing cholangitisassociated cholangiocarcinoma [J]. J Surg Res, 1999; 84(1): 8893.
21.	Werner H, Song JJ, Have M, et al. Homozygous deletions inactivate DCC, but not MADH4/DPC4/SMAD4, in a subset of pancreatic and biliary cancers [J]. Genes Chromosomes Cancer, 2000; 27(4): 353357.
22.	Ito Y, Takeda T, Sasaki Y, et al. Bcl2 expression in cholangiocellular carcinoma is inversely correlated with biologically aggressive phenotypes [J]. Oncology, 2000; 59(1): 6367.
23.	Tipsmark C. Identification of FXYD protein genes in a teleost: tissuespecific expression and response to salinity change [J]. Am J Physiol Regul Integr Comp Physiol, 2008; 294(4): R1367R1378.
24.	李尹雄. 基因克隆技术 [A]. 见：卢圣栋. 现代分子生物学实验技术 [M]. 北京: 高等教育出版社, 1993: 293294.
25.	黎孟枫译. 聚合酶链式反应体外扩增DNA [A]. 见：金冬雁, 黎孟枫主译. 分子克隆实验指南 [M]. 第2版. 北京: 科学出版社, 1999: 674675.
26.	周隽. 核苷酸顺序测定技术 [A]. 见：陈赛娟. 人类基因组研究基本技术 [M]. 北京: 人民军医出版社, 2002: 166167.
27.	申宗侯. 蛋白质的转运、加工与修饰 [A]. 见：陈惠黎. 生物大分子的结构与功能 [M]. 上海: 上海医科大学出版社, 1999: 196197.
28.	Geering K. FXYD proteins: new regulators of NaKATPase [J]. Am J Physiol Renal Physiol, 2006; 290(2): F241F250.
29.	来鲁华. 蛋白质的结构预测与分子设计 [M]. 北京: 北京大学出版社, 1993: 7981.
30.	万涛, 孙涛, 吴加金. 蛋白顺序性抗原决定簇的多参数综合预测 [J]. 中国免疫学杂志, 1997; 13(6): 329333.
31.	冯健男, 万涛, 吴加金, 等. 基于三维结构的抗原决定簇预测 [J]. 分子科学学报, 1999; 15(2): 112115.
32.	杨思禹. 胆道的功能形态及生理 [A]. 见：张经济, 连至诚, 许冠荪, 等. 消化道生理及病理生理学 [M]. 广州: 广东科技出版社, 1997: 885923.
33.	Nathanson MH, Boyer JL. Mechanisms and regulation of bile secretion [J]. Hepatology, 1991; 14(3): 551566.
34.	Strazzabosco M. New insights into cholangiocyte physiology [J]. J Hepatology, 1997; 27(5): 945952.
35.	Cameron IL, Smith NKR, Pool TB, et al. Intracellural concentration of sodium and other elements are related to mitogenesis and oncogenesis in vivo [J]. Cancer Res, 1980; 40(5): 14931500.
36.	Nagy I, Lustyik G, Lukács G, et al. Correlation of malignancy with the intracellular Na+: K+ ratio in human thyroid tumors [J]. Cancer Res, 1983; 43(11): 53955402.
37.	帝振宇, 赵青川, 何勇, 等. 钠离子微电流探测仪在胆道肿瘤诊断中的应用 [J]. 中国医学工程, 2006; 14(6): 619621.
38.	Morrison BW, Moorman R, Kowdley GC, et al. Mat8, a novel phospholemmanlike protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes [J]. J Biol Chem, 1995; 270(5): 21762182.
39.	Kayed H, Kleeff J, Kolb A, et al. FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth [J]. In J Cancer, 2006; 118(1): 4354.
40.	窦春青, 岳鑫, 周宁新. hFXYD6反义核酸对胆管癌细胞体外增殖和侵袭能力的影响 [J]. 世界华人消化杂志, 2007; 15(9): 929935.
41.	刘军桂, 周宁新, 张效东. FXYD6 shRNA表达载体的构建及其在体外对胰腺癌细胞增殖的影响 [J]. 生物技术通讯, 2009; 20(4): 458461.

1. Patel T. Worldwide trends in mortality from biliary tract malignancies [J]. BMC Cancer, 2002; 2: 1014.
2. 邹声泉. 胆管癌外科治疗的现状与展望 [J]. 中国普外基础与临床杂志, 2008; 15(2): 7780.
3. Hsing AW, Gao YT, Devesa SS, et al. Rising incidence of biliary tract cancers in Shanghai, China [J]. Int J Cancer, 1998; 75(3): 368370.
4. 周宁新, 黄志强, 刘永雄, 等. 肝外胆道癌全国调查1 098例分析 [J]. 中华外科杂志, 1990; 28(9): 516521.
5. Grainge MJ, West J, SolaymaniDodaran M, et al. The antecedents of biliary cancer: a primary care casecontrol study in the United Kingdom [J]. Br J Cancer, 2009; 100(1): 178180.
6. 万涛, 倪彬, 刘建伟, 等. 人胆管癌相关基因CCRL2/FXYD6大片段克隆与鉴定 [J]. 中华实验外科杂志, 2003; 20(5): 393395.
7. 肖梅, 周宁新, 高丽杰, 等. 半定量检测胆管癌组织中人fxyd6基因的表达 [J]. 中国普外基础与临床杂志, 2008; 15(2): 8387.
8. http: // www.ncbi.nlm.nic.gov/genbank.
9. Sweadner KJ, Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression [J]. Genomics, 2000; 68(1): 4156.
10. 周宁新, 黄志强, 张文智, 等. 402例肝门部胆管癌临床分型、手术方式与远期疗效的综合分析 [J]. 中华外科杂志, 2006; 44(23): 15991603.
11. Hsing AW, Sakoda LC, Rashid A, et al. Variants in inflammation genes and the risk of biliary tract cancers and stones: a populationbased study in China [J]. Cancer Res, 2008; 68 (15): 64426452.
12. Bishop JM. Molecular themes in oncogenesis [J]. Cell, 1991; 64(2): 235248.
13. Moolgavkar SH, Luebeck EG. Multistage carcinogenesis and the incidence of human cancer [J]. Genes Chromosomes Cancer, 2003; 38(4): 302306.
14. Vogelstein B, Kinzler KW. The multistep nature of cancer [J]. Trends Genet, 1993; 9(4): 138141.
15. Levi S, UrbanoIspizua A, Gill R, et al. Multiple Kras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerse chain reaction technique [J]. Cancer Res, 1991; 51(13): 34973502.
16. Yoshida S, Todoroki T, Ichikawa Y, et al. Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers [J]. Cancer Res, 1995; 55(13): 27562760.
17. Lee CS. Ras p21 protein immunoreactivity and its relationship to p53 expression and prognosis in gallbladder and extrahepatic biliary carcinoma [J]. Eur J Surg Oncol, 1997; 23(3): 233237.
18. Hahn SA, Bartsch D, Schroers A, et al. Mutations of the DPC4/Smad4 gene in biliary tract carcinoma [J]. Cancer Res, 1998; 58(6): 11241126.
19. Tannapfel A, Engeland K, Weinans L, et al. Expression of p73, a novel protein related to the p53 tumour suppressor p53, and apoptosis in cholangiocellular carcinoma of the liver [J]. Br J Cancer, 1999; 80(7): 10691074.
20. Ahrendt SA, Eisenberger CF, Yip L, et al. Chromosome 9p21 loss and p16 inactivation in primary sclerosing cholangitisassociated cholangiocarcinoma [J]. J Surg Res, 1999; 84(1): 8893.
21. Werner H, Song JJ, Have M, et al. Homozygous deletions inactivate DCC, but not MADH4/DPC4/SMAD4, in a subset of pancreatic and biliary cancers [J]. Genes Chromosomes Cancer, 2000; 27(4): 353357.
22. Ito Y, Takeda T, Sasaki Y, et al. Bcl2 expression in cholangiocellular carcinoma is inversely correlated with biologically aggressive phenotypes [J]. Oncology, 2000; 59(1): 6367.
23. Tipsmark C. Identification of FXYD protein genes in a teleost: tissuespecific expression and response to salinity change [J]. Am J Physiol Regul Integr Comp Physiol, 2008; 294(4): R1367R1378.
24. 李尹雄. 基因克隆技术 [A]. 见：卢圣栋. 现代分子生物学实验技术 [M]. 北京: 高等教育出版社, 1993: 293294.
25. 黎孟枫译. 聚合酶链式反应体外扩增DNA [A]. 见：金冬雁, 黎孟枫主译. 分子克隆实验指南 [M]. 第2版. 北京: 科学出版社, 1999: 674675.
26. 周隽. 核苷酸顺序测定技术 [A]. 见：陈赛娟. 人类基因组研究基本技术 [M]. 北京: 人民军医出版社, 2002: 166167.
27. 申宗侯. 蛋白质的转运、加工与修饰 [A]. 见：陈惠黎. 生物大分子的结构与功能 [M]. 上海: 上海医科大学出版社, 1999: 196197.
28. Geering K. FXYD proteins: new regulators of NaKATPase [J]. Am J Physiol Renal Physiol, 2006; 290(2): F241F250.
29. 来鲁华. 蛋白质的结构预测与分子设计 [M]. 北京: 北京大学出版社, 1993: 7981.
30. 万涛, 孙涛, 吴加金. 蛋白顺序性抗原决定簇的多参数综合预测 [J]. 中国免疫学杂志, 1997; 13(6): 329333.
31. 冯健男, 万涛, 吴加金, 等. 基于三维结构的抗原决定簇预测 [J]. 分子科学学报, 1999; 15(2): 112115.
32. 杨思禹. 胆道的功能形态及生理 [A]. 见：张经济, 连至诚, 许冠荪, 等. 消化道生理及病理生理学 [M]. 广州: 广东科技出版社, 1997: 885923.
33. Nathanson MH, Boyer JL. Mechanisms and regulation of bile secretion [J]. Hepatology, 1991; 14(3): 551566.
34. Strazzabosco M. New insights into cholangiocyte physiology [J]. J Hepatology, 1997; 27(5): 945952.
35. Cameron IL, Smith NKR, Pool TB, et al. Intracellural concentration of sodium and other elements are related to mitogenesis and oncogenesis in vivo [J]. Cancer Res, 1980; 40(5): 14931500.
36. Nagy I, Lustyik G, Lukács G, et al. Correlation of malignancy with the intracellular Na+: K+ ratio in human thyroid tumors [J]. Cancer Res, 1983; 43(11): 53955402.
37. 帝振宇, 赵青川, 何勇, 等. 钠离子微电流探测仪在胆道肿瘤诊断中的应用 [J]. 中国医学工程, 2006; 14(6): 619621.
38. Morrison BW, Moorman R, Kowdley GC, et al. Mat8, a novel phospholemmanlike protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes [J]. J Biol Chem, 1995; 270(5): 21762182.
39. Kayed H, Kleeff J, Kolb A, et al. FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth [J]. In J Cancer, 2006; 118(1): 4354.
40. 窦春青, 岳鑫, 周宁新. hFXYD6反义核酸对胆管癌细胞体外增殖和侵袭能力的影响 [J]. 世界华人消化杂志, 2007; 15(9): 929935.
41. 刘军桂, 周宁新, 张效东. FXYD6 shRNA表达载体的构建及其在体外对胰腺癌细胞增殖的影响 [J]. 生物技术通讯, 2009; 20(4): 458461.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Full-Length cDNA Cloning of FXYD6 Gene Related to Cholangiocarcinoma with Bioinformatic Localization of Its Functional Domain

Abstract Full text Figures/Tables Video References Cited by

Format

Content