转移相关基因BRMS1、CD44V6在口腔鳞癌中的表达及其意义研究
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摘要
目的：研究肿瘤转移抑制基因BRMS1及细胞粘附分子CD44V6在口腔鳞癌组织(OSCC)及癌旁组织中的表达水平，并探讨其表达规律及与临床病理学关系。鉴于恶性肿瘤的转移是涉及多种基因变化的一系列复杂过程，通过研究这两种肿瘤转移相关基因的不同表达及相互关系等方面，为促进OSCC诊断治疗水平的提高和预后评价提供分子生物学检测指标，并为进一步研究BRMS1这一新近发现的转移抑制基因奠定理论和实践基础。
方法：搜集人OSCC及癌旁粘膜等新鲜标本并贮存于-80℃超低温冰箱，采用半定量逆转录聚合酶链式反应(RT—PCR)技术检测38份OSCC、20份癌旁正常黏膜组织中BRMS1mRNA及CD44V6mRNA的表达情况，即将RT-PCR扩增的基因mRNA凝胶电泳图像输入美国Kodak凝胶分析系统，应用1D Image Analysis软件对电泳图灰度值进行表达强度分析，用细胞因子表达强度除以β-actin表达强度，从而得到相对半定量值。然后，从肿瘤患者的年龄、性别、组织分化程度、临床分期、瘤体大小、生长方式及有无转移等方面分组比较这两种基因的表达差异，并比较OSCC与癌旁粘膜标本中两基因的表达差异。
结果：OSCC标本组织BRMS1mRNA表达量比癌旁粘膜组织明显偏低，其差异具有统计学意义(P＜0.05)。38例OSCC病人从性别、年龄、病理学分级、
Objective: To investigate and analyze the expressions of BRMS1 (breast
cancer metastasis suppressor 1, BRMS1)mRNA and CD44V6mRNA in oral squamous cell carcinoma (OSCC) and normal oral mucous membrane near the tumors, and to explore their relationships of the expressions as well as the clinicopathological factors. Analyzing their relationships will accelerate and improve the level of diagnosis and treatment, even estimate prognosis of the OSCC patients, which could 'provide two significant targets of molecular biology and could establish theoretical and practical foundation for deeply studying the new gene BRMS1.
Methods: At first, these OSCC samples and the normal oral mucous
membranes near the tumors were collected and reserved immediately in -80 ℃ refrigeratory. Then the expressions of BRMS1mRNA and CD44V6mRNA in 38 cases of OSCC tissues and 20 cases of normal oral mucous membranes near the tumors were carried out by an approach of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). By using 1 D Image Analysis software, the electrophoresis
方法：搜集人OSCC及癌旁粘膜等新鲜标本并贮存于-80℃超低温冰箱，采用半定量逆转录聚合酶链式反应(RT—PCR)技术检测38份OSCC、20份癌旁正常黏膜组织中BRMS1mRNA及CD44V6mRNA的表达情况，即将RT-PCR扩增的基因mRNA凝胶电泳图像输入美国Kodak凝胶分析系统，应用1D Image Analysis软件对电泳图灰度值进行表达强度分析，用细胞因子表达强度除以β-actin表达强度，从而得到相对半定量值。然后，从肿瘤患者的年龄、性别、组织分化程度、临床分期、瘤体大小、生长方式及有无转移等方面分组比较这两种基因的表达差异，并比较OSCC与癌旁粘膜标本中两基因的表达差异。
结果：OSCC标本组织BRMS1mRNA表达量比癌旁粘膜组织明显偏低，其差异具有统计学意义(P＜0.05)。38例OSCC病人从性别、年龄、病理学分级、
Objective: To investigate and analyze the expressions of BRMS1 (breast
cancer metastasis suppressor 1, BRMS1)mRNA and CD44V6mRNA in oral squamous cell carcinoma (OSCC) and normal oral mucous membrane near the tumors, and to explore their relationships of the expressions as well as the clinicopathological factors. Analyzing their relationships will accelerate and improve the level of diagnosis and treatment, even estimate prognosis of the OSCC patients, which could 'provide two significant targets of molecular biology and could establish theoretical and practical foundation for deeply studying the new gene BRMS1.
Methods: At first, these OSCC samples and the normal oral mucous
membranes near the tumors were collected and reserved immediately in -80 ℃ refrigeratory. Then the expressions of BRMS1mRNA and CD44V6mRNA in 38 cases of OSCC tissues and 20 cases of normal oral mucous membranes near the tumors were carried out by an approach of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). By using 1 D Image Analysis software, the electrophoresis
引文
1. Welch DR, Wei LL. Genetic and epigenetic regulation of human breast cancer progression and metastasis[J]. Endocr Relat Cancer, 1998, 5(2): 155-197.
2. Steeg PS, Bevilacqua G, Kopper L, et al. Evidence for a novel gene associated with low tumor metastatic potential[J]. J Natl Cancer Inst, 1988, 80(3): 200-204.
3. Liotta LA, Mandler R, Murano G, et al. Tumor cell antocrine motility factor[J]. Proc Natl Acad Sci USA, 1986, 83(10): 3302-3306.
4．高进，章静波，主编．癌的侵袭与转移基础与临床[M]．北京：科学技术出版社，2003：66-166．
5. Welch DR, Steeg PS, Rinker-Schaeffer CW. Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis[J]. Breast Cancer Res, 2000, 2(6): 408-416.
6. Seraj MJ, Samant RS, Verderame MF, et al. Functional evidence for a novel human breast carcinoma metastasis suppressor BRMS1 encoded at chromosome 11q13[J]. Cancer Res, 2000, 60(11): 2764-2769.
7. Saunders MM, Seraj MJ, Li Z, et al. Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication[J]. Cancer Res, 2001, 61 (5): 1765-1767.
8. Samant RS, Debies MT, Shevde LA, et al. Identification and characterization of the murine ortholog(brmsl) of breast-cancer metastasis suppressor 1 (BRMS1) [J]. Int J cancer, 2002, 97(1):15-20.
9. Shevde LA, Samant RS, Goldberg SF, et al. Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1 [J]. Exp Cell Res, 2002, 273(2): 229-39.
10. Seraj MJ, Harding MA, Gildea JJ, et al. The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines[J]. Clin Exp Metastasis, 2000, 18(6): 519-525.
11. Cromer A, Carles A, Millon R, et al. Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis[J]. Oncogene, 2004, 23(14): 2484-2498.
12. Stark AM, Tongers K, Maass N, et al. Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases[J]. J Cancer Res Clin Oncol, 2005,131(3): 191-198.
13. 张殊,林其德,狄文。BRMS1 和fascin基因在卵巢上皮癌中的表达及其意义 [J].现代妇产科进展, 2003, 12(3): 167-170.
14. Meehan WJ, Welch DR. Breast cancer metastasis suppressor 1: update[J]. Clin Exp Metastasis, 2003,20(1): 45-50.
15. Forastiere A, Koch W, Trotti A, et al. Head and neck cancer [J]. N Engl J Med, 2001, 345(26): 1890-1900.
16. Gao AC, Lon W, Dong JT, et al. CD44 is a metastasis suppressor gene for prostatic cancer located on human chromsome 11p1 3[J]. Cancer Res, 1997, 57(2): 846-849.
17. Matsumura Y, Sugiyama M, Matsumura, et al. Unusal retention of intron in CD44 gene transcripts in bladder cancer provides new diagnostic and dinical oncologicalopportunites[J]. J Pathol, 1995, 17(7): 11-20.
18. Naor D, Sionov RV, Ish-Shalom D. CD44: structure, function and association with the malignant process [J]. Adv Cancer Res, 1997, 71(3): 241-319.
19. Tolg C, Hofmann M, Herrlich P, et al, Splicing choice from ten variant exons establishes CD44 variability [J]. Nucleic Acids Res, 1993,21(5): 1225-1229.
20. Hogerkorp CM, Bilke S, Breslin T, et al. CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analyzed by DNA microarrays. Blood, 2003, 101(6): 2307-2313.
21. Tempfer C, Losch A,Heinzl H ,et al. Prognostic value of in imunohistochemically detected CD44 isoforms CD44v5,CD44v6 and CD44v7-8 in human breast cancer[J]. Eur J Cancer, 1996, 32(11): 2023-2025.
22. Charpin C, Garcia S, Bouvier C, et al. Automated and quantitative immunocyto- chemical assays of CD44v6 in breast carcinomas[J]. Hum Pathol, 1997, 28(30): 289-296.
23. Yamamichi K, Uehara Y, Kitamura N, et al. Increased expression of CD44v6 mRNA significantly correlates with distant metastasis and poor prognosis in gastric cancer[J]. Int J Cancer, 1998, 79(3): 256-262.
24. Satio H, Tsujitani S, Katano K, et al. Serum concentration of CD44 variant 6 and its relation to prognosis in patients with gastric carcinoma[J]. Cancer, 1998, 83(6): 1094-1101.
25. Tempfer C, Haeusler G, Kaider A, et al. The prognostic value of CD44 isoform expression in endometrial cancer[J]. Br J Cancer, 1998, 77(7): 1137-1139.
26. Hirata T, Fukuse T, Naiki H, et al. Expression of CD44 variant exon 6 in stage I non-small cell lung carcinoma as a prognostic factor [J]. Cancer Res, 1998, 58(6): 1108-1110.
27. Takahashi S. Kimoto N, Orita S, et al. Relationship between CD44 expression and differentiation of human prostate adenocarcinomas[J]. Cancer Lett, 1998, 129(1): 97-102.
28. Gu J, Daa T, Kashima K, et al. Expression of splice variants of CD44 in thyroid neoplasms derive from follicular cells[J]. Pathol Int, 1998,48(3): 184-190.
29. Soukka T, salmi M, Joesuu H, et al. Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells[J]. Cancer Res, 1997, 57(11): 2281-2289.
30. Hudson DL, Speight PM, Watt FM. Altered expression of CD44 isoforms in squamous-cell carcinomas and cell lines derived from them[J]. Int J Cancer, 1996, 66(4): 457-463.
31. Piffko J, Bankfalvi A, Klauke K, et al. Unaltered strong immunohistochemical expression of CD44-v6 and -v5 isoforms during development and progression of oral squamous cell carcinomas [J]. J Oral Pathol Med, 1996, 25(9): 502-506.
32. Bahar R, Kunishi M, Kayada Y, et al. CD44 variant 6 (CD44v6) expression as a progression marker in benign, premalignant and malignant oral epithelial tissues[J]. Int J Oral Maxillofac Surg, 1997, 26(6): 443-446.
33. Kunishi M, Kayada Y, Yoshiga K. Down-regulated expression of CD44 variant 6 in oral squamous cell carcinomas and its relationship to regional lymph node metastasis[J]. Int J Oral Maxillofac Surg, 1997, 26(4): 280-283.
34．高岩，刘井岚，庞淑珍．癌前病变口腔上皮中细胞黏附分子CD44V3和CD44V6表达的改变[J]．中华口腔医学杂志，1999，34(5)：286-288．
35. Nicole LW, Van H, Guus A.M.S, et al. Characterization of CD44v6 isoforms in head-and-neck squamous-cell carcinoma[J]. Int J Cancer, 1999, 82(6): 837-845.
36. Soukka T, Salmi M, Joensuu H, et al. Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells[J]. Cancer Res, 1997, 57(11): 2281-2289.
37. Matsumura Y, Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation[J]. Lancet, 1992, 340(8827): 1053-1058.
38. Haynes BF, Liao HX, Patton KL. The transmembrane hyaluronate receptor (CD44): multiple functions, multiple forms[J]. Cancer Cells, 1991, 3(9): 347-350.
39. Gunthert U, Hofmann M, Rudy W, et al. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells[J]. Cell, 1991, 65 (1): 13-24.
40. Sy MS, Guo YJ, Stamenkovic Ⅰ. Distinct effects of two CD44 isoforms on tumor growth in vivo[J]. J Exp Med, 1991, 174(4): 859-66.
41．刘健，浦剑虹，顾水平，等．CD44V6在口腔鳞癌组织中的表达及意义[J]．江苏医药，2005，31(2)：159．
42．陈德建，昌桂英，尹格平，等．口腔颌面部恶性肿瘤CD44V6的表达及临床意义[J]．中华口腔医学杂志，2003，38(2)：84．
43．卢勇，李昌龙，周志瑜，等．颊癌中肿瘤转移抑制基因nm23mRNA的表达：Ⅰ．Northern斑点杂交研究[J]．华西口腔医学志，1997，15(2)：143-145．
44．龚莉，何志秀，卢勇，等．颊癌中肿瘤转移抑制基因nm23mRNA的表达：Ⅱ．定量逆转录多聚酶链式反应检测[J]．华西口腔医学杂志，1997，15(2)：147-149．
45. Steeg PS. Metastasis supressors alter the signal transduction of cancer cells[J]. Nat Rev Cancer, 2003, 3(1): 55-63.
46. Hartsough MT, Clare SE, Nair M. Elevation of breast carcinoma Nm23-H1 metastasis suppressor gene expression and reduced motility by DNA methylation inhibition[J]. Cancer Res, 2001, 61 (5): 2320-2327.
47. Akita H, Lizuka A, Hashimoto Y. Induction of KAI-1 expression in metastatic cancer cells by phorbol esters[J]. Cancer Lett, 2000, 153(1-2): 79-83.
48. Mashimo T, Bandyopadhyay S, Goodarzi G. Activation of the tumor metastasis suppressor gene, KAI1, by etoposide is mediated by p53 and c-Jun genes[J]. Biochem Biophys Res Commun, 2000, 274(2): 370-376.
49．吴秉铨，方伟岗，主编．肿瘤转移机制及其阻断扩散的基础和临床[M]．杭州：浙江科学技术出版社，2005：17—21．
2. Steeg PS, Bevilacqua G, Kopper L, et al. Evidence for a novel gene associated with low tumor metastatic potential[J]. J Natl Cancer Inst, 1988, 80(3): 200-204.
3. Liotta LA, Mandler R, Murano G, et al. Tumor cell antocrine motility factor[J]. Proc Natl Acad Sci USA, 1986, 83(10): 3302-3306.
4．高进，章静波，主编．癌的侵袭与转移基础与临床[M]．北京：科学技术出版社，2003：66-166．
5. Welch DR, Steeg PS, Rinker-Schaeffer CW. Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis[J]. Breast Cancer Res, 2000, 2(6): 408-416.
6. Seraj MJ, Samant RS, Verderame MF, et al. Functional evidence for a novel human breast carcinoma metastasis suppressor BRMS1 encoded at chromosome 11q13[J]. Cancer Res, 2000, 60(11): 2764-2769.
7. Saunders MM, Seraj MJ, Li Z, et al. Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication[J]. Cancer Res, 2001, 61 (5): 1765-1767.
8. Samant RS, Debies MT, Shevde LA, et al. Identification and characterization of the murine ortholog(brmsl) of breast-cancer metastasis suppressor 1 (BRMS1) [J]. Int J cancer, 2002, 97(1):15-20.
9. Shevde LA, Samant RS, Goldberg SF, et al. Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1 [J]. Exp Cell Res, 2002, 273(2): 229-39.
10. Seraj MJ, Harding MA, Gildea JJ, et al. The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines[J]. Clin Exp Metastasis, 2000, 18(6): 519-525.
11. Cromer A, Carles A, Millon R, et al. Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis[J]. Oncogene, 2004, 23(14): 2484-2498.
12. Stark AM, Tongers K, Maass N, et al. Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases[J]. J Cancer Res Clin Oncol, 2005,131(3): 191-198.
13. 张殊,林其德,狄文。BRMS1 和fascin基因在卵巢上皮癌中的表达及其意义 [J].现代妇产科进展, 2003, 12(3): 167-170.
14. Meehan WJ, Welch DR. Breast cancer metastasis suppressor 1: update[J]. Clin Exp Metastasis, 2003,20(1): 45-50.
15. Forastiere A, Koch W, Trotti A, et al. Head and neck cancer [J]. N Engl J Med, 2001, 345(26): 1890-1900.
16. Gao AC, Lon W, Dong JT, et al. CD44 is a metastasis suppressor gene for prostatic cancer located on human chromsome 11p1 3[J]. Cancer Res, 1997, 57(2): 846-849.
17. Matsumura Y, Sugiyama M, Matsumura, et al. Unusal retention of intron in CD44 gene transcripts in bladder cancer provides new diagnostic and dinical oncologicalopportunites[J]. J Pathol, 1995, 17(7): 11-20.
18. Naor D, Sionov RV, Ish-Shalom D. CD44: structure, function and association with the malignant process [J]. Adv Cancer Res, 1997, 71(3): 241-319.
19. Tolg C, Hofmann M, Herrlich P, et al, Splicing choice from ten variant exons establishes CD44 variability [J]. Nucleic Acids Res, 1993,21(5): 1225-1229.
20. Hogerkorp CM, Bilke S, Breslin T, et al. CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analyzed by DNA microarrays. Blood, 2003, 101(6): 2307-2313.
21. Tempfer C, Losch A,Heinzl H ,et al. Prognostic value of in imunohistochemically detected CD44 isoforms CD44v5,CD44v6 and CD44v7-8 in human breast cancer[J]. Eur J Cancer, 1996, 32(11): 2023-2025.
22. Charpin C, Garcia S, Bouvier C, et al. Automated and quantitative immunocyto- chemical assays of CD44v6 in breast carcinomas[J]. Hum Pathol, 1997, 28(30): 289-296.
23. Yamamichi K, Uehara Y, Kitamura N, et al. Increased expression of CD44v6 mRNA significantly correlates with distant metastasis and poor prognosis in gastric cancer[J]. Int J Cancer, 1998, 79(3): 256-262.
24. Satio H, Tsujitani S, Katano K, et al. Serum concentration of CD44 variant 6 and its relation to prognosis in patients with gastric carcinoma[J]. Cancer, 1998, 83(6): 1094-1101.
25. Tempfer C, Haeusler G, Kaider A, et al. The prognostic value of CD44 isoform expression in endometrial cancer[J]. Br J Cancer, 1998, 77(7): 1137-1139.
26. Hirata T, Fukuse T, Naiki H, et al. Expression of CD44 variant exon 6 in stage I non-small cell lung carcinoma as a prognostic factor [J]. Cancer Res, 1998, 58(6): 1108-1110.
27. Takahashi S. Kimoto N, Orita S, et al. Relationship between CD44 expression and differentiation of human prostate adenocarcinomas[J]. Cancer Lett, 1998, 129(1): 97-102.
28. Gu J, Daa T, Kashima K, et al. Expression of splice variants of CD44 in thyroid neoplasms derive from follicular cells[J]. Pathol Int, 1998,48(3): 184-190.
29. Soukka T, salmi M, Joesuu H, et al. Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells[J]. Cancer Res, 1997, 57(11): 2281-2289.
30. Hudson DL, Speight PM, Watt FM. Altered expression of CD44 isoforms in squamous-cell carcinomas and cell lines derived from them[J]. Int J Cancer, 1996, 66(4): 457-463.
31. Piffko J, Bankfalvi A, Klauke K, et al. Unaltered strong immunohistochemical expression of CD44-v6 and -v5 isoforms during development and progression of oral squamous cell carcinomas [J]. J Oral Pathol Med, 1996, 25(9): 502-506.
32. Bahar R, Kunishi M, Kayada Y, et al. CD44 variant 6 (CD44v6) expression as a progression marker in benign, premalignant and malignant oral epithelial tissues[J]. Int J Oral Maxillofac Surg, 1997, 26(6): 443-446.
33. Kunishi M, Kayada Y, Yoshiga K. Down-regulated expression of CD44 variant 6 in oral squamous cell carcinomas and its relationship to regional lymph node metastasis[J]. Int J Oral Maxillofac Surg, 1997, 26(4): 280-283.
34．高岩，刘井岚，庞淑珍．癌前病变口腔上皮中细胞黏附分子CD44V3和CD44V6表达的改变[J]．中华口腔医学杂志，1999，34(5)：286-288．
35. Nicole LW, Van H, Guus A.M.S, et al. Characterization of CD44v6 isoforms in head-and-neck squamous-cell carcinoma[J]. Int J Cancer, 1999, 82(6): 837-845.
36. Soukka T, Salmi M, Joensuu H, et al. Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells[J]. Cancer Res, 1997, 57(11): 2281-2289.
37. Matsumura Y, Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation[J]. Lancet, 1992, 340(8827): 1053-1058.
38. Haynes BF, Liao HX, Patton KL. The transmembrane hyaluronate receptor (CD44): multiple functions, multiple forms[J]. Cancer Cells, 1991, 3(9): 347-350.
39. Gunthert U, Hofmann M, Rudy W, et al. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells[J]. Cell, 1991, 65 (1): 13-24.
40. Sy MS, Guo YJ, Stamenkovic Ⅰ. Distinct effects of two CD44 isoforms on tumor growth in vivo[J]. J Exp Med, 1991, 174(4): 859-66.
41．刘健，浦剑虹，顾水平，等．CD44V6在口腔鳞癌组织中的表达及意义[J]．江苏医药，2005，31(2)：159．
42．陈德建，昌桂英，尹格平，等．口腔颌面部恶性肿瘤CD44V6的表达及临床意义[J]．中华口腔医学杂志，2003，38(2)：84．
43．卢勇，李昌龙，周志瑜，等．颊癌中肿瘤转移抑制基因nm23mRNA的表达：Ⅰ．Northern斑点杂交研究[J]．华西口腔医学志，1997，15(2)：143-145．
44．龚莉，何志秀，卢勇，等．颊癌中肿瘤转移抑制基因nm23mRNA的表达：Ⅱ．定量逆转录多聚酶链式反应检测[J]．华西口腔医学杂志，1997，15(2)：147-149．
45. Steeg PS. Metastasis supressors alter the signal transduction of cancer cells[J]. Nat Rev Cancer, 2003, 3(1): 55-63.
46. Hartsough MT, Clare SE, Nair M. Elevation of breast carcinoma Nm23-H1 metastasis suppressor gene expression and reduced motility by DNA methylation inhibition[J]. Cancer Res, 2001, 61 (5): 2320-2327.
47. Akita H, Lizuka A, Hashimoto Y. Induction of KAI-1 expression in metastatic cancer cells by phorbol esters[J]. Cancer Lett, 2000, 153(1-2): 79-83.
48. Mashimo T, Bandyopadhyay S, Goodarzi G. Activation of the tumor metastasis suppressor gene, KAI1, by etoposide is mediated by p53 and c-Jun genes[J]. Biochem Biophys Res Commun, 2000, 274(2): 370-376.
49．吴秉铨，方伟岗，主编．肿瘤转移机制及其阻断扩散的基础和临床[M]．杭州：浙江科学技术出版社，2005：17—21．