人肝癌组织中nm23-H1基因表达及其cDNA基因突变的研究
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摘要
目的 检测肿瘤转移抑制基因nm23-H1在原发性肝细胞癌(hepatocellular carcinoma，HCC)中的表达及其基因突变情况，探讨nm23-H1在HCC发生发展中的作用及肝癌细胞转移的分子生物学机制。方法 应用半定量逆转录聚合酶链反应(RT-PCR)技术及单链构象多态法(single strand conformation polymorphism，SSCP)，对30例肝癌组织、30例癌旁组织和16例正常肝组织中nm23-H1基因的表达情况及cDNA基因突变情况进行检测和分析。结果 30例肝癌患者的癌组织、相应癌旁组织及正常组织中nm23-H1基因阳性表达率均为100％，nm23-H1基因阳性表达率在这三者之间无差异；②nm23-H1基因在肝癌组织、癌旁组织、正常组织中的表达水平分别为1.619±0.407((?)±s)、1.302±0.304((?)±s)及1.232±0.386((?)±s)。统计学分析表明在这三种组织中nm23-H1基因的表达水平有显著差异(P＜0.01)。肝癌组织中nm23-H1基因的表达水平明显低于癌旁组织(P＜0.05)和正常组织(P＜0.01)。该基因在癌旁组织和正常组织中的表达水平无显著差异(P＞0.05)；③低分化肝癌组织中nm23-H1基因表达水平低于高、中分化肝癌组织中nm23-H1基因表达水平(P＜0.05)；④有卫星转移灶的和无卫星灶的肝癌组织中nm23-H1基因表达水平无显著差异(P＞0.05)；⑤肝癌组织中nm23-H1基因表达水平与患者的年龄、性别无显著相关性(P值均大于0.05)；⑥30例肝癌组织及相应的癌旁组织中未发现1例存在nm23-H1 cDNA基因突变。结论 ①原发性肝细胞癌中nm23-H1基因阴性表达率很低；②nm23-H1基因在肝癌组织中的表达水平明显低于癌旁组织和正常组织，提示nm23-H1失活与肝癌发生相关；③原发性肝细胞癌中nm23-H1表达水平与肝癌恶性程度有明显相关性，提示nm23-H1变异可以作为患者预后的指标；④肝癌组织中nm23-H1表达水平与卫星转移灶的有无及患者的性别、年龄均无明显相关性；⑤肝癌组织及癌旁组织中nm23-H1 cDNA基因突变率很低。
Objective To detect the expression of nm23-H1 and its cDNA gene mutation in hepatocellular carcinoma (HCC), and study the role of nm23-H1 in the pathogenesis of HCC and elucidate the molecular biology mechanism of hepatocellular carcinoma metastasis. Methods Using semi-quantitive reverse transcriptase-polymerase chain reaction (RT-PCR) and single strand conformation polymorphism (SSCP) , the expression variation and cDNA gene mutation of nm23-Hl gene were detected in 30 HCC tissues, their corresponding adjacent liver tissues and in normal liver tissues of 16 non-liver disease patients. Results 漏the expression rate of nm23-Hl RNA was 100% in the HCC tissues , their corresponding adjacent tissues and in the normal liver tissues. There were no significant difference in the expression rate of nm23-H1 gene among the three kind of tissues(P>0. 05); (2)The expression level of nm23-Hl gene in the HCC tissues, their adjacent tissues and normal liver tissues was 1.619 0.407( x s), 1.302 0.343( x s) and 1.232 0.386(
x s), respectively. The expression level of nm23-H1 gene in the HCC tissues was singnificantly lower than that in the adjacent and normal liver tissues (P <0.01); (3) The expression level of nm23-H1 gene in the low-differentiated HCC was much lower than that in the high and middle differentiated HCC tissues (P< 0.01); (4) The expression level of nm23-Hl gene in HCC tissues with satellite focuses was not significantly different from that without satellite focuses (P > 0. 05) ; (5) The expression quantity of nm23-H1 gene in HCC tissues was not closely related to HCC patients' age and gender (P>0. 05) ; (6) None of the nm23-Hl gene mutation was found in all the hepatocellular carcinoma samples. Conclusion (1)The negative expression rate of nm23-H1 in the HCC tissues is very low; (2) The expression level of nm23-H1 in the HCC tissues is much lower than that in the adjacent and normal liver tissues, which indicates that the inactivation of nm23-H1 is associated with the
pathogenesis of HCC; (3)The expression level of nm23-Hl gene is highly associated with the pathological differentiation of HCC, which indicates that the alternation of nm23-Hl can serve as a prognostic indicators in the progression of HCC; (4)The expression level of nm23-Hl gene in HCC tissues was not closely related to HCC patients' age, gender and whether there are satellite focuses in HCC tissues; (5) The cDNA gene mutation rate of nm23-Hl in the HCC tissues is very low.
Objective To detect the expression of nm23-H1 and its cDNA gene mutation in hepatocellular carcinoma (HCC), and study the role of nm23-H1 in the pathogenesis of HCC and elucidate the molecular biology mechanism of hepatocellular carcinoma metastasis. Methods Using semi-quantitive reverse transcriptase-polymerase chain reaction (RT-PCR) and single strand conformation polymorphism (SSCP) , the expression variation and cDNA gene mutation of nm23-Hl gene were detected in 30 HCC tissues, their corresponding adjacent liver tissues and in normal liver tissues of 16 non-liver disease patients. Results 漏the expression rate of nm23-Hl RNA was 100% in the HCC tissues , their corresponding adjacent tissues and in the normal liver tissues. There were no significant difference in the expression rate of nm23-H1 gene among the three kind of tissues(P>0. 05); (2)The expression level of nm23-Hl gene in the HCC tissues, their adjacent tissues and normal liver tissues was 1.619 0.407( x s), 1.302 0.343( x s) and 1.232 0.386(
x s), respectively. The expression level of nm23-H1 gene in the HCC tissues was singnificantly lower than that in the adjacent and normal liver tissues (P <0.01); (3) The expression level of nm23-H1 gene in the low-differentiated HCC was much lower than that in the high and middle differentiated HCC tissues (P< 0.01); (4) The expression level of nm23-Hl gene in HCC tissues with satellite focuses was not significantly different from that without satellite focuses (P > 0. 05) ; (5) The expression quantity of nm23-H1 gene in HCC tissues was not closely related to HCC patients' age and gender (P>0. 05) ; (6) None of the nm23-Hl gene mutation was found in all the hepatocellular carcinoma samples. Conclusion (1)The negative expression rate of nm23-H1 in the HCC tissues is very low; (2) The expression level of nm23-H1 in the HCC tissues is much lower than that in the adjacent and normal liver tissues, which indicates that the inactivation of nm23-H1 is associated with the
pathogenesis of HCC; (3)The expression level of nm23-Hl gene is highly associated with the pathological differentiation of HCC, which indicates that the alternation of nm23-Hl can serve as a prognostic indicators in the progression of HCC; (4)The expression level of nm23-Hl gene in HCC tissues was not closely related to HCC patients' age, gender and whether there are satellite focuses in HCC tissues; (5) The cDNA gene mutation rate of nm23-Hl in the HCC tissues is very low.
引文
1 易基群，杨晓明，曾波航，等．肿瘤转移抑制基因nm23-H1 mRNA在乳腺癌中的转录表达．现代临床医学生物工程学杂志，2003，9(2)：85-87
2 Aoyagi K, Koufuji K, Kodama I. Immunohistochemical analysis of erbB-2 and nm23 expression in gastric carcinoma. Nippon Shokakibyo Gakkai Zasshi ,1996, (9):613-619
3 Iizuka N, Tangoku A, Hazama S, et al. nm23-H1 gene as a molecular switch between the free floating and adherent states of gastric cancer cells. Cancer Lett, 2001, 1:651-671
4 Iizuka N, Tangoku A, Hayashi H, et al. The association between nm23-H1 expression and survival in patients with esophageal squamous cell carcinoma. Cancer Lett, 1999,12:139-144
5 杨毅军，曹道俊，许菊萍，等．青年人大肠癌nm23基因表达及意义．河南肿瘤杂志，2000，13(2)：87-88
6 Volm M, Mattern J, Koomgi R. Association between nm23-H1 expression ,proliferation and apotosis in non-small cell lung carcinoma. Clin Exp metastasis, 1998,16(7):595-602
7 陈琼，陈合群，李国虎．原发性肺癌nm23基因mRNA表达的研究．中国现代医学杂志，2001，11(4)：18-21
8 王忠民，常德安．上皮性卵巢癌中nm23-H1基因突变．肿瘤，2002，22(3)：209-212
9 吕志勇，吕怀盛，陈福宝，等．nm23-H1基因产物在膀胱移行细胞癌的表达及临床意义．宁夏医学杂志，2002，24(5)：259-261
10 孔宪国，张元芳，丁强，等．肾癌nm23-H1 mRNA表达与浸润转移的关系．中华肿瘤杂志，2000，19：88
11 Shimada M, Taguchi K, Hasegawa H. nm23-H1 expression in intrahepatic or extrahepatic metastases of hepatocellular carcinoma. Liver, 1998,5:337-342
12 Lin LI, Lee PH, Wu CM. Significance of nm23 mRNA expression in human hepatocellular carcinoma. Anticancer Res, 1998,1B:541-546
13 赵爱志，窦羽峰，李开宗，等．肝细胞癌中血管内皮生长因子及nm23-H1蛋白的表达．第四军医大学学报，2000，21(11)：136-137
14 马文峰，翟秀岩，长崛熏，等．抗转移基因nm23-H1在多发性肝癌中的表达．中国医
科大学学报，2001，30(6)：421-422
15 Matthias Engel, Birgit Theisinger, Thomas Seib, et al. High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages. Int. J. Cancer, 1993,55:373-379
16 Steeg PS, Bevilacqua, Kopper L, et al. Evidence for a novel gene associated with low tumor metastatic potential. J. Natl. Cancer Inst, 1988, 80:200
17 Biggs J, Hersperger E, Steeg PS, et al. A droscphila gene that is homologous to a mammalian gene associated tumor metastasis codes for a nucleoside diphosphate kinase. Cell, 1990, 63:933
18 Steeg PS, Bevilacqua G, Pozzatti R, et al. Alteres expression of nm23 a gene associated with low tumor metastatic potential, during adenovirus Z Ela inhibition of experimental metastasis. Cancer Res, 1988,48(22):6550-6554
19 Leone A, Flatow U, King C, et al. Reduced tumor incidence metastatic potential and cytokine responsiveness of nm23 transfected melanoma cells. Cell, 1991,65(1):25-35
20 Leone A, Flatow U, Van Houtte K, et al. Transfection of human nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: relationship to growth colonization metastatic potential and enzymatic activity. Cancer, 1994,79(12):2913-2922
21 Price JE, Polyzos A, Zhang RD, et al. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res, 1990, 50 (3) : 717-721
22 Fujimoto Y, Ohtake T, Nishimori H. Reduced expression and rare genomic altern of nm23-H1 in human hepatocellular carcinoma and hepatoma cell lines. J Gastroenterol, 1998,3:368-375
23 Boix L, Bruix J, Campo E, et al. nm23-H1 expression and disease recurrence after surgical resection of small hepatocellular carcinoma. Gastroenterology, 1994,107(2): 486-491
24 Zheng XY, Ling ZY, Tang ZY. The abundance of nm23-H1 mRNA is related with in situ microenvironment and intrahepatic metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res, 1998,3:337-341
25 Chen X, Dai Y, Yang J. Expression of metastasis suppressor gene nm23 in human hepatocellular carcinoma. Chung Hua Ping Li Hsueh Tsa Chih, 1996,2:76-78
26 Leone A, McBride OW, Weston A, et al. Somatic allelic deletion of nm23 in human cancer. Cancer Res, 1991,51(9):2490-2493
27 Cohn KH, Wang FS, Desoto-Lapaix F, et al. Association of nm23-H1 allelic deletions with distant metastasis in colorectal carcinoma. Lancet, 1991, 238(8769): 722-724
28 黄培林，金月玲，黄照权，等．nm23-H1基因突变及异常表达与大肠癌转移相关性的研究．东南大学学报(医学版)，2002，21(1)：44-48
29 Leone A, Seeger RC, Hong CM, et al. Evidence for nm23 RNA overexpression, DNA amplification and mutation in aggressive childhood neuroblastomas. Oncogene, 1993,8(4):855-865
30 陈军，周清华，覃扬，等．人肺癌中nm23等位基因缺失的研究．中国肺癌杂志，2000，3(1)：8-13
31 于景翠，李晓敏，傅松滨．肺癌基因组nm23-H1基因的扩增及异常分析．哈尔滨医科大学学报，2000，34(1)：6-7
32 王国丽，杜传书，林群娣．非小细胞肺癌淋巴结转移与nm23-H1基因突变及mRNA表达异常关系研究．中国结核和呼吸杂志，1997，20(6)：340-343
33 王国丽，杜传书．人原发性乳腺癌及肺癌中乳腺癌抑制基因突变．中山医科大学学报，1996，17：95
34 刘伦旭，周清华，石应康，等．人肺癌组织中nm23-H1基因突变研究．中国肺癌杂志，2000，3(3)：313-317
2 Aoyagi K, Koufuji K, Kodama I. Immunohistochemical analysis of erbB-2 and nm23 expression in gastric carcinoma. Nippon Shokakibyo Gakkai Zasshi ,1996, (9):613-619
3 Iizuka N, Tangoku A, Hazama S, et al. nm23-H1 gene as a molecular switch between the free floating and adherent states of gastric cancer cells. Cancer Lett, 2001, 1:651-671
4 Iizuka N, Tangoku A, Hayashi H, et al. The association between nm23-H1 expression and survival in patients with esophageal squamous cell carcinoma. Cancer Lett, 1999,12:139-144
5 杨毅军，曹道俊，许菊萍，等．青年人大肠癌nm23基因表达及意义．河南肿瘤杂志，2000，13(2)：87-88
6 Volm M, Mattern J, Koomgi R. Association between nm23-H1 expression ,proliferation and apotosis in non-small cell lung carcinoma. Clin Exp metastasis, 1998,16(7):595-602
7 陈琼，陈合群，李国虎．原发性肺癌nm23基因mRNA表达的研究．中国现代医学杂志，2001，11(4)：18-21
8 王忠民，常德安．上皮性卵巢癌中nm23-H1基因突变．肿瘤，2002，22(3)：209-212
9 吕志勇，吕怀盛，陈福宝，等．nm23-H1基因产物在膀胱移行细胞癌的表达及临床意义．宁夏医学杂志，2002，24(5)：259-261
10 孔宪国，张元芳，丁强，等．肾癌nm23-H1 mRNA表达与浸润转移的关系．中华肿瘤杂志，2000，19：88
11 Shimada M, Taguchi K, Hasegawa H. nm23-H1 expression in intrahepatic or extrahepatic metastases of hepatocellular carcinoma. Liver, 1998,5:337-342
12 Lin LI, Lee PH, Wu CM. Significance of nm23 mRNA expression in human hepatocellular carcinoma. Anticancer Res, 1998,1B:541-546
13 赵爱志，窦羽峰，李开宗，等．肝细胞癌中血管内皮生长因子及nm23-H1蛋白的表达．第四军医大学学报，2000，21(11)：136-137
14 马文峰，翟秀岩，长崛熏，等．抗转移基因nm23-H1在多发性肝癌中的表达．中国医
科大学学报，2001，30(6)：421-422
15 Matthias Engel, Birgit Theisinger, Thomas Seib, et al. High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages. Int. J. Cancer, 1993,55:373-379
16 Steeg PS, Bevilacqua, Kopper L, et al. Evidence for a novel gene associated with low tumor metastatic potential. J. Natl. Cancer Inst, 1988, 80:200
17 Biggs J, Hersperger E, Steeg PS, et al. A droscphila gene that is homologous to a mammalian gene associated tumor metastasis codes for a nucleoside diphosphate kinase. Cell, 1990, 63:933
18 Steeg PS, Bevilacqua G, Pozzatti R, et al. Alteres expression of nm23 a gene associated with low tumor metastatic potential, during adenovirus Z Ela inhibition of experimental metastasis. Cancer Res, 1988,48(22):6550-6554
19 Leone A, Flatow U, King C, et al. Reduced tumor incidence metastatic potential and cytokine responsiveness of nm23 transfected melanoma cells. Cell, 1991,65(1):25-35
20 Leone A, Flatow U, Van Houtte K, et al. Transfection of human nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: relationship to growth colonization metastatic potential and enzymatic activity. Cancer, 1994,79(12):2913-2922
21 Price JE, Polyzos A, Zhang RD, et al. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res, 1990, 50 (3) : 717-721
22 Fujimoto Y, Ohtake T, Nishimori H. Reduced expression and rare genomic altern of nm23-H1 in human hepatocellular carcinoma and hepatoma cell lines. J Gastroenterol, 1998,3:368-375
23 Boix L, Bruix J, Campo E, et al. nm23-H1 expression and disease recurrence after surgical resection of small hepatocellular carcinoma. Gastroenterology, 1994,107(2): 486-491
24 Zheng XY, Ling ZY, Tang ZY. The abundance of nm23-H1 mRNA is related with in situ microenvironment and intrahepatic metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res, 1998,3:337-341
25 Chen X, Dai Y, Yang J. Expression of metastasis suppressor gene nm23 in human hepatocellular carcinoma. Chung Hua Ping Li Hsueh Tsa Chih, 1996,2:76-78
26 Leone A, McBride OW, Weston A, et al. Somatic allelic deletion of nm23 in human cancer. Cancer Res, 1991,51(9):2490-2493
27 Cohn KH, Wang FS, Desoto-Lapaix F, et al. Association of nm23-H1 allelic deletions with distant metastasis in colorectal carcinoma. Lancet, 1991, 238(8769): 722-724
28 黄培林，金月玲，黄照权，等．nm23-H1基因突变及异常表达与大肠癌转移相关性的研究．东南大学学报(医学版)，2002，21(1)：44-48
29 Leone A, Seeger RC, Hong CM, et al. Evidence for nm23 RNA overexpression, DNA amplification and mutation in aggressive childhood neuroblastomas. Oncogene, 1993,8(4):855-865
30 陈军，周清华，覃扬，等．人肺癌中nm23等位基因缺失的研究．中国肺癌杂志，2000，3(1)：8-13
31 于景翠，李晓敏，傅松滨．肺癌基因组nm23-H1基因的扩增及异常分析．哈尔滨医科大学学报，2000，34(1)：6-7
32 王国丽，杜传书，林群娣．非小细胞肺癌淋巴结转移与nm23-H1基因突变及mRNA表达异常关系研究．中国结核和呼吸杂志，1997，20(6)：340-343
33 王国丽，杜传书．人原发性乳腺癌及肺癌中乳腺癌抑制基因突变．中山医科大学学报，1996，17：95
34 刘伦旭，周清华，石应康，等．人肺癌组织中nm23-H1基因突变研究．中国肺癌杂志，2000，3(3)：313-317