微卫星不稳定性与食管癌发生的相关性研究
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摘要
目的：人类错配修复基因的突变可诱发癌基因的激活或抑癌基因的失活，从而启动细胞癌变。本文通过对散发性食管癌患者，鳞状细胞癌和腺癌组织中微卫星不稳定性的分析，了解其在食管癌患者中的比例，探讨微卫星不稳定性与食管癌发病的关系；并进一步对微卫星不稳定性阳性的标本进行RIZ基因的分析，以便找到食管癌发生过程中的目标基因，为食管癌的早期诊断和治疗提供更有效的依据。
方法：所有组织标本均取材于河北医科大学第四医院胸外科散发性食管癌患者手术切除后的大体标本。取肿瘤组织以及边缘切口处正常组织（距离肿瘤组织至少5cm处），立即置入液氮罐速冻，而后迅速保存于-80℃冰箱。采用经典方法提取食管的肿瘤组织及正常食管组织的基因组DNA。应用聚合酶链反应（PCR）技术扩增微卫星Marker。通过7%变性聚丙烯酰胺凝胶电泳进行微卫星不稳定性分析，与正常标本对比，找出异常条带。另外，对微卫星不稳定性阳性患者的基因组DNA进行RIZ基因的检测，结合PCR-SSCP银染方法确定该抑癌基因与食管癌的发生是否具有相关性。
结果：1. 对80例散发性食管癌标本，提取基因组DNA，进行扩增，Bat-26扩增产物进行微卫星不稳定性分析，经变性聚丙烯酰胺凝胶电泳分析，微卫星不稳定性
（microsatellite instability, MSI）阳性患者15例，阳性率为18.75%；2. MSI与组织学分型的关系：在52例食管鳞状细胞癌患者中有8例MSI阳性，阳性率为15.4%；23例腺癌患者中MSI阳性有2例，阳性率为8.7%；5例小细胞癌患者中均有MSI阳性，阳性率为100%；经SAS统计学软件分析，食管肿瘤的不同组织病理学分型与微卫星不稳定性有显著相关性（p<0.05），小细胞癌患者的MSI的发生率显著高于鳞状细胞癌和腺癌患者。3. MSI与性别、年龄、及肿瘤转移的关系：51例男性食管癌患者中有10例发生MSI，阳性率为19.6%；29例女性食管癌患者中5例发生MSI，阳性率为17.2%，经SAS统计学软件分析，MSI的发生与患者的性别之间无显著差异（p>0.05）。28例55岁以下食管癌患者中有4例发生MSI，阳性率为14.2%；52例55岁以上患者中有11例发生MSI，阳性率为21.1%，经SAS统计学检验，MSI的发生与年龄之间无显著的相关性（p>0.05）；无淋巴结转移者MSI阳性有8（43）例，阳性率为18.6%；淋巴结转移者MSI阳性有7（37）例，阳性率为18.9%，经SAS统计学检验，MSI的发生率与肿瘤进展程度关系不大（p>0.05）；4. 对于微卫星不稳定性阳性的15例患者进行抑癌基因RIZ的检测，经PCR-SSCP银染分析，RIZ基因有6例发生突变，条带出现异常改变，突变率为40%。由此可见，RIZ基因的突变与MSI的发生有密切联系，推断RIZ可能是MSI发生的目标基因，通过移码突变或基因缺失导致PR区域的功能受损，参与食管癌的发生和发展。
结论：1. 在检测的正常食管癌组织中（包括肿瘤病人
癌旁正常食管粘膜，非肿瘤病人正常食管粘膜），Bat-26 MS DNA长度基本一致，电泳条带无明显变化，均显示较好的单态性。因而选取这一位点研究MSI时可以不必每例肿瘤标本均设立自身正常对照。然而，为排除假阳性出现，可以将所有MSI阳性标本的邻近正常组织再作一次检测，如果相应的肿瘤标本与之相比仍有差异，则说明该患者为MSI阳性。2. 在80例散发性食管癌标本中，DNA扩增产物经7%变性聚丙烯酰胺凝胶电泳分析，有15例出现微卫星不稳定性结果，阳性率为18.75%。3. 本实验证实了MSI与食管癌的发生有密切的关系。经统计学SAS分析表明，MSI与临床病理分型有显著相关性，即小细胞癌的MSI发生率显著高于鳞状细胞癌和腺癌；而其它临床资料，如食管癌患者的性别、年龄、淋巴结转移情况等均与MSI的发生无显著相关。4. 通过对MSI阳性患者进行RIZ基因的扩增，产物经SSCP银染分析，结果表明RIZ基因的改变与食管癌的发生有显著相关性，提示RIZ基因可能是食管癌发生发展过程中的目标基因。RIZ基因中有两个多聚腺嘌呤序列A（8）和A（9）发生移码突变，造成终止密码子提前出现，产生截短型蛋白，使错配修复蛋白的性状发生改变，错配修复蛋白功能降低，造成DNA碱基错配率增高，从而诱发细胞癌变，导致癌症的发生。因此对于已发现存在遗传性错配修复基因的患者，检测其家族中的其他成员，确认突变携带者，不但有助于对癌症易感个体重点进行临床监测和临床症状的早期诊断，随着进一步研究的深入，RIZ基因将在人类肿瘤的诊断、治疗中将发挥更大的作用。本研究结果提示，RIZ基因是食管癌发展过程中的一个重
要基因，该基因将为食管癌的诊断和基因治疗提供了广阔的前景。
Objective: As mutations of the human mismatch repair (MMR) gene can lead to the activation of oncogenes or inactivation of cancer suppressor gene which intiate the process of cell cancerization. In this paper, we analyzed the microsatellite instability in sporadic squamous cell carcinoma (SCC) and Barrett's adenocarcinoma (BA) to investigate the percentage of MSI and the relation of tumorgenesis. In MSI positive samples, we analyzed the RIZ gene in order to find the target gene in the esophageal cancerization. It can provide more effective basis for earlier diagnosis and therapy of esophageal cancer.
Methods: All resected samples for the experiment was obtained from the patients with sporadic esophageal cancer of the department of chest surgery in Hebei Medical University No. 4 Hospital. Resected tumor and its edge as well as normal tissue(apart from tumor tissue at least 5centimetre) were collected and stored at -80℃ refrigeratory quickly. Tumor and normal tissue DNA were obtained from eighty patients with sporadic esophageal cancer using standard extraction techniques. Microsatellite sequences at BAT-26 was amplified by polymerase chain reaction (PCR)
reaction. Amplified products were analyzed by 7% acrylamide gels electrophoresis for microsatellite analysis. Microsatellite instability was scored as present when an abnormal band occurred in the tumor sample when compared to the corresponding normal DNA sample. In addition, we studied RIZ mutations in sporadic esophageal cancer with microsatellite instability. We studied the mutation in the two coding polyadenosine tracks of RIZ by PCR-SSCP silver stain in order to identify the correlation of the suppressor gene and the esophageal cancerization.
Results: 1.Genetic DNA was obtained from eighty patients of sporadic esophageal cancer and was amplified with microsatellite marker BAT-26. Amplified products were analyzed by 7% denaturing acrylamide gels electrophoresis. Fifteen of eighty cancers were detected as unstable. Microsatellite instability (MSI) is observed in 18.75% of esophageal cancer. 2. The relationship between MSI and histological type: MSI was identified in seven of fifty-two sporadic squamous cell carcinoma (SCC) patients (15.4%), while MSI was observed in two of twenty-three sporadic Barrett's adenocarcinoma (BA) patients(8.7%). Five small-cell carcinoma patients were all found MSI (100%). The results analyzed by using SAS software showed that the relationship between MSI and histological type is statistically significant (p<0.05, χ2 test), that is to say, the incidence of small-cell carcinoma is significantly higher than patients of
squamous cell carcinoma (SCC) and Barrett's adenocarcinoma (BA). 3. The relationship between MSI and patients' sex, age and tumor metastasis: MSI was observed in ten of fifty-one male esophageal cancer patients (19.6%), while MSI was identified in five of twenty-nine female patients (17.2%). The presence of MSI did not correlate with patient sex by using SAS software (p>0.05, χ2 test). MSI was found in four of twenty-eight patients less than fifty-five years old (14.2%), while MSI was observed in eleven of fifty-two patients more than fifty-five years old (21.1%). With the analysis of SAS, there is no association with the patient age (p>0.05). MSI was identified in eight of forty-three patients with no lymph node metastasis (18.6), while MSI was observed in seven of thirty-seven patients with lymph node metastasis (18.9%). The incidence of MSI did not correlate with the tumor progress (p>0.05); 4. We studied the RIZ mutation in fifteen MSI patients. Mutations in the two coding polyadenosine tracks of RIZ were found in six of fifteen MSI patients by SSCP silver stain(40%) with abnormal bands. Therefore, mutations of RIZ were a close association with the presence of MSI. The result suggested that RIZ gene was possible a target gene of esophageal cancerization with impairment of the PR domain-mediated function through either frameshift mutation or genomic deletion.
Conclusions: 1. We detected
方法：所有组织标本均取材于河北医科大学第四医院胸外科散发性食管癌患者手术切除后的大体标本。取肿瘤组织以及边缘切口处正常组织（距离肿瘤组织至少5cm处），立即置入液氮罐速冻，而后迅速保存于-80℃冰箱。采用经典方法提取食管的肿瘤组织及正常食管组织的基因组DNA。应用聚合酶链反应（PCR）技术扩增微卫星Marker。通过7%变性聚丙烯酰胺凝胶电泳进行微卫星不稳定性分析，与正常标本对比，找出异常条带。另外，对微卫星不稳定性阳性患者的基因组DNA进行RIZ基因的检测，结合PCR-SSCP银染方法确定该抑癌基因与食管癌的发生是否具有相关性。
结果：1. 对80例散发性食管癌标本，提取基因组DNA，进行扩增，Bat-26扩增产物进行微卫星不稳定性分析，经变性聚丙烯酰胺凝胶电泳分析，微卫星不稳定性
（microsatellite instability, MSI）阳性患者15例，阳性率为18.75%；2. MSI与组织学分型的关系：在52例食管鳞状细胞癌患者中有8例MSI阳性，阳性率为15.4%；23例腺癌患者中MSI阳性有2例，阳性率为8.7%；5例小细胞癌患者中均有MSI阳性，阳性率为100%；经SAS统计学软件分析，食管肿瘤的不同组织病理学分型与微卫星不稳定性有显著相关性（p<0.05），小细胞癌患者的MSI的发生率显著高于鳞状细胞癌和腺癌患者。3. MSI与性别、年龄、及肿瘤转移的关系：51例男性食管癌患者中有10例发生MSI，阳性率为19.6%；29例女性食管癌患者中5例发生MSI，阳性率为17.2%，经SAS统计学软件分析，MSI的发生与患者的性别之间无显著差异（p>0.05）。28例55岁以下食管癌患者中有4例发生MSI，阳性率为14.2%；52例55岁以上患者中有11例发生MSI，阳性率为21.1%，经SAS统计学检验，MSI的发生与年龄之间无显著的相关性（p>0.05）；无淋巴结转移者MSI阳性有8（43）例，阳性率为18.6%；淋巴结转移者MSI阳性有7（37）例，阳性率为18.9%，经SAS统计学检验，MSI的发生率与肿瘤进展程度关系不大（p>0.05）；4. 对于微卫星不稳定性阳性的15例患者进行抑癌基因RIZ的检测，经PCR-SSCP银染分析，RIZ基因有6例发生突变，条带出现异常改变，突变率为40%。由此可见，RIZ基因的突变与MSI的发生有密切联系，推断RIZ可能是MSI发生的目标基因，通过移码突变或基因缺失导致PR区域的功能受损，参与食管癌的发生和发展。
结论：1. 在检测的正常食管癌组织中（包括肿瘤病人
癌旁正常食管粘膜，非肿瘤病人正常食管粘膜），Bat-26 MS DNA长度基本一致，电泳条带无明显变化，均显示较好的单态性。因而选取这一位点研究MSI时可以不必每例肿瘤标本均设立自身正常对照。然而，为排除假阳性出现，可以将所有MSI阳性标本的邻近正常组织再作一次检测，如果相应的肿瘤标本与之相比仍有差异，则说明该患者为MSI阳性。2. 在80例散发性食管癌标本中，DNA扩增产物经7%变性聚丙烯酰胺凝胶电泳分析，有15例出现微卫星不稳定性结果，阳性率为18.75%。3. 本实验证实了MSI与食管癌的发生有密切的关系。经统计学SAS分析表明，MSI与临床病理分型有显著相关性，即小细胞癌的MSI发生率显著高于鳞状细胞癌和腺癌；而其它临床资料，如食管癌患者的性别、年龄、淋巴结转移情况等均与MSI的发生无显著相关。4. 通过对MSI阳性患者进行RIZ基因的扩增，产物经SSCP银染分析，结果表明RIZ基因的改变与食管癌的发生有显著相关性，提示RIZ基因可能是食管癌发生发展过程中的目标基因。RIZ基因中有两个多聚腺嘌呤序列A（8）和A（9）发生移码突变，造成终止密码子提前出现，产生截短型蛋白，使错配修复蛋白的性状发生改变，错配修复蛋白功能降低，造成DNA碱基错配率增高，从而诱发细胞癌变，导致癌症的发生。因此对于已发现存在遗传性错配修复基因的患者，检测其家族中的其他成员，确认突变携带者，不但有助于对癌症易感个体重点进行临床监测和临床症状的早期诊断，随着进一步研究的深入，RIZ基因将在人类肿瘤的诊断、治疗中将发挥更大的作用。本研究结果提示，RIZ基因是食管癌发展过程中的一个重
要基因，该基因将为食管癌的诊断和基因治疗提供了广阔的前景。
Objective: As mutations of the human mismatch repair (MMR) gene can lead to the activation of oncogenes or inactivation of cancer suppressor gene which intiate the process of cell cancerization. In this paper, we analyzed the microsatellite instability in sporadic squamous cell carcinoma (SCC) and Barrett's adenocarcinoma (BA) to investigate the percentage of MSI and the relation of tumorgenesis. In MSI positive samples, we analyzed the RIZ gene in order to find the target gene in the esophageal cancerization. It can provide more effective basis for earlier diagnosis and therapy of esophageal cancer.
Methods: All resected samples for the experiment was obtained from the patients with sporadic esophageal cancer of the department of chest surgery in Hebei Medical University No. 4 Hospital. Resected tumor and its edge as well as normal tissue(apart from tumor tissue at least 5centimetre) were collected and stored at -80℃ refrigeratory quickly. Tumor and normal tissue DNA were obtained from eighty patients with sporadic esophageal cancer using standard extraction techniques. Microsatellite sequences at BAT-26 was amplified by polymerase chain reaction (PCR)
reaction. Amplified products were analyzed by 7% acrylamide gels electrophoresis for microsatellite analysis. Microsatellite instability was scored as present when an abnormal band occurred in the tumor sample when compared to the corresponding normal DNA sample. In addition, we studied RIZ mutations in sporadic esophageal cancer with microsatellite instability. We studied the mutation in the two coding polyadenosine tracks of RIZ by PCR-SSCP silver stain in order to identify the correlation of the suppressor gene and the esophageal cancerization.
Results: 1.Genetic DNA was obtained from eighty patients of sporadic esophageal cancer and was amplified with microsatellite marker BAT-26. Amplified products were analyzed by 7% denaturing acrylamide gels electrophoresis. Fifteen of eighty cancers were detected as unstable. Microsatellite instability (MSI) is observed in 18.75% of esophageal cancer. 2. The relationship between MSI and histological type: MSI was identified in seven of fifty-two sporadic squamous cell carcinoma (SCC) patients (15.4%), while MSI was observed in two of twenty-three sporadic Barrett's adenocarcinoma (BA) patients(8.7%). Five small-cell carcinoma patients were all found MSI (100%). The results analyzed by using SAS software showed that the relationship between MSI and histological type is statistically significant (p<0.05, χ2 test), that is to say, the incidence of small-cell carcinoma is significantly higher than patients of
squamous cell carcinoma (SCC) and Barrett's adenocarcinoma (BA). 3. The relationship between MSI and patients' sex, age and tumor metastasis: MSI was observed in ten of fifty-one male esophageal cancer patients (19.6%), while MSI was identified in five of twenty-nine female patients (17.2%). The presence of MSI did not correlate with patient sex by using SAS software (p>0.05, χ2 test). MSI was found in four of twenty-eight patients less than fifty-five years old (14.2%), while MSI was observed in eleven of fifty-two patients more than fifty-five years old (21.1%). With the analysis of SAS, there is no association with the patient age (p>0.05). MSI was identified in eight of forty-three patients with no lymph node metastasis (18.6), while MSI was observed in seven of thirty-seven patients with lymph node metastasis (18.9%). The incidence of MSI did not correlate with the tumor progress (p>0.05); 4. We studied the RIZ mutation in fifteen MSI patients. Mutations in the two coding polyadenosine tracks of RIZ were found in six of fifteen MSI patients by SSCP silver stain(40%) with abnormal bands. Therefore, mutations of RIZ were a close association with the presence of MSI. The result suggested that RIZ gene was possible a target gene of esophageal cancerization with impairment of the PR domain-mediated function through either frameshift mutation or genomic deletion.
Conclusions: 1. We detected
引文
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2 Thibodeau SN, Bren G Schaid D, et al. Micrsatellite instability in cancer of proximal colon. Science, 1993, 260: 816~819
3 Fishel R, Lescoe MK, Rao MRS, et al. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell, 1993, 75: 1027~1038
4 Gonzalez~zuleata M, Ruppert JM, Tokino K, et al. Microsatellite instability in bladder cancer. Cancer Res, 1993, 53: 5620~5623
5 Meltzer SJ, Yin J, Manin B, et al. Microsatellity instability occurs frequently and in both diploid and aneuploid cell populations of Barrett's-associated esophageal adenocarcinomas. Cancer Res, 1994, 54: 3379~3382
6 Tomatis L, Aitio A, Day NE, et al. Cancer causes occurrence and control. IARC Sci Publ, 1990, 100:55~56
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30 Liu B, Darsons R, Papadopoulos N, et al. Analysis of mismatch repair gene in hereditary non-polyposis colorectal cancer patients. Nature Medine, 1996, 2: 169~173
2 Thibodeau SN, Bren G Schaid D, et al. Micrsatellite instability in cancer of proximal colon. Science, 1993, 260: 816~819
3 Fishel R, Lescoe MK, Rao MRS, et al. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell, 1993, 75: 1027~1038
4 Gonzalez~zuleata M, Ruppert JM, Tokino K, et al. Microsatellite instability in bladder cancer. Cancer Res, 1993, 53: 5620~5623
5 Meltzer SJ, Yin J, Manin B, et al. Microsatellity instability occurs frequently and in both diploid and aneuploid cell populations of Barrett's-associated esophageal adenocarcinomas. Cancer Res, 1994, 54: 3379~3382
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