人肝癌p57~(kip2)启动子区甲基化及基因组不稳定性与mRNA和蛋白表达关系研究
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摘要
目的:为阐明p57~(kip2)基因异常与人肝细胞癌(Hepatocellular careinoma,HCC)发生发展的关系,本文从mRNA及蛋白质水平检测肝癌组织中p57~(kip2)基因的表达;并从基因结构分析肝癌组织中p57~(kip2)基因的杂合性缺失(Loss of heterozygosity,LOH)、微卫星不稳定性(Microsatelliteinstability,MSI)及甲基化状况,进而探讨其间的相互关系,以评价p57~(kip2)基因异常在肝癌发生及演进过程中作用与地位。
方法:选取正常肝组织、癌周肝组织和肝细胞肝癌组织各30例,采用原位杂交和免疫组化技术,分别检测p57~(kip2)基因mRNA的表达状况和p57~(kip2)蛋白的表达水平的改变。对p57~(kip2)所在染色体11p15.5区域的二个微卫星位点(D11S2351和D115569)运用PCR-聚丙烯酰胺凝胶电泳技术进行杂合性缺失和微卫星不稳定性的检测。应用甲基化特异性PCR(methylation-specific PCR,MSP)对p57~(kip2)启动子区CpG岛进行甲基化分析。对p57~(kip2)mRNA、蛋白表达和甲基化之间的关系进行分析。
结果:1.原位分子杂交技术显示:正常肝组织组未见表达,癌周肝组织组与肝癌组阳性表达率均为26.7%(8/30)。p57~(kip2)mRNA在不同分化程度的肝癌中的阳性表达率分别为高分化组0%,中分化组20.8%,低分化组60%。p57~(kip2)mRNA表达在正常组与癌周肝组织组,正常肝组织组与肝癌组之间比较差异有统计学意义。癌周肝组织组与肝癌组比较差异无统计学意义。高、中和低分化三组不同分化程度的肝癌之间p57~(kip2)mRNA表达差异有统计学意义。
2.免疫组织化学法显示:正常肝组织组未见表达,癌周肝组织组与肝癌组阳性表达率均为56.67%(17/30)。p57~(kip2)蛋白表达在正常组与癌周肝组织组,正常组与肝癌组之间比较差异有统计学意义。癌周肝组织组与肝癌组比较差异无统计学意义。p57~(kip2)蛋白在不同分化程度的肝癌中的阳性表达率分别为高分化组0%,中分化组54.1%,低分化组80%,p57~(kip2)蛋白的表达在不同分化程度的肝癌之间差异有统计学意义。
3.采用PCR-聚丙烯酰胺凝胶电泳检测结果:在30例肝癌组织标本中,仅在D11S569位点检测到1例杂合性缺失,在D11S2351和D11S569两个位点上共检测到4例微卫星不稳定,其中肝癌组微卫星位点D11S2351发生MSI与p57~(kip2)mRNA表达有正相关性但无生物学意义,与p57~(kip2)蛋白表达呈负相关性有生物学意义。
4.MSP检测结果:在30例肝癌组织标本中检测到6例p57~(kip2)启动子甲基化,其p57~(kip2)启动子区甲基化与p57~(kip2)mRNA和蛋白表达异常没有关联性。
结论:1.p57~(kip2)mRNA和蛋白表达异常提示p57~(kip2)在原发性肝癌发生发展过程中可能不具有肿瘤抑制基因特性。
2.与p57~(kip2)基因同一区域2个微卫星位点(D11S2351和D11S569)不是p57~(kip2)基因LOH和MSI的频发位点。
3.LOH和MSI可能不是导致p57~(kip2)mRNA和蛋白表达异常的原因。
4.甲基化也不是导致p57~(kip2)mRNA和蛋白表达异常的原因。
Objective:In order to research the relation between abnormal expression of p57~(kip2) gene and incidence of HCC,this article examine the expression of p57~(kip2) from level of mRNA & protein,and the loss of hererozygosity(LOH),microsatellite instability(MSI) and methylation from level of DNA in HCC.
Methods:The expression state of mRNA and protein of p57~(kip2) gene were detected in situ hybridization and Immunohistochemistry staining,and the LOH and MSI of two microsatellite loci(D11S2351 and D11S569) located on chromosome 11p15.5 region were detected using PCR-polyacrylamide gel electrophoresis technology.p57~(kip2) promoter region CpG island methylation were also analysised by methylation-specific PCR (methylation-specific PCR,MSP).Further,we analysis the relationship among expression of p57~(kip2) mRNA and protein,LOH,MSI and methylation.
Results:1.Expression of p57~(kip2) mRNA:There were no expression in normal liver tissue.Expressions both in pericancerous cirrhosis and hepatocellular carcinoma were 26.67%(8/30).The rate of expression of p57~(kip2)mRNA at different levels in HCC were 0%in high differentiated HCC group, 20.8%in well-differentiated groups,60%in poorly differentiated groups.There were statistically significant in the expression of p57~(kip2)mRNA between the groups of normal liver and the pericancerous cirrhosis,the normal liver and HCC.But there were not statistically significant between the groups of pericancerous cirrhosis and HCC. The expression of p57~(kip2) mRNA have statistical significance in three differentiated groups.
2.Expression of p57~(kip2) protein:There were no expressions in normal liver tissue.Expressions in precancerous cirrhosis and hepatocellular carcinoma were 56.67%(17/30).The rate of expression of p57~(kip2) protein at different levels in HCC were 0%in high differentiated HCC group,54.1%in well-differentiated groups,80%in poorly differentiated groups.There were statistically significant in the expression of p57~(kip2) protein between the groups of normal liver and the pericancerous cirrhosis,the normal liver and HCC.But there were not statistically significant between the groups of pericancerous cirrhosis and HCC. The expression of p57~(kip2) protein have statistical significance in three differentiated groups.
3.LOH and MSI detected:Only 1 case of LOH were identified in 30 cases on one microsatellite locies(D11S569),and 4 case of MSI were showed in both 2 microsatellite locies.There was correlation between the MSI of D11S2351 locies and the expression of p57~(kip2)mRNA and protein.
4.MSP detected:There were 6 HCC of hypermethylation in 30 cases,but there were not correlation between hypermethylation and the expression of p57~(kip2)mRNA and protein.
Conclusions:1.The disorder expressions of p57~(kip2)mRNA and protein indicated that p57~(kip2) may not be as a tumor suppressor gene in hepatocarcinogenesis and prognosis.
2.The two microsatellite loci where the p57~(kip2) gene is located in were not the frequent loci for LOH and MSI.
3.LOH and MSI may not be one of the reasons that lead to the loss of p57~(kip2)mRNA and protein in hepatocarcingenesis and prognosis.
4.The hypermethylation of p57~(kip2) might not lead to the abnormal expression of p57~(kip2) mRNA and protein.
方法:选取正常肝组织、癌周肝组织和肝细胞肝癌组织各30例,采用原位杂交和免疫组化技术,分别检测p57~(kip2)基因mRNA的表达状况和p57~(kip2)蛋白的表达水平的改变。对p57~(kip2)所在染色体11p15.5区域的二个微卫星位点(D11S2351和D115569)运用PCR-聚丙烯酰胺凝胶电泳技术进行杂合性缺失和微卫星不稳定性的检测。应用甲基化特异性PCR(methylation-specific PCR,MSP)对p57~(kip2)启动子区CpG岛进行甲基化分析。对p57~(kip2)mRNA、蛋白表达和甲基化之间的关系进行分析。
结果:1.原位分子杂交技术显示:正常肝组织组未见表达,癌周肝组织组与肝癌组阳性表达率均为26.7%(8/30)。p57~(kip2)mRNA在不同分化程度的肝癌中的阳性表达率分别为高分化组0%,中分化组20.8%,低分化组60%。p57~(kip2)mRNA表达在正常组与癌周肝组织组,正常肝组织组与肝癌组之间比较差异有统计学意义。癌周肝组织组与肝癌组比较差异无统计学意义。高、中和低分化三组不同分化程度的肝癌之间p57~(kip2)mRNA表达差异有统计学意义。
2.免疫组织化学法显示:正常肝组织组未见表达,癌周肝组织组与肝癌组阳性表达率均为56.67%(17/30)。p57~(kip2)蛋白表达在正常组与癌周肝组织组,正常组与肝癌组之间比较差异有统计学意义。癌周肝组织组与肝癌组比较差异无统计学意义。p57~(kip2)蛋白在不同分化程度的肝癌中的阳性表达率分别为高分化组0%,中分化组54.1%,低分化组80%,p57~(kip2)蛋白的表达在不同分化程度的肝癌之间差异有统计学意义。
3.采用PCR-聚丙烯酰胺凝胶电泳检测结果:在30例肝癌组织标本中,仅在D11S569位点检测到1例杂合性缺失,在D11S2351和D11S569两个位点上共检测到4例微卫星不稳定,其中肝癌组微卫星位点D11S2351发生MSI与p57~(kip2)mRNA表达有正相关性但无生物学意义,与p57~(kip2)蛋白表达呈负相关性有生物学意义。
4.MSP检测结果:在30例肝癌组织标本中检测到6例p57~(kip2)启动子甲基化,其p57~(kip2)启动子区甲基化与p57~(kip2)mRNA和蛋白表达异常没有关联性。
结论:1.p57~(kip2)mRNA和蛋白表达异常提示p57~(kip2)在原发性肝癌发生发展过程中可能不具有肿瘤抑制基因特性。
2.与p57~(kip2)基因同一区域2个微卫星位点(D11S2351和D11S569)不是p57~(kip2)基因LOH和MSI的频发位点。
3.LOH和MSI可能不是导致p57~(kip2)mRNA和蛋白表达异常的原因。
4.甲基化也不是导致p57~(kip2)mRNA和蛋白表达异常的原因。
Objective:In order to research the relation between abnormal expression of p57~(kip2) gene and incidence of HCC,this article examine the expression of p57~(kip2) from level of mRNA & protein,and the loss of hererozygosity(LOH),microsatellite instability(MSI) and methylation from level of DNA in HCC.
Methods:The expression state of mRNA and protein of p57~(kip2) gene were detected in situ hybridization and Immunohistochemistry staining,and the LOH and MSI of two microsatellite loci(D11S2351 and D11S569) located on chromosome 11p15.5 region were detected using PCR-polyacrylamide gel electrophoresis technology.p57~(kip2) promoter region CpG island methylation were also analysised by methylation-specific PCR (methylation-specific PCR,MSP).Further,we analysis the relationship among expression of p57~(kip2) mRNA and protein,LOH,MSI and methylation.
Results:1.Expression of p57~(kip2) mRNA:There were no expression in normal liver tissue.Expressions both in pericancerous cirrhosis and hepatocellular carcinoma were 26.67%(8/30).The rate of expression of p57~(kip2)mRNA at different levels in HCC were 0%in high differentiated HCC group, 20.8%in well-differentiated groups,60%in poorly differentiated groups.There were statistically significant in the expression of p57~(kip2)mRNA between the groups of normal liver and the pericancerous cirrhosis,the normal liver and HCC.But there were not statistically significant between the groups of pericancerous cirrhosis and HCC. The expression of p57~(kip2) mRNA have statistical significance in three differentiated groups.
2.Expression of p57~(kip2) protein:There were no expressions in normal liver tissue.Expressions in precancerous cirrhosis and hepatocellular carcinoma were 56.67%(17/30).The rate of expression of p57~(kip2) protein at different levels in HCC were 0%in high differentiated HCC group,54.1%in well-differentiated groups,80%in poorly differentiated groups.There were statistically significant in the expression of p57~(kip2) protein between the groups of normal liver and the pericancerous cirrhosis,the normal liver and HCC.But there were not statistically significant between the groups of pericancerous cirrhosis and HCC. The expression of p57~(kip2) protein have statistical significance in three differentiated groups.
3.LOH and MSI detected:Only 1 case of LOH were identified in 30 cases on one microsatellite locies(D11S569),and 4 case of MSI were showed in both 2 microsatellite locies.There was correlation between the MSI of D11S2351 locies and the expression of p57~(kip2)mRNA and protein.
4.MSP detected:There were 6 HCC of hypermethylation in 30 cases,but there were not correlation between hypermethylation and the expression of p57~(kip2)mRNA and protein.
Conclusions:1.The disorder expressions of p57~(kip2)mRNA and protein indicated that p57~(kip2) may not be as a tumor suppressor gene in hepatocarcinogenesis and prognosis.
2.The two microsatellite loci where the p57~(kip2) gene is located in were not the frequent loci for LOH and MSI.
3.LOH and MSI may not be one of the reasons that lead to the loss of p57~(kip2)mRNA and protein in hepatocarcingenesis and prognosis.
4.The hypermethylation of p57~(kip2) might not lead to the abnormal expression of p57~(kip2) mRNA and protein.
引文
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