TOP2A在胃癌中的表达及TOP2A低表达胃腺癌细胞模型的建立
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摘要
[目的]由于拓扑异构酶Ⅱα(TopoisomeraseⅡα)基因TOP2A在染色体分离、配对、浓缩、结构形成及改变DNA超螺旋结构有重要的作用,并且又是很多肿瘤化疗药物的作用靶点,因此它在肿瘤治疗中引起了特殊的关注。我科基因芯片结果发现胃腺癌组织中的TOP2A明显高表达,本课题拟通过病例调查,明确TOP2A在人胃癌组织中的表达特征,并进一步建立TOP2A基因低表达的胃腺癌细胞模型,明确抑制TOP2A蛋白表达对其生物学行为的影响,为将来探讨TOP2A蛋白表达对topoⅡ抑制剂的疗效的预测作用提供良好的基础。
[方法]本研究共分两部分:第一部分,首先从2004年-2008年从本院就诊的胃癌患者中筛选出做TOP2A蛋白免疫组化染色患者200例,根据其TOP2A蛋白表达量的高低及分化程度、病变部位分组进行统计分析;其次,用WesternBloting(WB)法检验胃癌细胞株MGC-803、BGC-823及乳腺癌细胞株MCF-7中TOP2A蛋白的表达。第二部分,设计构建针对TOP2A基因的shRNA(smallhairpin RNA,短发夹RNA)重组表达质粒pSRZTopⅡα并转染至G3T-hi包装细胞,收集绿色荧光蛋白表达阳性的包装细胞病毒上清,测定病毒滴度,感染三株目的细胞BGC-823、MGC-803及MCF-7,流式分选阳性克隆,扩增并培养。WB检测三株目的细胞感染重组逆转录病毒pSRZTopⅡα后细胞内TOP2A的表达情况,并用细胞生长曲线的方法记录感染病毒前后细胞生长状态及活性的变化。
[结果]第一部分,200例TOP2A蛋白表达阳性患者其表达量的高低与患者年龄、性别、胃癌分化程度及胃癌部位并无相关性:胃腺癌细胞BGC-823、MGC-803和乳腺癌细胞MCF-7的总蛋白进行Western bloting检测,均有TOP2A的表达。第二部分,酶切及测序鉴定pSRZTopⅡα质粒构建成功,pSRZTopⅡα表达质粒转染至G3T-hi包装细胞后收集病毒上清,病毒滴度测定结果最高达1.87×10~7,由此证实逆转录病毒构建成功;目的细胞感染病毒并分选后进行荧光显微镜观察,64h的荧光蛋白表达比率较17h明显增高,证实逆转录病毒感染目的细胞成功,三株目的细胞感染后TOP2A蛋白表达较阴性对照组明显降低,胃腺癌细胞MGC-803细胞生长趋势较未感染组和阴性对照组明显减慢,各时间点细胞计数值明显降低。
[结论]1.靶向TOP2A的shRNA能瞬时抑制TOP2A蛋白的表达:2.靶向抑制TOP2A基因的表达可以相对抑制肿瘤细胞的生长;3.首次构建低表达TOP2A的腺癌细胞模型成功。
[Objective]TOP2A is of particular interest with respect to cancer therapeutics, because it is vital in the segregation of newly replicated chromosome pairs,in chromosome condensation,in forming chromosome scaffolds,and in altering DNA superhelicity,and even more importantly,it is also the molecular target for a large group of clinically relevant anti-cancer drugs termed topolI-inhibitors.We found the higher level of TOP2A in the gastric adenocarcinoma according to the gene chip technology.Our research was prepared to identify the distinction of TOP2A in the human gastric carcinoma tissue.Then we can identify the effect between different expression of TOP2A to the biological behaviour of gastric adenocarcinoma cell by constructing low-expression model.It will provide us satisfactory foundation for approaching the prediction effect of the expression of TOP2A to the therapeutic effect.
[Methods]1.200 gastric adenocarcinoma cases in 1972-2008 who had immunohistochemistry results ofTopoisomeraseⅡαwere collected and analysised by different expression of TopoisomeraseⅡα/differentiation/position of the tumor, the expression of topoisomeraseⅡαin the MGC-803/BGC-823/MCF-7 cells was detected by WB.2.Construct the recombinant retrovirus plasmid of pSRZTopⅡαand transfect the plamid into packaging cell G3T-hi,collect the virus supematant of packaging cells and detect the virus titer.Infect the target cell BGC-823、MGC-803 and MCF-7 with supernatant and separatethe positive one to incubate.Detect the expression of topoisomeraseⅡαin target cells before after infected by recombinate retrovirus pSRZTopⅡαby WB,and record the difference of cell activity and proliferativeability by cell growth curve.
[Result]1.The immunohistochemistry results of 200 cases were all positive, there showed no significant difference in expression of TopoisomeraseⅡαwith sex/age of patients or the differentiation/position of gastric adenocarcinoma.The expression of TOP2A gene found in three adenocarcinoma cells by WB.2.The enzyme digestion and sequencing results showed the construction of plasmid pSRZTopⅡαis succeed.After transfection,collect the supematant and get the highest level of 1.87×10~7 in virus titer.It showed the success of constructing retrovirus.It was obviously that the GFP ratio of cells at the 64h was higher than that at 17h after FACS.So the infection of retrovirus was succeed.It showed lower expression of topoisomeraseⅡαin the target cells than negative control group after infection.The gastric carcinoma cell MGC-803 grew lower after infection and the cell counts in experiment group were lower than negative control group.
[Conelusion]1.ShRNA target TOP2A can inhibit the expression of TopoisomeraseⅡαtransiently 2.Inhibition TOP2A gene can inhibit the growth of tumor cells relatively.3.At the first time,the cell model of low expression of topoisomeraseⅡαwas successfully constructed.
[方法]本研究共分两部分:第一部分,首先从2004年-2008年从本院就诊的胃癌患者中筛选出做TOP2A蛋白免疫组化染色患者200例,根据其TOP2A蛋白表达量的高低及分化程度、病变部位分组进行统计分析;其次,用WesternBloting(WB)法检验胃癌细胞株MGC-803、BGC-823及乳腺癌细胞株MCF-7中TOP2A蛋白的表达。第二部分,设计构建针对TOP2A基因的shRNA(smallhairpin RNA,短发夹RNA)重组表达质粒pSRZTopⅡα并转染至G3T-hi包装细胞,收集绿色荧光蛋白表达阳性的包装细胞病毒上清,测定病毒滴度,感染三株目的细胞BGC-823、MGC-803及MCF-7,流式分选阳性克隆,扩增并培养。WB检测三株目的细胞感染重组逆转录病毒pSRZTopⅡα后细胞内TOP2A的表达情况,并用细胞生长曲线的方法记录感染病毒前后细胞生长状态及活性的变化。
[结果]第一部分,200例TOP2A蛋白表达阳性患者其表达量的高低与患者年龄、性别、胃癌分化程度及胃癌部位并无相关性:胃腺癌细胞BGC-823、MGC-803和乳腺癌细胞MCF-7的总蛋白进行Western bloting检测,均有TOP2A的表达。第二部分,酶切及测序鉴定pSRZTopⅡα质粒构建成功,pSRZTopⅡα表达质粒转染至G3T-hi包装细胞后收集病毒上清,病毒滴度测定结果最高达1.87×10~7,由此证实逆转录病毒构建成功;目的细胞感染病毒并分选后进行荧光显微镜观察,64h的荧光蛋白表达比率较17h明显增高,证实逆转录病毒感染目的细胞成功,三株目的细胞感染后TOP2A蛋白表达较阴性对照组明显降低,胃腺癌细胞MGC-803细胞生长趋势较未感染组和阴性对照组明显减慢,各时间点细胞计数值明显降低。
[结论]1.靶向TOP2A的shRNA能瞬时抑制TOP2A蛋白的表达:2.靶向抑制TOP2A基因的表达可以相对抑制肿瘤细胞的生长;3.首次构建低表达TOP2A的腺癌细胞模型成功。
[Objective]TOP2A is of particular interest with respect to cancer therapeutics, because it is vital in the segregation of newly replicated chromosome pairs,in chromosome condensation,in forming chromosome scaffolds,and in altering DNA superhelicity,and even more importantly,it is also the molecular target for a large group of clinically relevant anti-cancer drugs termed topolI-inhibitors.We found the higher level of TOP2A in the gastric adenocarcinoma according to the gene chip technology.Our research was prepared to identify the distinction of TOP2A in the human gastric carcinoma tissue.Then we can identify the effect between different expression of TOP2A to the biological behaviour of gastric adenocarcinoma cell by constructing low-expression model.It will provide us satisfactory foundation for approaching the prediction effect of the expression of TOP2A to the therapeutic effect.
[Methods]1.200 gastric adenocarcinoma cases in 1972-2008 who had immunohistochemistry results ofTopoisomeraseⅡαwere collected and analysised by different expression of TopoisomeraseⅡα/differentiation/position of the tumor, the expression of topoisomeraseⅡαin the MGC-803/BGC-823/MCF-7 cells was detected by WB.2.Construct the recombinant retrovirus plasmid of pSRZTopⅡαand transfect the plamid into packaging cell G3T-hi,collect the virus supematant of packaging cells and detect the virus titer.Infect the target cell BGC-823、MGC-803 and MCF-7 with supernatant and separatethe positive one to incubate.Detect the expression of topoisomeraseⅡαin target cells before after infected by recombinate retrovirus pSRZTopⅡαby WB,and record the difference of cell activity and proliferativeability by cell growth curve.
[Result]1.The immunohistochemistry results of 200 cases were all positive, there showed no significant difference in expression of TopoisomeraseⅡαwith sex/age of patients or the differentiation/position of gastric adenocarcinoma.The expression of TOP2A gene found in three adenocarcinoma cells by WB.2.The enzyme digestion and sequencing results showed the construction of plasmid pSRZTopⅡαis succeed.After transfection,collect the supematant and get the highest level of 1.87×10~7 in virus titer.It showed the success of constructing retrovirus.It was obviously that the GFP ratio of cells at the 64h was higher than that at 17h after FACS.So the infection of retrovirus was succeed.It showed lower expression of topoisomeraseⅡαin the target cells than negative control group after infection.The gastric carcinoma cell MGC-803 grew lower after infection and the cell counts in experiment group were lower than negative control group.
[Conelusion]1.ShRNA target TOP2A can inhibit the expression of TopoisomeraseⅡαtransiently 2.Inhibition TOP2A gene can inhibit the growth of tumor cells relatively.3.At the first time,the cell model of low expression of topoisomeraseⅡαwas successfully constructed.
引文
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[1]Devesa SS,Blot WJ,Fraumeni JF Jr.Changing patterns in the incidence of esophageal and gastric carcinoma in the United States.Cancer,1998;83:2049-2053
[2]Devesa SS,Fraumeni JF Jr.The rising incidence of gastric cardia cancer.J Natl Cancer Inst 1999;91:747-749
[3]Wang LD,Zheng S,Zheng ZY,et al.Primary adenocarcinomas of lower esophagus,esophagogastric junction and gastric cardia:in special reference to China.World J Gastroenterol 2003;9(6):1156-1164
[4]Sharma P.Cancer of the esophagogastric junction:epiderniology and pathogenesis.J Gastrointest Surg,2003;6:516-517
[5]Valerie W.Rusch.Are cancers of the esophagus,gastroesophageal junction,and cardia one disease,two,or several? Seminars in Oncology,Vol 31,No 4(August),2004:pp 444-449
[6]Siewert JR,Feith M,Werner M,et al.Adenocarcinoma of the esophagogastric junction.Results of surgical therapy based on anatomical/topographic classification in 1,002consecutive patients.Ann Surg,2000;232:353-361
[7]Parkin D,Pisani P,Ferley J,et al.Global cancer statistics.CA Cancer J Clin 1999;49:33-64.
[8]Devesa SS,Blot WJ,Frammeni JF.Changing patterns in the incidence of esophageal and gastric carcinoma in the United States.Cancer 2005;83:2049-2053.
[9]Wu AH,Wan P,Bernstein L.A multiethnic population-based study of smoking,alcohol and body size and risk of adenocarcinomas of the stomach and esophagus(United States).Cancer Causes Control,2001;12:721-732
[10]Eksteen JA,Latchford A,Thomas SJ,et al.Commentary:regional variations in oesophageal and gastric cardia cancers--implications and practice.Int J Epidemiol,2001;30:1425-1427
[11]Ectors N,Driessen A,De Hertog G,et al.Is Adenocarcinoma of the Esophagogastric Junction or Cardia Different From Barrett Adenocarcinoma? Arch Pathol Lab Med.2005 Feb;129(2):183-5.
[12]Gao C-M,Takezaki T,Wu J-Z,et al:Glutathione-S-transferases M1(GSTM1) and GSTT1genotype,smoking,consumption of alcohol and tea and risk of esophageal and stomach cancers:A case-control study of a high-incidence area of Jiangsu Province,China.Cancer Lett,2002;188:95-102
[13]Taniee P,Martel-Planche G,Maurici D,et al:Molecular and clinical differences between adenocarcinomas of the esophagus and of the gastric cardia.Am J Pathol,2001;158:33-40