非甾体类抗炎药活化基因-1在胃癌中的表达及其真核表达载体的构建
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摘要
目的:
1.研究非甾体类抗炎药活化基因- 1(nonsteroidal anti- inflammatory drug - activated gene-1 ,NAG-1)在胃癌组织中的表达及与胃癌临床病理特征之间的关系,探讨NAG-1在胃癌发生、发展和侵袭中的作用。
2.构建NAG-1的真核表达载体pEGFP-N1/NAG-1,为体外研究NAG-1基因功能建立实验基础。
方法:
1.采用半定量逆转录聚合酶链反应(RT-PCR)法检测76例原发性胃癌患者癌组织及相应胃正常黏膜组织中NAG-1mRNA的表达;
2.采用免疫组织化学S-P方法检测胃癌及正常胃粘膜组织中NAG-1蛋白的表达情况;
3.以正常胃粘膜组织cDNA为模板,用RT-PCR技术扩增NAG-1基因片段,将该片段插入表达载体pEGFP-N1中,并通过蓝白斑实验、酶切、PCR和DNA测序刷选和鉴定阳性克隆。
结果:
1.正常组织和胃癌组织中均表达NAG-1mRNA,胃癌组织中的相对含量为0.5563±0.2181,显著低于胃正常组织的0.9059±0.0751(P<0.05)。高、中分化胃癌组织中的NAG-1mRNA相对含量为0.6701±0.1637,显著高于低、未分化胃癌组织的0.4005±0.1859(p<0.05)。不同浸润深度、有无淋巴结转移、不同临床分期的胃癌组织间NAG-1mRNA相对含量的差异均无统计学意义(p>0.05)。
2.胃癌组织的NAG-1蛋白的表达程度显著于低于胃正常粘膜组织(X2=47.186 ,p=0.000<0.05)。细胞分化程度低的癌组织NAG-1蛋白表达程度显著低于分化程度高的癌组织(X2= 7.506,p=0.006<0.05)。不同性别、有无淋巴结转移、不同临床分期的胃癌组织间NAG-1蛋白表达程度的差异均无统计学意义(p值>0.05)。
3.对阳性克隆进行PCR、酶切及DNA测序鉴定,证实NAG-1目的片段已正确插入表达载体。
结论:
1. NAG-1mRNA在胃癌组织中的表达与浸润深度、淋巴结转移、TNM分期无关,而与肿瘤的分化程度相关,提示NAG-1mRNA的表达程度影响胃癌的分化。
2. NAG-1蛋白的表达与肿瘤分化程度相关,NAG-1蛋白质在胃癌组织中的表达与浸润深度、淋巴结转移、TNM分期无关,提示NAG-1蛋白质的表达可能影响胃癌的分化。
3. NAG-1mRNA在胃癌组织低于正常胃组织中的表达,NAG-1蛋白在胃癌组织中的表达明显低于它们在正常胃组织中的表达,提示NAG-1的表达改变可能与胃癌发生与发展有关。
4.成功构建pEGFP-N1/NAG-1重组质粒,为进一步研究NAG-1基因功能建立实验基础。
【Objective】:
1.To study the expression of nonsteroidal anti - inflammatory drug - activated gene-1 in human gastric cancer and its relationship with the clinical staging ,differntiation degree ,and lymphatic metastasis of gastric cancer.
2.To clone a fragment of NAG-1 gene into an expression vector pEGFP-N1 for expressing NAG-1 protein in vitro.
【Methods】:
1.The express of NAG-1 mRNA was detected by semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in the cancer tissues and corresponding adjacent normal tissues in 76 gastric cancer patients.
2.The expression of NAG-1 protein expression in human gastric carcinoma tissues and normal gastric tissues were detected by S-P immunohistochemistry.
3.Using nomal gastic tissue cDNA as template,amplified the NAG-1 gene with RT-PCR.Insert this fragment into the expression vector pEGFP-N1,and identified the positive clone with white and blue assay,endonuclease,PCR and DNA sequenceing identification.
【Results】:
1.NAG-1 mRNA was positive in both nomal tissues and gastric cancer tissues; expression in gastric cancer tissue significantly was lower than that in nomal tissues(0.5563±0.2181 vs 0.9059±0.0751,P<0.05).NAG-1mRNA expression in well and moderately differentiated cancer cells was significantly higher than those of poorly and non-differerntiated cancer cells(0.6701±0.1637 vs 0.4005±0.1859,p<0.05) , but the expression was not associated with the depth of tumor invasion,lymph node metastasis , and clinical stage (p>0.05).
2Immuno-histochemistry showed the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues (p<0.05);and the expression was not relationship with the gender, lymph node metastasis and clinical stage (all p >0.05).
3. The expression of NAG-1 mRNA in gastric cancer tissues was lower than that in nomal tissues ;the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues.they suggest that the expression of NAG-1 may relationship with the development and progression of gastic cancer.
4. The positive clons were identified with PCR,endonuclease and DNA sequencing identification,and they proved that the target fragment of NAG-1 gene had been inserted into expression vector pEGFP-N1 rightly.
【Conclusion】:
1. The expression of NAG-1mRNA in gastric carcinoma tissues and normal gastric tissues had statistical difference;The expression of NAG-1mRNA had no correlation with the depth of tumor invasion、lymph node metastasis and TNM stage. It is associated with the differentiation of tumor. That suggest the carcinogenesis、progression and invasion of gastric carcinoma had correlation with the NAG-1mRNA expression.
2. The expression of NAG-1 protein was significant lower in gastric carcinoma than normal gastric tissues. The expression of NAG-1 protein was associated with differentiation grades.That show NAG-1 protein suppressed the processes of carcinogenesis、progression and invasion of gastric carcinoma. Downregulation of NAG-1 protein promoted the processes of carcinogenesis、progression、invasion and metastasis of gastric carcinoma,Which made the gastric carcinoma cells more malignant and more invasive
3. Cloning NAG-1 recombinant plasmid lays the foundation for expressing NAG-1 protein in vitro.
1.研究非甾体类抗炎药活化基因- 1(nonsteroidal anti- inflammatory drug - activated gene-1 ,NAG-1)在胃癌组织中的表达及与胃癌临床病理特征之间的关系,探讨NAG-1在胃癌发生、发展和侵袭中的作用。
2.构建NAG-1的真核表达载体pEGFP-N1/NAG-1,为体外研究NAG-1基因功能建立实验基础。
方法:
1.采用半定量逆转录聚合酶链反应(RT-PCR)法检测76例原发性胃癌患者癌组织及相应胃正常黏膜组织中NAG-1mRNA的表达;
2.采用免疫组织化学S-P方法检测胃癌及正常胃粘膜组织中NAG-1蛋白的表达情况;
3.以正常胃粘膜组织cDNA为模板,用RT-PCR技术扩增NAG-1基因片段,将该片段插入表达载体pEGFP-N1中,并通过蓝白斑实验、酶切、PCR和DNA测序刷选和鉴定阳性克隆。
结果:
1.正常组织和胃癌组织中均表达NAG-1mRNA,胃癌组织中的相对含量为0.5563±0.2181,显著低于胃正常组织的0.9059±0.0751(P<0.05)。高、中分化胃癌组织中的NAG-1mRNA相对含量为0.6701±0.1637,显著高于低、未分化胃癌组织的0.4005±0.1859(p<0.05)。不同浸润深度、有无淋巴结转移、不同临床分期的胃癌组织间NAG-1mRNA相对含量的差异均无统计学意义(p>0.05)。
2.胃癌组织的NAG-1蛋白的表达程度显著于低于胃正常粘膜组织(X2=47.186 ,p=0.000<0.05)。细胞分化程度低的癌组织NAG-1蛋白表达程度显著低于分化程度高的癌组织(X2= 7.506,p=0.006<0.05)。不同性别、有无淋巴结转移、不同临床分期的胃癌组织间NAG-1蛋白表达程度的差异均无统计学意义(p值>0.05)。
3.对阳性克隆进行PCR、酶切及DNA测序鉴定,证实NAG-1目的片段已正确插入表达载体。
结论:
1. NAG-1mRNA在胃癌组织中的表达与浸润深度、淋巴结转移、TNM分期无关,而与肿瘤的分化程度相关,提示NAG-1mRNA的表达程度影响胃癌的分化。
2. NAG-1蛋白的表达与肿瘤分化程度相关,NAG-1蛋白质在胃癌组织中的表达与浸润深度、淋巴结转移、TNM分期无关,提示NAG-1蛋白质的表达可能影响胃癌的分化。
3. NAG-1mRNA在胃癌组织低于正常胃组织中的表达,NAG-1蛋白在胃癌组织中的表达明显低于它们在正常胃组织中的表达,提示NAG-1的表达改变可能与胃癌发生与发展有关。
4.成功构建pEGFP-N1/NAG-1重组质粒,为进一步研究NAG-1基因功能建立实验基础。
【Objective】:
1.To study the expression of nonsteroidal anti - inflammatory drug - activated gene-1 in human gastric cancer and its relationship with the clinical staging ,differntiation degree ,and lymphatic metastasis of gastric cancer.
2.To clone a fragment of NAG-1 gene into an expression vector pEGFP-N1 for expressing NAG-1 protein in vitro.
【Methods】:
1.The express of NAG-1 mRNA was detected by semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in the cancer tissues and corresponding adjacent normal tissues in 76 gastric cancer patients.
2.The expression of NAG-1 protein expression in human gastric carcinoma tissues and normal gastric tissues were detected by S-P immunohistochemistry.
3.Using nomal gastic tissue cDNA as template,amplified the NAG-1 gene with RT-PCR.Insert this fragment into the expression vector pEGFP-N1,and identified the positive clone with white and blue assay,endonuclease,PCR and DNA sequenceing identification.
【Results】:
1.NAG-1 mRNA was positive in both nomal tissues and gastric cancer tissues; expression in gastric cancer tissue significantly was lower than that in nomal tissues(0.5563±0.2181 vs 0.9059±0.0751,P<0.05).NAG-1mRNA expression in well and moderately differentiated cancer cells was significantly higher than those of poorly and non-differerntiated cancer cells(0.6701±0.1637 vs 0.4005±0.1859,p<0.05) , but the expression was not associated with the depth of tumor invasion,lymph node metastasis , and clinical stage (p>0.05).
2Immuno-histochemistry showed the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues (p<0.05);and the expression was not relationship with the gender, lymph node metastasis and clinical stage (all p >0.05).
3. The expression of NAG-1 mRNA in gastric cancer tissues was lower than that in nomal tissues ;the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues.they suggest that the expression of NAG-1 may relationship with the development and progression of gastic cancer.
4. The positive clons were identified with PCR,endonuclease and DNA sequencing identification,and they proved that the target fragment of NAG-1 gene had been inserted into expression vector pEGFP-N1 rightly.
【Conclusion】:
1. The expression of NAG-1mRNA in gastric carcinoma tissues and normal gastric tissues had statistical difference;The expression of NAG-1mRNA had no correlation with the depth of tumor invasion、lymph node metastasis and TNM stage. It is associated with the differentiation of tumor. That suggest the carcinogenesis、progression and invasion of gastric carcinoma had correlation with the NAG-1mRNA expression.
2. The expression of NAG-1 protein was significant lower in gastric carcinoma than normal gastric tissues. The expression of NAG-1 protein was associated with differentiation grades.That show NAG-1 protein suppressed the processes of carcinogenesis、progression and invasion of gastric carcinoma. Downregulation of NAG-1 protein promoted the processes of carcinogenesis、progression、invasion and metastasis of gastric carcinoma,Which made the gastric carcinoma cells more malignant and more invasive
3. Cloning NAG-1 recombinant plasmid lays the foundation for expressing NAG-1 protein in vitro.
引文
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[1] Kim KS,Baek SJ,Flake GP,et al. Expressinon and regulation of nnosterodial anti-inflammatory drug-activated gene(NAG-1) in human and mouse tissue Gastroenterology,2002,12:1388-1398
[2] Brown DA,Ward RL,Buckhaults P,Brown DA,et al.MIC-1 serum level and genotype:associations with progress and pognosis of colorectal carcinoma.[J]Clinical Cancer Res,2003,9:2642-2650.
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[5] Karan D,Chen SJ, Johansson SL,et al.Dysregulated expression of MIC-1/PDF in human prostate tumor cells.Biochem Biophys Res Commun,2003,598-604.
[6] Lee DH,Yang Y,Lee SJ,e tal.Macrophage inhibitory cytokine-1 induces the invasiveness of gastric cancer cells by up-regulating the urokinase-type plasminogen activator system, Cancer Res,2003,4648-4655.
[7] Park JY,Park KH.,Bang S,et al .Expression of nonsteroidal anti-infammatory drug-activated gene-1 (NAG-1) inversely correlates with tumor progression in gastric adenomas and carcinomas [J]Cancer Res Clin Oncol ,2008, 134: 1029–1035
[1] Kim S,Baek SJ, Flake GP, et al,Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in humanand mouse tissue.[J]. Gastroenterology, 2002;122:1388–98.
[2] Baek SJ , Kim KS , Nixon JB , et al . Cyclooxygenase inhibitors regulate the expression of a TGF -βsuperfamily member that has proapoptotic and antitumorigenic activities .[J ] Mol Pharmacol , 2001 , 59:901 - 908.
[3] Baek Sj and Horowitz Jm , Molecular cloning and characterization of human nonsteroridal anti-inflammatory drug-activated gene promoter.Basal transcription is mediated by Sp1 and Sp3.[ J] Biol chem,2001.33384-33392
[4] Julie AK, Lucia MS, James RL, et al. p53 controls prostate-derived factor/macrophage inhibitory cytokine/NSAID-activated gene-1 expression in response to cell density,DNA damage and hypoxia through diverse mechanisms,[J]Cancer Letters,2009, 277:38-47。
[5] Lim JH.,Park JW,Min DS. ,et al. NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells ,[J]Apoptosis ,2007,12:411–421
[6] Kathy KW, Cho CH and Ko KS.,A novel anticancer effect of Astragalus saponins: Transcriptional activation of NSAID-activated gene,[ J]. Cancer,2009, 125:1082–1091
[7] Pang RP , Zhou JG, Zeng ZR , et al .Celecoxib induces apoptosis in COX- 2 deficient human gastric cancer cells through Akt/GSK3β/NAG - 1 pathway [ J ] . Cancer Lett , 2007 , 251: 268 - 277.
[8] Shim M and Thomas E,Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism,[J]Mol Cancer Ther, 2008,7:421-428
[9] Shim M, Eling TE, Protein kinase C-dependent regulation of NAG-1/placental bone morphogenic protein/MIC-1 expression in LNCaP prostate carcinoma cells, J. Biol.Chem. 2005,280 :18636–18642.
[10] Lee SH, Cekanova M and Baek SJ,Multiple Mechanisms Are Involved in 6-Gingerol-Induced Cell Growth Arrest and Apoptosis in Human Colorectal Cancer Cells ,[J]mole carc ,2008,47:197–208
[11] Baek S.J, Kim J.S, Nixon J.B, et al.Expression of NAG-1, a transforming growth factor-beta superfamily member, by troglitazone requires the early growth response gene EGR-1,[ J]. Biol. Chem. 2004,279: 6883–6892.
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[1] Kim S,Baek SJ, Flake GP, et al,Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in humanand mouse tissue.[J]. Gastroenterology, 2002;122:1388–98.
[2] Baek SJ , Kim KS , Nixon JB , et al . Cyclooxygenase inhibitors regulate the expression of a TGF -βsuperfamily member that has proapoptotic and antitumorigenic activities .[J ] Mol Pharmacol , 2001 , 59:901 - 908.
[3] Baek Sj and Horowitz Jm , Molecular cloning and characterization of human nonsteroridal anti-inflammatory drug-activated gene promoter.Basal transcription is mediated by Sp1 and Sp3.[ J] Biol chem,2001.33384-33392
[4] Julie AK, Lucia MS, James RL, et al. p53 controls prostate-derived factor/macrophage inhibitory cytokine/NSAID-activated gene-1 expression in response to cell density,DNA damage and hypoxia through diverse mechanisms,[J]Cancer Letters,2009, 277:38-47。
[5] Lim JH.,Park JW,Min DS. ,et al. NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells ,[J]Apoptosis ,2007,12:411–421
[6] Kathy KW, Cho CH and Ko KS.,A novel anticancer effect of Astragalus saponins: Transcriptional activation of NSAID-activated gene,[ J]. Cancer,2009, 125:1082–1091
[7] Pang RP , Zhou JG, Zeng ZR , et al .Celecoxib induces apoptosis in COX- 2 deficient human gastric cancer cells through Akt/GSK3β/NAG - 1 pathway [ J ] . Cancer Lett , 2007 , 251: 268 - 277.
[8] Shim M and Thomas E,Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism,[J]Mol Cancer Ther, 2008,7:421-428
[9] Shim M, Eling TE, Protein kinase C-dependent regulation of NAG-1/placental bone morphogenic protein/MIC-1 expression in LNCaP prostate carcinoma cells, J. Biol.Chem. 2005,280 :18636–18642.
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[12] Hiroki Y, Hideki K,et al. Nonsteroidal Anti-inflammatory Drug-activated Gene(NAG-1/GDF15) Expression Is Increased by the Histone Deacetylase Inhibitor Trichostatin A,[J] bilo chem ,2008, 48:251-259
[13] Cekanova M, Lee SH, Donnell R, et al. Nonsteroidal Anti-inflammatory Drug-Activated Gene-1 Expression Inhibits Urethane-Induced Pulmonary Tumorigenesis in Transgenic Mice ,[J]Cancer Prev Res, 2009;2:231-239
[14] Jang TJ, Kang HJ, Kim JR ,et al. Non-steroidal anti-inflammatory drug activated gene (NAG-1) expression is closely related to death receptor-4 and -5induction, which may explain sulindac sulfide induced gastric cancer cell apoptosis,[J]Carcinogenesis,2004,.25:1853-1858.
[15] Liu T., Bauskin AR., Zaunders J, et al . Macrophage inhibitorycytokine 1 reduces cell adhesion and induces apoptosis in prostate cancer cells. [J]Cancer Res. 2003, 63:5034-5040.
[16] Kim KS, Baek SJ, Flake GP, et al. Expression and regulation of nonsteroidal anti-inflammatory drugactivated gene (NAG-1) in human and mouse tissue.[J] Gastroenterology ,2002;122:1388–1398.
[17] Park JY , Park KH , Bang S, et al, Expression of nonsteroidal anti-inXammatory drug-activatedgene-1 (NAG-1) inversely correlates with tumor progression in gastric adenomas and carcinomas, [J] Cancer Res Clin Oncol ,2008,134:1029–1035
[18] Newman D, Sakaue M, Koo JS, et al. Differential regulation of nonsteroidal anti-inflammatory drug-activated gene in normal human tracheobronchial epithelial and lung carcinoma cells by retinoids. [J]Mol Pharmacol,2003,63:557–564.
[19] Brown DA, Ward RL, Buckhaults P et al. MIC-1 serum level and genotype: associations with progress and prognosis of colorectal carcinoma. [J]Clin Cancer Res, 2003,9:2642–2650.
[20] Welsh JB, Sapinoso LM, Su AI et al. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.[J] Cancer Res 2001;61:5974–5978.
[21] Karan D, Chen SJ, Johansson SL, et al. Dysregulated expression of MIC-1/PDF in human prostate tumor cells. [J]Biochem Res,2003;305:598–604.