胃腺癌组织差异表达蛋白质的鉴定与分析
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摘要
细胞发生恶性变化具有复杂的分子机制,常涉及到多基因、多途径的病理改变过程,细胞总蛋白质表达谱的改变最能真实地反应细胞的这些病理变化。本研究着眼于胃腺癌组织总蛋白质表达谱的改变,应用蛋白质组学技术寻找与鉴定正常胃组织与胃腺癌组织之间差异表达的蛋白质,从而选择其中一种对其致癌作用文献报道很少以及功能未明确的差异表达的蛋白质,应用western blot和免疫组化方法,探索它作为胃腺癌生物标志物的可能性,并利用基因克隆技术构建它的真核表达载体、利用基因转染技术初步探讨它的致胃癌作用。
本研究第一部分从临床获得10例胃腺癌患者的胃腺癌组织及与其配对的相同患者的正常胃粘膜组织,首先应用双相电泳技术对正常胃组织及胃腺癌组织的总蛋白质表达谱进行了对比分析,发现了42个表达差异至少两倍的蛋白质点;接着应用质谱技术对差异蛋白质点进行分析,成功地鉴定出42种不同的蛋白质。其中,胃腺癌组织表达下调的有垂体腺苷酸环化酶激活肽(PACAP protein,PACAP)等29种;胃腺癌组织表达上调的有波形蛋白(vimentin)等13种。
本研究第二部分应用western blot和免疫组化方法进一步验证已经质谱鉴定的差异表达的蛋白质:胃腺癌组织表达降低的人类蛋白酶体激活剂PA28β亚单位(proteasome activator hPA28 suunit beta,PA28β)。应用这两种方法分别检测40例冻存和石蜡包埋的胃腺癌组织及与其配对的正常胃组织PA28β的表达。发现PA28β在28例胃腺癌组织中低表达,低表达率为70.0%(28/40,western blot法);免疫组化结果也显示,PA28β在38例正常胃黏膜固有层胃腺细胞中表达,但在29例胃腺癌细胞中,PA28β表达降低或缺乏表达(29/41,72.5%)。初步结果表明PA28β可作为胃腺癌的生物标志物。
本研究的第三部分对PA28β的致胃癌作用进行初步探讨。首先通过RT-PCR,获得PA28β全长cDNA序列,构建PA28β基因的真核表达载体,并转染入胃腺癌MKN-45细胞中。通过MTT测定方法、Brdu掺入实验和克隆形成实验,研究转染PA28β基因前后细胞增殖的变化;应用软琼脂集落形成实验研究转染PA28β基因前后MKN-45细胞的致瘤性(恶性度)的变化。结果显示MKN-45细胞转染PA28β基因后,细胞增殖减弱,致瘤性减弱,统计学结果表明与未转染对照及空载体对照比较,差异非常显著(P<0.01)。初步结果提示:PA28β可导致细胞增殖和恶性程度的变化,其表达下降与胃腺癌的发生发展有关,相关机制有待进一步研究。
It has been well established that cancer development possesses complicated molecular mechanism, which usually involves multiple genetic changes and effects from the environment. This complicated interplay of factors leading to neoplastic progression is ultimately reflected in the protein expression profiles of cells. In this study, we tried to identify the change of protein expression profiles of gastric adenocarcinomas (GA) with the purpose to find differentially expressed proteins between cancer and normal mucosa tissue using a proteomics approach, thereby to identify a cancer associated protein which was few investigated previously for further study. The further study included to validate it as a potential candidate of GA biomarker using both western blot and immunohistochemistry assays, and to preliminarily investigate its role in the carcinogenesis of GA using a gene cloning techniques by constructing its mammalian expression vector, and then using a gene transfection technique to introduce it into cells.
In the first part of this study, ten protein expression profiles of GA and paired non-neoplastic mucosa tissues were analyzed by 2-dimensional gel electrophoresis (2-D PAGE) . Forty-two protein spots that were differentially expressed by 2-fold or greater between cancer and normal mucosa tissue were excised and identified by MALDI-TOF mass spectrometry(MS). This generated 42 distinct proteins that were differentially expressed at least two-fold between the tissues. 29 of these proteins displayed decreased expression in cancer tissue, such as PACAP protein; while 13 were over expressed in the cancer tissue, such as vimentin.
In the second part of this study, human proteasome activator PA28 suunit beta (PA28β) which was decreased expression in GA tissue observed by 2-D PAGE and identified by MS was chosen for further verification using both western blot and immunohistochemistry assays. 40 cases GA and paired non-neoplastic tissues were examined by both the assays with an anti PA28βantibody. PA28βwas found down-expressed in 28 samples of cancer tissue (28/40, 70.0%) when detected by western blot. The results of immunohistochemistry confirmed that PA28βwas expressed in 38 cases of paired non-neoplastic tissues with positive cytoplasmic staining in gastric gland cells of lamina propria of gastric mucosa of these samples, and was indeed down-expressed or absent in 29 cases of GA (29/40, 72.5%). The preliminary results indicate PA28βmay be used as a novel biomarker for GA.
In the third part of this study, the role of PA28βin the carcinogenesis of GA was investigated. The full length cDNA sequences of PA28βwere obtained by RT-PCR. pcDNA3.1-PA28βmammalian expression vector was constructed, and was transfected into MKN-45 gastric adenocarcinomas cells. The activity of cell proliferation before and after transfected with PA28βgene was detected by MTT assay, Brdu labeling assay and colony formation assay. The tumorigenicity of the MKN-45 cells before and after transfected with PA28βgene was tested by the colony formation in soft agar assay. The results showed that both the proliferation activity and the tumorigenicity of MKN-45 cells were decreased after the cells had been transfected with PA28βgene. The analystical results of statistics indicated the discrepancy was very significant between MKN-45 cells transfected with pcDNA3.1-PA28βvector and pcDNA3.1/hygro(+) empty vector (P<0.01), and also between MKN-45 cells transfected with pcDNA3.1- PA28βvector and untransfected MKN-45 cells(P<0.01). The preliminary results indicate PA28βmight cause the changes of cell proliferation activity and tumorigenicity, and it participates the carcinogenesis of GA with an unclear molecular mechanism which needs to be further investigated.
本研究第一部分从临床获得10例胃腺癌患者的胃腺癌组织及与其配对的相同患者的正常胃粘膜组织,首先应用双相电泳技术对正常胃组织及胃腺癌组织的总蛋白质表达谱进行了对比分析,发现了42个表达差异至少两倍的蛋白质点;接着应用质谱技术对差异蛋白质点进行分析,成功地鉴定出42种不同的蛋白质。其中,胃腺癌组织表达下调的有垂体腺苷酸环化酶激活肽(PACAP protein,PACAP)等29种;胃腺癌组织表达上调的有波形蛋白(vimentin)等13种。
本研究第二部分应用western blot和免疫组化方法进一步验证已经质谱鉴定的差异表达的蛋白质:胃腺癌组织表达降低的人类蛋白酶体激活剂PA28β亚单位(proteasome activator hPA28 suunit beta,PA28β)。应用这两种方法分别检测40例冻存和石蜡包埋的胃腺癌组织及与其配对的正常胃组织PA28β的表达。发现PA28β在28例胃腺癌组织中低表达,低表达率为70.0%(28/40,western blot法);免疫组化结果也显示,PA28β在38例正常胃黏膜固有层胃腺细胞中表达,但在29例胃腺癌细胞中,PA28β表达降低或缺乏表达(29/41,72.5%)。初步结果表明PA28β可作为胃腺癌的生物标志物。
本研究的第三部分对PA28β的致胃癌作用进行初步探讨。首先通过RT-PCR,获得PA28β全长cDNA序列,构建PA28β基因的真核表达载体,并转染入胃腺癌MKN-45细胞中。通过MTT测定方法、Brdu掺入实验和克隆形成实验,研究转染PA28β基因前后细胞增殖的变化;应用软琼脂集落形成实验研究转染PA28β基因前后MKN-45细胞的致瘤性(恶性度)的变化。结果显示MKN-45细胞转染PA28β基因后,细胞增殖减弱,致瘤性减弱,统计学结果表明与未转染对照及空载体对照比较,差异非常显著(P<0.01)。初步结果提示:PA28β可导致细胞增殖和恶性程度的变化,其表达下降与胃腺癌的发生发展有关,相关机制有待进一步研究。
It has been well established that cancer development possesses complicated molecular mechanism, which usually involves multiple genetic changes and effects from the environment. This complicated interplay of factors leading to neoplastic progression is ultimately reflected in the protein expression profiles of cells. In this study, we tried to identify the change of protein expression profiles of gastric adenocarcinomas (GA) with the purpose to find differentially expressed proteins between cancer and normal mucosa tissue using a proteomics approach, thereby to identify a cancer associated protein which was few investigated previously for further study. The further study included to validate it as a potential candidate of GA biomarker using both western blot and immunohistochemistry assays, and to preliminarily investigate its role in the carcinogenesis of GA using a gene cloning techniques by constructing its mammalian expression vector, and then using a gene transfection technique to introduce it into cells.
In the first part of this study, ten protein expression profiles of GA and paired non-neoplastic mucosa tissues were analyzed by 2-dimensional gel electrophoresis (2-D PAGE) . Forty-two protein spots that were differentially expressed by 2-fold or greater between cancer and normal mucosa tissue were excised and identified by MALDI-TOF mass spectrometry(MS). This generated 42 distinct proteins that were differentially expressed at least two-fold between the tissues. 29 of these proteins displayed decreased expression in cancer tissue, such as PACAP protein; while 13 were over expressed in the cancer tissue, such as vimentin.
In the second part of this study, human proteasome activator PA28 suunit beta (PA28β) which was decreased expression in GA tissue observed by 2-D PAGE and identified by MS was chosen for further verification using both western blot and immunohistochemistry assays. 40 cases GA and paired non-neoplastic tissues were examined by both the assays with an anti PA28βantibody. PA28βwas found down-expressed in 28 samples of cancer tissue (28/40, 70.0%) when detected by western blot. The results of immunohistochemistry confirmed that PA28βwas expressed in 38 cases of paired non-neoplastic tissues with positive cytoplasmic staining in gastric gland cells of lamina propria of gastric mucosa of these samples, and was indeed down-expressed or absent in 29 cases of GA (29/40, 72.5%). The preliminary results indicate PA28βmay be used as a novel biomarker for GA.
In the third part of this study, the role of PA28βin the carcinogenesis of GA was investigated. The full length cDNA sequences of PA28βwere obtained by RT-PCR. pcDNA3.1-PA28βmammalian expression vector was constructed, and was transfected into MKN-45 gastric adenocarcinomas cells. The activity of cell proliferation before and after transfected with PA28βgene was detected by MTT assay, Brdu labeling assay and colony formation assay. The tumorigenicity of the MKN-45 cells before and after transfected with PA28βgene was tested by the colony formation in soft agar assay. The results showed that both the proliferation activity and the tumorigenicity of MKN-45 cells were decreased after the cells had been transfected with PA28βgene. The analystical results of statistics indicated the discrepancy was very significant between MKN-45 cells transfected with pcDNA3.1-PA28βvector and pcDNA3.1/hygro(+) empty vector (P<0.01), and also between MKN-45 cells transfected with pcDNA3.1- PA28βvector and untransfected MKN-45 cells(P<0.01). The preliminary results indicate PA28βmight cause the changes of cell proliferation activity and tumorigenicity, and it participates the carcinogenesis of GA with an unclear molecular mechanism which needs to be further investigated.
引文
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18. 邹建湘 陈玉龙 王立东. 胃癌发病机制的研究. 新消化病学杂志,1997;5(3):180-181
19. Stadtlander CTK-H and Waterbor JW. Molecular epidemiology, pathogenesis and prevention of gastric cancer. Carcinogenesis. 1999, 20:2195-2207
20. Kakeji Y, Maehara Y, Koga T et al. Gastric cancer with high telomerase activity shows rapid development and invasiveness. Oncol Rep 2001, 8:107-110.
21. Watari J, Tanaka A, Tanabe H, et al. K-ras mutations and cell kinetics in Helicobacter pylori associated gastric intestinal metaplasia: a comparison before and after eradication in patients with chronic gastritis and gastric cancer. J Clin Pathol. 2007,60:921-926.
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24. Yano T, Doi T, Ohtsu A, et al. Comparison of HER2 gene amplification assessed by fluorescence in situ hybridization and HER2 protein expression assessed by immunohistochemistry in gastric cancer. Oncol Rep. 2006,15(1):65-71
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7. Danesh J. Helicobacter pylori and gastric cancer: systematic review of the epidemiological studies. Aliment Pharmacol Ther 1999, 13(7):851-856
8. Juhasz M, Herszenyi L, Tulassay Z, et al. Helicobacter pylori and molecular mechanisms of gastric carcinogenesis: targets for prevention and therapy. Expert Rev Anticancer Ther 2004, 97-103.
9. Crabtree JE, Wyatt JI, Trejdosiewicz LK et al. Interleukin 8 expression in Helicobacter pylori infected, normal and neoplastic gastroduodenal mucosa. J Clin Pathol 1994, 47(1):61-66.
10. Tsuji S, Kawano S, Tsujii M et al. Helicobacter pylori stimulates inflammatory nitric oxide production. Cancer Lett 1996, 108(2):195-200。
11. Schandl L, Malfertheiner P, Ebert MP. Prevention of gastric cancer by Helicobacter pylori eradication? Results from clinical intervention studies. Digest.Dis. 2002, 20:18-22.
12. Hsing AW, Hansson LE, Mclaughlin JK, et al. Pernicious anemia and subsequent cancer: a population based cohort study. Cancer, 1993:71:745-750.
13.International Agency for Research on Cancer Infection with Helicobacter pylori In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol.61, Schistosomes, Liver Flukes and . Helicobacter pylori IARC, Lyon, 1994, p177-240.
14. Sasaki H, Igaki H, Ishizuka T, et al. Presence of Streptococcus DNA sequence in surgical specimens of gastric cancer. Jpn J Cancer Res 1995, 86:791-794.
15. Shibata D and Weiss LM. Epstein-Barr virus-associated gastric adenocarcinoma. Am J Pathol 1992, 140:769-774.
16. Stalnikowicz R and Benbassat J. Risk of gastric cancer after gastric surgery for benign disorders. Arch Intern Med 1990, 150:2022-2026
17. Ilson DH. Adjuvant therapy for noncolorectal cancers. Curr Opin Oncol 2001, 13:287-290.
18. 邹建湘 陈玉龙 王立东. 胃癌发病机制的研究. 新消化病学杂志,1997;5(3):180-181
19. Stadtlander CTK-H and Waterbor JW. Molecular epidemiology, pathogenesis and prevention of gastric cancer. Carcinogenesis. 1999, 20:2195-2207
20. Kakeji Y, Maehara Y, Koga T et al. Gastric cancer with high telomerase activity shows rapid development and invasiveness. Oncol Rep 2001, 8:107-110.
21. Watari J, Tanaka A, Tanabe H, et al. K-ras mutations and cell kinetics in Helicobacter pylori associated gastric intestinal metaplasia: a comparison before and after eradication in patients with chronic gastritis and gastric cancer. J Clin Pathol. 2007,60:921-926.
22. Kim IJ, Park JH, Kang HC, et al. Mutational analysis of BRAF and K-ras in gastric cancers: absence of BRAF mutations in gastric cancers. Hum Genet. 2003,114(1):118-120.
23. Kozma L, Kiss I, Hajdú J, et al. C-myc amplification and cluster analysis in human gastric carcinoma. Anticancer Res. 2001, 21(1B):707-710.
24. Yano T, Doi T, Ohtsu A, et al. Comparison of HER2 gene amplification assessed by fluorescence in situ hybridization and HER2 protein expression assessed by immunohistochemistry in gastric cancer. Oncol Rep. 2006,15(1):65-71
25. Belyavskaya VA, Vardosanidze VK, Smirnova OY, et al. Genetic status of p53 in stomach cancer: somatic mutations and polymorphism of codon 72. Bull Exp Biol Med. 2006,141(2):243-246.
26. Al-Moundhri MS, Nirmala V, Al-Hadabi I, et al. The prognostic significance of p53, p27 kip1, p21 waf1, HER-2/neu, and Ki67 proteins expression in gastric cancer: a clinicopathological and immunohistochemical study of 121 Arab patients. J Surg Oncol. 2005,91(4):243-252.
27. Cho JH, Noguchi M, Ochiai A, et al. Loss of heterozygosity of multiple tumor suppressor genes in human gastric cancers by polymerase chain reaction. Lab Invest. 1996,74 (4):835-341.
28. 武和平. 胃癌的研究近况. 中华现代内科学杂志. 2007, 6(4):517-519.
29. Yoo NJ, Lee SH, Jeong EG, et al. Expression of phosphorylated caspase-9 in gastric carcinomas. APMIS. 2007,115(4):354-359.
30. Rodriguez-Sanjuan JC, Fontalba A, Mayorga M, et al. A novel mutation in the E-cadherin gene in the first family with hereditary diffuse gastric cancer reported in Spain. Eur J Surg Oncol. 2006,32(10):1110-1113.
31. Oshima CT, Wonraht DR, Catarino RM, et al. Estrogen and progesterone receptors in gastric and colorectal cancer. Hepatogastroenterology. 1999,46 (30):3155-3158.
32. Krupp G, Bonatz G, Parwaresch R. Telomerase, immortality and cancer. Biotechnol. Ann Rev, 2000, 6:103-140.
33. Beghelli S, de Manzoni G, Barbi S, et al. Microsatellite instability in gastric cancer is associated with better prognosis in only stage II cancers. Surgery. 2006,139 (3):347-356.
34. Regalo G, Canedo P, Suriano G, et al. C/EBPbeta is over-expressed in gastric carcinogenesis and is associated with COX-2 expression. J Pathol. 2006, 210(4):398-404
35. Wu CY, Wu MS, Chen YJ, et al. Clinicopathological significance of MMP-2 and TIMP-2 genotypes in gastric cancer. Eur J Cancer. 2007, 43 (4):799-808.