【作者】 刘震雄；

【导师】 吴开春； 王新；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 【背景】胃癌是世界范围尤其是亚洲地区的常见恶性肿瘤,其发病机制目前尚不完全明确。研究显示,环氧合酶-2(Cyclooxygenase 2, COX-2)在多数胃癌组织及细胞系中表达异常增高,它可以促进胃癌侵袭转移及血管的形成,并且与胃癌的低生存率密切相关。而抑制COX-2的活性可以抑制胃癌相关的血管生成,而且能抑制胃癌细胞增殖,使凋亡增加,体内成瘤减小,前列腺素合成受到抑制。这提示COX-2与胃癌的发生发展关系密切,但其机制并不清楚。蛋白质组学是近年来出现的能够全貌性研究差异蛋白质表达的方法,双向电泳联合质谱鉴定技术是目前蛋白质组学研究的主流技术[1],能够有效地比较不同蛋白质组之间的差异,是查找特征性功能蛋白质、筛选蛋白效应分子和疾病相关标志物的有力手段。在本课题研究中,我们利用蛋白质组学的方法对COX-2siRNA及空载体转染的SGC7901人胃癌细胞系进行比较蛋白质组学分析,以筛选与COX-2导致胃癌发生、发展密切相关的蛋白质,并对下游分子进行功能鉴定,还利用全基因组芯片和转录因子芯片对COX-2的下游效应分子进行了高通量的筛选,从而为揭示COX-2在胃癌发生发展中的作用机理、确定新的胃癌基因治疗靶点及提供新的胃癌疫苗候选分子提供坚实的实验基础和理论依据。【目的】1、筛选COX-2促进胃癌发生发展的相关分子;2、研究目的分子15-羟基前列腺素脱氢酶与COX-2在胃癌中的相关性;3、研究15-羟基前列腺素脱氢酶在胃癌中的表达与临床病理特征及临床分期的关系,探讨其在胃癌发生和进展中的意义;4、观察15-羟基前列腺素脱氢酶对胃癌细胞恶性表型的影响。【方法】1、采用双向凝胶电泳技术、银染显色及PDQuest软件分析筛选COX-2siRNA及空载体转染的SGC7901胃癌细胞系中差异表达的蛋白质,而后用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对表达存在显著性变化的蛋白质进行检测,将所得蛋白质肽质量指纹图谱(PMF)行生物信息学分析;2、应用Western blot和免疫细胞化学的方法检测COX-2和15-PGDH在COX-2siRNA载体和COX-2全长载体转染的SGC7901胃癌细胞系中的表达;3、采用免疫组织化学和Western blot技术检测COX-2和15-PGDH在胃癌组织中的表达特征;4、分析COX-2与15-PGDH表达的相关性以及它们的表达与临床病理参数的关系;5、通过基因重组方法构建15-PGDHsiRNA及15-PGDH全长载体,并分别稳定转染MKN45和SGC7901胃癌细胞系;6、应用MTT实验、流式细胞术、平板克隆实验、软琼脂克隆形成实验以及裸鼠体内成瘤实验的方法研究15-PGDH对胃癌细胞生长和体内成瘤能力的影响;7、采用全基因组表达谱芯片和转录因子活性谱芯片筛选COX-2下游效应分子。【结果】1、蛋白质组学筛选出14个差异蛋白质Western blot技术检测COX-2siRNA载体转染SGC7901胃癌细胞后COX-2的表达,结果显示,COX-2siRNA能显著下调COX-2的表达,其中以第1号siRNA的抑制效率最高。因此,我们采用第1号siRNA转染的SGC7901胃癌细胞系作为研究对象。将细胞系样品总蛋白质进行双向电泳,通过软件分析后,对22个表达明显改变的蛋白质点用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析鉴定,获得14个蛋白质的相应肽质量指纹图谱(PMF)。其中,对比空载体转染的SGC7901细胞,COX-2siRNA转染的SGC7901细胞系中有7个蛋白显著下调,7个蛋白显著上调。对PMF进行数据库检索,生物信息学分析鉴定出在COX-2siRNA转染的SGC7901细胞系中下调的7个蛋白质是:细胞分裂周期蛋白16(Cell division cycle protein 16),肌动蛋白γ(Gamma Actin),14-3-3蛋白σ(14-3-3 protein sigma),波形蛋白(Vimentin),71kDa热休克同源蛋白(Heat shock cognate 71kDa protein),组胺-N-甲基转移酶(Histamine N-methyltransferase) , Ras相关蛋白Rab-3B(Ras-related protein Rab-3B);而上调的7个蛋白质是:乳酸酰谷胱苷肽裂解酶(Lactoylglutathione lyase) , 15-羟基前列腺素脱氢酶(15-hydroxyprostaglandin dehydrogenase [NAD+]),补体成分1q亚成分结合蛋白(Complement component 1q subcomponent binding protein),生长分化因子2(Growth differentiation factor 2),热休克70kDa蛋白5(Heat shock 70kDa protein 5),磷脂酰肌醇转移蛋白β亚型(Phosphatidylinositol transfer protein beta isoform),应激蛋白70(Stress-70 protein)。其中15-羟基前列腺素脱氢酶(15-PGDH)是在COX-2siRNA转染的胃癌细胞中显著上调的蛋白质之一,我们用Western blot及免疫细胞化学检测证实了这一结果。2、15-PGDH在胃癌中表达降低并与COX-2表达呈负相关我们应用Western blot技术检测8例胃癌手术组织标本中COX-2与15-PGDH的表达,发现与相应的癌旁组织相比,在胃癌组织中COX-2表达显著升高,而15-PGDH的蛋白表达水平明显降低甚至缺失;我们应用免疫组化技术检测15-PGDH在55例胃癌患者手术标本的连续石蜡切片中的表达,发现与正常胃粘膜上皮相比,15-PGDH在胃癌组织中表达显著降低甚至缺失,而COX-2表达显著增高;进一步分析COX-2及15-PGDH的表达与胃癌临床病理特征的关系,发现15-PGDH在低分化胃癌中明显低于高分化胃癌中的表达(p<0.05);15-PGDH在TNMⅢ期和Ⅳ期患者的胃癌组织中的染色明显低于于在Ⅰ期和Ⅱ期组织中的表达(p<0.05);存在淋巴结转移的胃癌组织中15-PGDH表达显著低于无淋巴结转移胃癌组织中的表达(p<0.05),而COX-2的表达与15-PGDH的表达模式相反。Spearsman相关性统计学分析显示15-PGDH的表达与COX-2呈显著负相关(rs=-0.564, p<0.01)。Western blot和免疫细胞化学的方法检测COX-2正义表达载体转染的SGC7901胃癌细胞系COX-2和15-PGDH的表达,结果显示COX-2正义载体能显著提高COX-2的表达水平,而15-PGDH在COX-2正义转染的胃癌细胞中显著下调。结果表明胃癌组织中15-PGDH的表达显著降低,在低分化、TNM晚期和有淋巴结转移的患者中表达更低,其表达与COX-2呈显著负相关,且上调COX-2能抑制15-PGDH的表达,提示在胃癌中15-PGDH受到了COX-2的负调节。3、外源性高表达15-PGDH可以降低胃癌细胞恶性表型,15-PGDH的特异性小干扰RNA可以促进胃癌细胞增殖。Western blot检测了SGC7901、AGS、BGC823、MKN45、MKN28五种胃癌细胞系和永生化正常胃粘膜上皮细胞GES中15-PGDH的表达,结果显示15-PGDH在胃癌细胞系中的表达均低于GES细胞,在MKN45细胞中的表达相对较高,在SGC7901细胞中表达相对更低。我们用以色列的Ido Wolf教授惠赠的15-PGDH正义表达载体稳定转染了SGC7901细胞,该正义载体能显著提高SGC7901中15-PGDH的表达。15-PGDH的正义转染可使SGC7901细胞体外增殖减慢,在平板和软琼脂上形成克隆的能力降低,裸鼠体内成瘤性也降低,瘤体积明显减小,生长减缓,流式细胞仪检测显示细胞阻滞于G1期。我们构建了15-PGDH的siRNA表达载体,并稳定转染了MKN45细胞,发现该siRNA能显著抑制15-PGDH的表达,并能够明显促进MKN45在体外的生长增殖,在平板和软琼脂中形成克隆的能力及裸鼠体内的成瘤能力增强。上述结果提示,15-PGDH参与了胃癌细胞的生长、增殖等多种生物学行为的变化,且提高15-PGDH的表达能降低胃癌细胞的恶性表型。4、差异表达的转录因子及差异表达基因的筛选利用核蛋白提取试剂盒(Pierce biotechnology)分别抽提COX-2siRNA和空载体转染的SGC7901胃癌细胞的核蛋白,并用核酸蛋白检测仪测定蛋白浓度。经转录因子活性谱芯片分析,以变化倍数1.5倍为标准判定差异表达的转录因子。结果发现,与对照细胞相比,SGC7901-COX-2/siRNA细胞中上调表达的转录因子有4个,下调表达的转录因子有2个。经过Trizol法抽提COX-2siRNA和空载体转染的SGC7901胃癌细胞的总RNA,检测显示所提取的RNA符合基因芯片测定的要求。经芯片杂交、图像采集及数据分析等步骤,以变化倍数2倍为标准判定差异表达基因。结果发现,与对照细胞相比,SGC7901-COX-2/siRNA细胞中上调表达的基因有1040个,下调表达的基因有234个。【结论】1、本研究通过比较蛋白组学技术筛选出不同COX-2表达水平胃癌细胞系的差异蛋白质,这些蛋白质涉及细胞周期调控、细胞运动、细胞分化、细胞增殖及蛋白质转运等诸多功能,可能在胃癌的发生、发展及其转移过程中起到重要的作用;2、进一步对差异蛋白质15-PGDH的研究显示15-PGDH在胃癌的发生和进展中起着抑癌基因的作用,并且受到COX-2的负调控,而且转染15-PGDH全长的表达载体能够提高胃癌细胞中15-PGDH的表达并在一定程度上逆转胃癌细胞的恶性表型;3、用基因芯片和转录因子芯片筛选出不同COX-2表达水平胃癌细胞系的差异表达基因及转录因子,对这些差异表达分子进一步研究将对揭示COX-2促进胃癌的机制研究有着重要意义。更多还原

【Abstract】 【Background】Gastric cancer is one of the most common malignant tumors in the world, especialy in Asia. But the pathogenesy is still unclear. COX-2, frequently detected over-expressed in gastric cancer tissues and cell lines, was believed to play a crucial role in promotion of proliferation and angiogenesis in gastric cancer. Up-regulation of COX-2 might facilitate invasion of gastric cancer and was significantly related to the low survival rate of gastric cancer patients. Reduced COX-2 activity could result in decreased angiogenesis, increased apoptosis and suppressed synthesis of prostaglandins. COX-2 was closed related to gastric carcinogenesis and progression, however, the underlying mechanisms are not fully understood. The proteomics approach provides a new tool to comparative studies of protein expression levels. Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis (MALDI-TOF MS) are principal techniques which could efficiently find differential expressing proteins and screen out the disease related markers. In this study, proteomic strategy was used to compare the differential expressing proteins between COX-2 siRNA stably transfected gastric cancer cells SGC7901 and the mock vector transfected SGC7901 cells. COX-2 modulated proteins were screened out and follow-up verification and functional studies were carried out to elucidate the mechanism of COX-2 related gastric carcinogenesis and progression, and provide solid experimental basis for the development of novel targets for the gastric cancer vaccine and gene therapy.【Objectives】(1) To screen out the COX-2 related molecules in gastric carcinogenesis and progression. (2) To investigate the relation between 15-PGDH and COX-2 in gastric cancer. (3) To study the expression status of 15-PGDH in gastric cancer and correlate it with clinicopathological parameters. (4) To explore the role of 15-PGDH in the malignant behavior of gastric cancer.【Methods】(1) 2-DE and the PDQuest software analysis were applied to compare the differential expression proteins of SGC7901 transfected with COX-2 siRNA vector. The differential protein dots were excised and further analyzed by MALDI-TOF-MS. Corresponding peptide mass figureprints (PMFs) were analyzed by bioinformatics analysis. (2) COX-2 and 15-PGDH expression in COX-2 siRNA vector transfectants and full-length cDNA vector transfectants were determined by Western blot and immunocytochemistry assay. (3) Immunohistochemistry and Western blot were used to determine the expression status of both COX-2 and 15-PGDH in gastric cancer tissues. (4) Statistic analyses were explored to value the correlation of COX-2 and 15-PGDH expression and their relations with the clinicopathological parameters. (5) 15-PGDHsiRNA vector and sense expression plasmid was constructed and transfected into the gastric cancer cell line MKN45 and SGC7901 with the mock vector as the control. The stable transfectants were screened out successfully. (6) The effects of 15-PGDH on the proliferation and in vivo tumor growth of gastric cancer cells were respectively investigated by MTT assay, plan plate colony formation assay, soft agar colony formation assay, flow cytometry and nude mice tumoration. (7) Screening candidate molecules regulated by COX-2 with oligonucleotide array-based transcription factor assay (OTAFA) and gene expression profiling microarray.【Results】1. 14 differential expressed proteins were identified by proteomics.Western blotting analysis confirmed that the stable SGC7901 clones transfected with COX-2siRNA showed lower COX-2 expression compared to empty vector-transfected SGC7901-pSilencer cells. Among them, the COX-2 expression of the first stable clone (siRNA1) was the lowest in all clones, so the first stable clone was used to do the further study. 2-DE was used to identify the differentially expressed proteins in the two cell lines. Twenty-two differentially expressed protein spots have been identified and subjected to MALDI-TOF MS. Fourteen PMFs were obtained and submitted to database query. Compared with SGC7901-pSilencer, the expressional levels of 7 proteins, namely Cell division cycle protein 16 homolog (CDC16), Actin cytoplasmic 2 (ACTG), 14-3-3 protein sigma (1433S), Vimentin (VIME), Heat shock cognate 71kDa protein (HSP7C), Histamine N-methyltransferase (HNMT) and Ras-related protein Rab-3B (RAB3B) decreased in SGC7901-COX-2/siRNA, while the expression levels of the other 7 proteins, namely Lactoylglutathione lyase (LGUL), 15-hydroxyprostaglandin dehydrogenase [NAD+] (15-PGDH), Complement component 1 Q subcomponent-binding protein precursor (C1QBP), Growth differentiation factor 2 precursor (GDF2), Heat shock 70kDa protein 5 (GRP78), Phosphatidylinositol transfer protein beta isoform (PIPNB) and Stress-70 protein (GRP75) increased in SGC7901-COX-2/siRNA. Further Western blot and immunocytochemical assay verified that the protein level of 15-PGDH was much higher in SGC7901-COX-2/siRNA with the lower expression of COX-2 than that in SGC7901-pSilencer.2. 15-PGDH expression was down-regulated in gastric cancer tissues and was negatively correlated to COX-2 expression.Expression levels of COX-2 and 15-PGDH were examined by Western blotting in gastric cancer tissues and adjacent non-tumor tissues taken from 8 patients. 15-PGDH expression was found decreased or even absent while COX-2 was over-expressed in gastric cancer tissues compared with the corresponding non-tumor tissues. Meanwhile, immunohistochemistry was conducted in 55 cases of gastric cancer specimens and normal mucosa. 15-PGDH expression was also found decreased or even absent while COX-2 was over-expressed in gastric cancer tissues compared with the corresponding non-tumor tissues. Further analysis about their relationship with clinicopathological parameters revealed that the level of 15-PGDH was significantly lower in patients of III-IV stage than in I-II stage (p<0.05). Its expression had significant difference among differentiation grade (p<0.05) and was related with lymph node metastasis (p<0.01). However, COX-2 expression just had the opposite pattern. Spearman analysis showed that significant negative correlation between COX-2 and 15-PGDH immunoreactivity with rs=-0.564 (p<0.01). Western blotting analysis confirmed that the stable SGC7901clones transfected with pcDNA3.1-COX-2 showed higher COX-2 expression and much lower 15-PGDH compared to empty vector-transfected SGC7901 cells. All these results demonstrated that 15-PGDH was down-regulated in gastric cancer tissues, especially decreased in those cases of lowly differentiation, late TNM stage and with lymph node metastasis. Expression of 15-PGDH has a negative correlation with that of COX-2. Up-regulation of COX-2 could suppress the expression of 15-PGDH in vitro, which was consistent with above findings by 2-DE.3. Exogenous upregulation of 15-PGDH could partially reverse the malignant phenotype of gastric cancer while knock-down of its expression by siRNA stimulate proliferation in gastric cancer cells.15-PGDH expression was detected in 5 kinds of human gastric cancer cell lines (SGC7901、AGS、BGC823、MKN45、MKN28) and human normal gastric epithelial cell line GES. Its expression decreased in all the gastric cancer cell lines compared to GES, especially with low level in SGC7901 cells. cDNA eukaryotic expression vector from Prof. Ido Wolf (Israel) was stably transfected into SGC7901 in order to up-regulate 15-PGDH expression. SGC7901 cells stably transfected with the 15-PGDH cDNA were found to exhibit significantly lower rate of proliferation than empty vector transfectants. 15-PGDH suppresses tumor cell growth in soft agar and tumor formation in athymic nude mice. Flow cytometry detected more cells blocked in G1 stage. Meanwhile, 15-PGDH siRNA vector was constructed and stably transfected into MKN45 cells which expressed relatively high level of 15-PGDH. MKN45 cells stably transfected with the 15-PGDHsiRNA were found to exhibit significantly proliferation and stimulate growth of MKN45 cells in soft agar and tumor formation in athymic nude mice than empty vector transfectants. In conclusion, 15-PGDH participates in many gastric cancer malignant behaviors such as proliferation. Exogenous upregulation of 15-PGDH could partially reverse the gastric cancer malignant phenotype.4. The candidate molecules regulated by COX-2 were screened with oligonucleotide array-based transcription factor assay (OTAFA) and gene expression profiling microarray.Nucleoprotein was extracted from the SGC7901 cells transfected with COX-2siRNA plasmid or empty vector with the kit of Pierce biotechnology. OATFA was then performed to detect the activity variation of transcription factors. Spot intensity 1.5 fold increased or decreased was used as the criteria for differential expression of transcription factors. Compared with the control cells, 4 up-regulated transcription factors and 2 down-regulated transcription factors were detected in SGC7901-COX-2/siRNA cells.Total RNA of SGC7901-COX-2/siRNA and SGC7901-pSilence cells was prepared using Trizol according to the manufacturer’s instructions. After array hybridization, image scanning and data analysis, candidates with the filtering criteria of intensity 2 fold increased or decreased were selected out. Compared to the control cells, 1040 up-regulated genes and 234 down-regulated genes were found in SGC7901-COX-2/siRNA cells.【Conclusions】(1) Differential expression proteins related to COX-2 mediated gastric carcinogenesis and progression were screened out. These proteins function in the process of cell cycle regulation, motility, differentiation, apoptosis, proliferation as well as protein transportation, and may play critical roles in gastric carcinogenesis, progression and metastasis. (2) Follow-up verification and functional study revealed that 15-PGDH is negatively regulated by COX-2, and may play its role in gastric carcinogenesis and progression as an onco-suppressor. Up-regulation of 15-PGDH expression by sense transfection could partially reverse the malignant phenotype of gastric cancer. (3) Differential expression genes and transcription factors were successfully screened out by gene expression profiling microarray and OTAFA. Further studies may yield novel clues for elucidating the mechanisms of COX-2 mediated gastric carcinogenesis.更多还原