【作者】 张钧；

【导师】 何超；

【作者基本信息】 浙江大学 ， 肿瘤学， 2010， 博士

【摘要】 为了降低肿瘤的死亡率或提高治疗的效果,许多研究致力于肿瘤关键生物标志物的发现和筛选。基因组和蛋白组学等新技术的应用为筛选新的肿瘤标志物或耐药相关分子提供了新的途径。尼克酰胺-N-甲基化酶(NNMT,EC 2.1.1.1)正是近年用蛋白组学和基因芯片技术比较不同癌组织和癌旁组织差异时发现的在癌组织中异常表达的蛋白质酶。与正常组织和肿瘤旁组织相比,NNMT在结直肠癌、甲状腺乳头状癌、肾透明细胞癌、口腔鳞状细胞癌、肺癌、膀胱癌、胃和胰腺等肿瘤组织有过表达,并在多种肿瘤患者的血清中升高。这些研究提示NNMT与肿瘤发生密切相关,推测NNMT可能参与肿瘤细胞的多种生物功能,与肿瘤的发生和发展有关,或可影响化、放疗效果,但对NNMT在肿瘤细胞中的过表达的意义或确切作用仍需细胞学实验证实。鉴于大肠癌是严重危害人类健康的恶性肿瘤之一,在西方发达国家,大肠癌死亡率仅次于肺癌居第二位,在我国的恶性肿瘤死因顺位中居第三、四位,是我国现阶段重点防治的8大癌症之一,并具有高发趋势,我们选择该肿瘤为本次研究的重点。目的：研究NNMT对结直肠癌细胞的生物学功能的影响；研究NNMT在肿瘤中的作用机制或分析其下游相关分子组成的模块或通路,NNMT与结直肠癌临床病理的关系。方法：1.通过分子克隆技术构建原核表达质粒p GEX-4T-2/NNMT和真核表达质粒pcDNA3.1/NNMT。2.利用基因工程制备NNMT蛋白,并作为免疫原免疫小鼠制备NNMT单克隆抗体,并建立基于此单克隆抗体的Western-blot、免疫组化等实验方法。3.采用RT-PCR和Western-blot分析多株结直肠癌细胞系的NNMT表达,筛选NNMT无／低表达和高表达株。4.把pcDNA3.1/NNMT转入COLO 320和SW480细胞株,用G418来筛选转入目的基因的细胞,建立相应的NNMT高表达肿瘤细胞模型和空质粒pcDNA3.1转染的对照模型。5.通过NNMT高表达肿瘤细胞模型,结合RNA干扰技术,MTT法分析NNMT对肿瘤细胞生长增殖和化疗药物敏感试验、流式细胞仪分析凋亡和细胞周期、用Transwell细胞迁移实验和Matrigel基质浸润实验研究NNMT对肿瘤细胞迁移和侵润等生物学功能的影响。6.用Affimetrix gene expression芯片比较NNMT基因导入前后大肠癌细胞的基因表达差异,分析NNMT下游效应分子和作用机制。7.采用免疫组化检测组织NNMT与结直肠癌的临床病理的关系。结果：1.构建了原核表达质粒p GEX-4T-2/NNMT和真核表达质粒pcDNA3.1/NNMT,并通过了酶切和测序验证。2.利用基因工程制备的NNMT蛋白作为免疫原免疫小鼠制备了能稳定分泌抗NNMT单克隆抗体的4个细胞株,该NNMT单克隆抗体具有很好的特异性和反应性,并建立了基于此单克隆抗体的Western-blot、免疫组化等实验方法。3.对多株大肠癌细胞系(HT-29、SW480、COLO 320、SW620、HCT116、HCE8693)的NNMT的mRNA和蛋白表达进行了分析, RT-PCR结果显示除SW480外,其余细胞均能检测到mRNA,其中COLO 320表达水平最高。Westernblot结果也显示NNMT蛋白在COLO 320细胞株表达量最高,其次为HCE8693和HT-29,SW480细胞株表达阴性,SW620、HCT116细胞株表达较弱。4.建立了相应的NNMT高表达肿瘤细胞模型和空质粒pcDNA3.1转染的对照模型。pcDNA3.1/NNMT转染SW480组NNMT mRNA水平较对照大幅上升,且转染pcDNA3.1/NNMT的SW480细胞NNMT蛋白表达阳性,而对照细胞阴性,提示NNMT基因导入成功,并有效表达；转染pcDNA3.1/NNMT的COLO 320细胞的NNMT蛋白表达较对照组高约1.621倍,也表明NNMT基因导入成功,并有效表达。5.MTT法检测了NNMT对细胞增殖的影响：从第72小时开始,转染NNMT基因的细胞株的490nm波长处光吸收值高于对照组,经方差分析有统计学差异(p<0.05)。直接选择SANTA CRUZ BIOTECHNOLOGY公司的NNMT siRNA(m):sc-61214,在SW480细胞,稳定转染pcDNA3.1/NNMT和空载体的SW480细胞进行了初步实验,结果显示转染NNMT基因的细胞株生长增殖能力高于对照组,但可被NNMT特异的siRNA抑制。6.MTT法检测NNMT对5-FU和奥沙利铂的影响。结果导入NNMT SW480细胞株对5-Fu耐药性较对照组细胞株强,SW480-pcDNA3.1/NNMT cell的IC50为16.8 mM, SW480-pcDNA3.1和SW480的IC50为8.8,9.2 mM；对奥沙利铂耐药性变化不大。7.对NNMT转染前后SW480细胞进行了Transwell细胞迁移实验和Matrigel基质浸润实验。实验结果显示SW480细胞和空载体SW480细胞几乎无细胞穿过,而pcDNA3.1/NNMT的SW480细胞则约有15-30个每低倍视野。8.细胞周期结果显示SW480细胞,稳定转染pcDNA3.1/NNMT和空载体的SW480细胞三组间G1期、G2期和S期的比例差别无统计学意义,在COL0320,COLO320-pcDNA3.1, COLO320-pcDNA3.1/NNMT细胞三组细胞也得到了同样的结果。9.细胞凋亡结果显示,SW480细胞组用5-FU的处理诱导24h后的凋亡变化显示SW480-pcDNA3.1/NNMT细胞的LR(右下象限)细胞数为9.12%,低于对照组15.66%、15.85%,提示NNMT具有抗凋亡功能。COLO320细胞组的凋亡分析结果也显示NNMT具有抗凋亡功能,COLO320-pcDNA3.1/NNMT细胞的LR(右下象限)细胞数为8.90%,低于对照组11.44%、12.57%,UR区(右上象限)的FITC+／PI+坏死细胞没有明显差别。10.我们用Affimetrix gene expression芯片比较NNMT基因导入前后大肠癌细胞株SW480基因表达差异,根据pcDNA3.1/NNMT的SW480细胞／SW480细胞,或pcDNA3.1/NNMT的SW480细胞,空载体的SW480细胞的基因表达差异超过2,同时考虑空载体的SW480细胞／SW480细胞,共选出33个候选基因。对上述基因进行GO功能分析发现具有差异表达的基因主要与物质代谢(脂类、糖)、细胞周期、细胞凋亡、细胞增殖调节、信号传导和细胞分化相关。其中上调的基因主要参与脂类代谢、氧化还原和电子传递。11.对109例原发灶、26例淋巴结转移灶的肿瘤组织进行了NNMT免疫组化分析,18例(16.5%)原发灶的肿瘤组织阳性,但淋巴结转移灶的肿瘤组织全阴性；阳性反应主要在肿瘤组织的细胞浆,高分化、低分化和黏液腺癌组织均有病例显示阳性,癌旁的正常组织阴性,NNMT在结直肠癌组织的表达与病人的临床病理因素的相关分析显示：NNMT的阳性率与肿瘤细胞的分化程度有关,在低分化腺癌中的阳性率为36.4%,高于高中分化腺癌(7.9%),P<0.01；NNMT的阳性率在不同年龄、性别、肿瘤的部位、TNM分期、DUKE’S分期组间差别无统计学意义,P>0.05。结论：1.从正常人肝脏组织中获得NNMT基因,并构建了原核表达质粒pGEX-4T-2/NNMT和真核表达质粒pcDNA3.1/NNMT；构建了SW480-pcDNA3.1, SW480-pcDNA3.1/NNMT, COLO320-pcDNA3.1和COLO320-pcDNA3.1/NNMT等细胞模型；利用基因工程制备的NNMT蛋白可作为免疫原免疫小鼠制备抗体、作为反应抗原建立ELISA或Western-Blot的阳性对照；制备了国内首株能稳定分泌抗NNMT单克隆抗体的细胞株,并建立了基于此单克隆抗体的Western-blot、免疫组化等实验方法,为研究NNMT提供了很好的工具。2.不同大肠癌细胞系的NNMT的mRNA和蛋白表达不一,COLO 320细胞株表达量最高,而SW480细胞株表达阴性。NNMT与结直肠癌的临床病理分析显示NNMT的阳性率与肿瘤细胞的分化程度有关,阳性的NNMT主要在低分化腺癌。NNMT的阳性率在不同年龄、性别、肿瘤的部位、TNM分期、DUKE’S分期组间差别无统计学意义,NNMT的阳性反应主要在肿瘤组织的细胞浆,这符合NNMT是胞浆酶的定位。NNMT对肿瘤的迁移和侵润的影响,临床病理结果和细胞实验结果并不一致,需进一步研究。3.细胞水平实验研究结果显示NNMT可能与肿瘤细胞的生物学功能密切相关：NNMT能促进肿瘤细胞增殖,NNMT量的提高也能促进肿瘤细胞增殖；NNMT能促进肿瘤细胞的迁移和侵袭能力；NNMT能通过某种途径抗凋亡；NNMT可能影响肿瘤细胞耐药。4.共选出33个候选基因,这些具有差异表达的基因主要与物质代谢(脂类、糖)、细胞周期、细胞凋亡、细胞增殖调节、信号传导和细胞分化相关。其中上调的基因主要参与脂类代谢、氧化还原和电子传递。我们推测NNMT可通过细胞膜的胆固醇合成从而影响细胞的功能；氧化还原和电子传递过程中产生的活性氧(ROS)也可能是引起细胞增殖的机制。需更多的工作来证实这些假设。更多还原

【Abstract】 There is an urgent need to screen and identify new biomarkers, which are warranted to provide more information on the tumor biology, chemotherapeutic effects, allowing a better prognostic and possibly predictive stratification of patients and reducing the mortality.Proteomic technologies and DNA microarray are providing the tools needed to discover and identify biomarkers associated with diverse diseases and biological processes. Nicotinamide N-methyltransferase (NNMT) was one of the potential tumor biomarkers identified recently by those technologies, in which NNMT in both mRNA and protein levels were found over-expressed in a wide range of tumors. When comparing normal tissue with cancer tissue, abnormal expression of NNMT has been reported in tumors such as papillary thyroid cancers, glioblastoma, stomach adenocarcinoma, colon cancer, renal carcinoma, oral squamous cell carcinoma, and lung cancer. NNMT was also found increased in serum of patients with cancer. Those results indicate that NNMT may be closely related to the biological function of tumour, and may influence the results of chemotherapy, thus become a potential biomarker for diagnosis of tumor and may have a potential role for predicting response to radiation therapy or chemotherapy. All these functions need to be proven by studying the influence of NNMT on tumor cell.As colorectal cancer was one of the most common malignancies worldwide and is considered to be the second or third leading cause of cancer death, thus colorectal cancer was chosen in this study.Objective:1.To understand the influence of NNMT on the biological function of tumor cell;2.To explore the mechanisms of the influence of NNMT on tumor;3.To evaluate the relationship between NNMT and clinical pathological parameter of patients with colorectal cancer.Methods:1. Molecular clone techniques were used to construct NNMT protocaryon and eukaryotic expression plasmids.2. The recombinant glutathione S-transferase (GST)-NNMT and NNMT were produced and purified by gene engineering, and were used for immunization of mice and making hybridomas secreting Ag-specific monoclonal antibodies. Western-blot and Immunohistological stain were established to detect NNMT based on the prepared monoclonal antibodies.3. RT-PCR and Western-blot were used to screen low or no NNMT expression and high expression colorectal cancer cell strains.4. To construct NNMT high expression cancer cell models, the eukaryotic expression plasmids, pcDNA3.1/NNMT was transferred into low or no NNMT expression and high expression colorectal cancer strains; G418 was used to screen the positive cell strains.5.Based on the above constructed cell models and RNAi technique, the influence of NNMT on the biological function of tumor cells such as cell cycle, cell apoptosis, cell proliferation, invasion and immigration, and response to chemotherapy were studied. 6. The Affymetrix gene chips (Human Genome U133 plus 2.0 Array) were used for mRNA expression profiling of the colorectal strains with NNMT transferred or not.7. Immunohistological stain was used to detect NNMT in cancer tissue, and the relationship between NNMT and clinical pathological parameter of patients with colorectal cancer was evaluatedResults:1. NNMT cDNA was cloned from normal human liver tissue, and was inserted into protocaryon and eukaryotic expression plasmids, which named p GEX-4T-2/ NNMT and pcDNA3.1/NNMT respectively.2. Four strains of hybridoma cells secreting anti-human NNMT McAbs were obtained. McAbs secreted by all the strains have high specificity and reactivity. Western-blot and Immunohistological stain to detect NNMT based on the prepared monoclonal antibodies were set up.3. NNMT expression of colorectal cancer cells such as HT-29. SW480、COLO 320、SW620、HCT116 and HCE8693 were detected by RT-PCR and Western-blot. The COLO320 expressed the highest NNMT for both mRNA and protein level while the HCE8693, HT-29,,SW620 and HCT116 shoewd decreased in level respectively, with SW480 showing the lowest.4. NNMT highly expression cancer cell models, SW480 -pcDNA3.1/NNMT and COLO320-pcDNA3.1/NNMT, were constructed. These cell models were confirmed expressing highly NNMT mRNA and protein level by RT-PCR and Western-blot. NNMT mRNA and protein level can be detected in SW480-pcDNA3.1/NNMT while negative in wild or control SW480.There was a 1.62 fold increase of NNMT protein in COLO320-pcDNA3.1/NNMT when comparing with wild or control SW480.5. The results of cell growth curve showed:Both SW480-pcDNA3.1/NNMT and COLO320-pcDNA3.1/NNMT cell grow or proliferate faster than the control cells, the significance can be detected since the point of 72 hour of culture; and the difference of growth speed can be inhibited by RNAi of NNMT.6.The influnce of NNMTon response to chemotherapy showed that SW480-pcDNA3.1/NNMT was more resistant to 5-Fu when comparing with control cells, the IC50 of SW480-pcDNA3.1/NNMT cell was 16.8 mM; 8.8 mM and 9.2 mM for SW480-pcDNA3.1and SW480 respectively. However, no signifant change was detected when analyzing with Oxaliplatin.7. Matrigel and Transwelltest showed only SW480-pcDNA3.1/NNMT can go through the gel and membrance with 15-30 cell/low power microscope field.8.There was no significant change of cell cycle transition (G1,G2 and S phase) among SW480, SW480-pcDNA3.1/NNMT and SW480-pcDNA3.1 were detected.The same results were obstained in COLO320, COLO320-pcDNA3.1 and COLO320-pcDNA3.1/NNMT cell.9. Flow Cytometer(FCM)was used to analyse cell apoptosis.After treating with 5-FU for 24 hours,the cell percentage of LR in SW480-pcDNA3.1/NNMT was 9.12%, which was lower than 15.66% in SW480-pcDNA3.1 and 15.85% in SW480. Thus anti-apoptosis of NNMT can be drawn in SW480-pcDNA3.1/NNMT model. The same change trend of cell percentage of LR observed in COLO320-pcDNA3.1/NNMT model.10. There were 33 genes 2-fold upregulated or downregulated when comparing SW480-pcDNA3.1/NNMT with SW480-pcDNA3.1.The changed genes were related to metabolism,cell cycle, proliferation, apoptosis,signal transduction, and cell differentiation.The most of the upregulated genes were related to cholesterol synthesis and oxidoreduction.11. The diagnostic and prognostic function of NNMT protein in colorectal cancer was evaluated by analyzing 109 colorectal cancer tissues and 26 lymphnode through immunohistochemical staining for NNMT by using 2F8 antibodies.18 cases (16.5%) were positive, and the positive NNMT was mostly related to the poorly-differentaited adenocarcinoma; 12 (36.4%) NNMT positivity was significantly higher in poorly-differentaited adenocarcinoma cells when compared with the highly or moderately-differentaited adenocarcinoma, (P<0.01). No correlations or associations between NNMT and clinico-pathological parameters such as age, gender, tumor location,TNM abd Dukes grade were detected,(P>0.05).Conclusion:1. Through this study, we successfully obtained a couple of important tools for studying NNMT function. The tools were protocaryon and eukaryotic expression plasmids of p GEX-4T-2/NNMT and pcDNA3.1/NNMT; hybridoma cells secreting anti-human NNMT McAbs with high specificity and reactivity; Western-blot and Immunohistological stain based on the prepared antibodies, NNMT highly expression cancer cell models, SW480-pcDNA3.1/NNMT and COLO320-pcDNA3.1/NNMT; GST-NNMT and NNMT protein.2. NNMT expression was different in the tested colorectal cancer cells. The COLO320 expressed the highest NNMT for both in mRNA and protein level while SW480 showed the lowest. In cancer tissue, positive NNMT were mostly related to the poorly-differentiated adenocarcinoma when compared with the highly or moderately-differentiated adenocarcinoma. However, there were no correlations or associations between NNMT and clinico-pathological parametersdrawn out on age, gender, tumor location, TNM and Dukes grade.3. NNMT may be closely related to the biological function of tumour:it may improve cell growth or proliferation rate; influence the results of chemotherapy; enhance the ability for invasion and immigration; and play a role as anti-apoptosis.4. A total of 33 genes related to metabolism,cell cycle,proliferation, apoptosis, signal transduction, and cell differentiation were found upregulated or downre-gulated.The most of the upregulated genes were related to cholesterol synthesis and oxidoreduction.Those changed gene related mechanisms may explain the influence of NNMT on tumor cells, however, more work needs to be done to prove it.更多还原