【作者】 李娟；

【导师】 白云；

【作者基本信息】 第三军医大学 ， 遗传学， 2012， 博士

【摘要】 食管癌是世界上致死率最高的恶性肿瘤之一，超过90%的食管癌都属于鳞状细胞癌。流行病学调查显示各种能引起慢性炎性刺激的因素均可能促进食管鳞状细胞癌的发生。近年来，食管癌的发病率呈逐年增加的趋势，但预后却不容乐观，5年生存率仅20-30%。因此研究其发病机理，寻找早期诊断及治疗的靶标具有非常重要的意义。NF-κB通路对肿瘤的发生发展有着广泛而重要的影响。研究显示NF-κB可以作用于肿瘤细胞影响其生长、凋亡及迁移侵袭等生物学过程，也可作用于肿瘤微环境中的炎性细胞，促进炎性因子的分泌，为肿瘤细胞的生长转移提供合适的环境。然而NF-κB对肿瘤的影响却不尽相同，有时扮演癌基因的角色，有时又发挥抑癌基因的作用，其中的机制至今尚不完全清楚。肿瘤的发生发展是一个多阶段、多因素参与的过程，涉及许多癌基因和抑癌基因的突变，不同类型或不同阶段的肿瘤细胞发生突变的基因也可能不同，NF-κB通路又常常与其他通路（如p53信号通路）发生交互作用来调控下游基因的表达。因此，细胞背景的不同可能会影响NF-κB对下游靶基因的调控从而引起对肿瘤发生发展效应的差异。研究发现食管鳞癌组织中存在NF-κB及下游靶基因的异常表达或活化。小鼠食管鳞癌发生模型中的研究显示食管上皮组织在发生恶性转化的早期已有炎性细胞因子的存在和NF-κB p65的活化，提示NF-κB很可能参与调控食管鳞癌的发生发展过程。由于食管鳞癌也常常发生p53基因的突变，而NF-κB和p53常有相互作用影响彼此对下游靶基因的调控。因此，探讨NF-κB在食管鳞癌发生发展中的作用及对不同食管鳞癌细胞的影响将有非常重要的价值。MicroRNA（miRNA）是近年来发现的一大类调控基因表达的非编码小RNA分子，主要通过与mRNA的3′非翻译区结合影响mRNA的稳定性和蛋白翻译过程。研究表明miRNA在炎症和肿瘤发生等多种病理生理过程中均发挥重要调控作用。目前的研究提示miRNA不仅作为基因表达的调控者调节其他基因的表达，其自身也受到经典的转录因子的调控，已发现miRNA参与多种癌基因或抑癌基因调控网络，包括p53、c-MYC及NF-κB等。然而目前有关NF-κB调控miRNA参与食管鳞癌发生发展的研究还未见报道。因此，本课题的研究目的旨在探讨NF-κB对食管鳞癌发生发展的影响及miRNA在其中的作用和机制。为此，本课题选用两株不同的食管鳞癌细胞—EC109和KYSE450作为实验对象（两株细胞的p53状态不同，EC109中为野生型，KYSE450中为突变型），首先研究NF-κB对不同食管鳞癌细胞生长、凋亡、增殖及周期的影响，进一步通过生物信息学方法和功能筛查的办法寻找食管鳞癌中可能参与NF-κB调控网络的miRNA分子，其中包括miR-34a和miR-330，最后探讨miR-34a和miR-330对食管鳞癌细胞生物学行为的影响及NF-κB对它们的调控和相关机制。本课题的主要研究内容和结果如下：1．NF-κB对不同食管鳞癌细胞的生物学行为影响不同。采用CCK8法、流式细胞术及Edu染色等方法研究NF-κB对食管鳞癌细胞生长、凋亡及增殖等生物学行为的影响。结果发现在EC109细胞中过表达NF-κB p65，可抑制细胞的生长，诱导G1、G2期细胞减少，S期细胞增多，抑制EC109细胞的增殖以及增加细胞凋亡的发生。而在KYSE450细胞中转染p65表达质粒，则有促进细胞生长的作用，对细胞周期的影响主要表现为S期细胞比例增加，对细胞的增殖有轻微的促进作用，而对细胞凋亡无明显影响。此外，采用荧光定量PCR技术还检测了过表达NF-κB p65对食管鳞癌细胞中一些周期和凋亡相关基因的表达改变。结果显示增加p65的表达水平可上调EC109细胞中多种周期素依赖的蛋白激酶抑制因子（CDKI），如p21、p27的表达，以及p53的蛋白水平。同时过表达p65还可降低EC109细胞中抗凋亡蛋白survivin的水平。而在KYSE450细胞中过表达p65除增加p21的蛋白水平，上述其他基因的表达没有明显变化。研究结果提示NF-κB对不同食管鳞癌细胞的生长、增殖和凋亡存在不同的调控，可能与细胞中p53状态的不同有关。2．miR-34a为NF-κB参与食管鳞癌细胞生物学行为调控的靶点。采用CCK8法、PI染色流式细胞检测和transwell实验等方法研究miR-34a对食管鳞癌细胞的生长、周期及迁移侵袭等生物学行为的影响。进一步采用荧光定量PCR、CHIP、EMSA和报告基因技术研究NF-κB对食管鳞癌细胞中miR-34a的表达调控及相关机制。研究发现过表达miR-34a可以诱导食管鳞癌细胞G1期阻滞，抑制细胞的生长，降低细胞的迁移和侵袭能力，提示miR-34a可能有抑制食管鳞癌发生发展的作用。过表达NF-κB p65可以促进食管鳞癌EC109细胞中miR-34a的表达，且这一作用发生在转录调控水平—NF-κB可以与miR-34a基因启动子区的κB位点结合促进基因的转录活性。而在KYSE450细胞中或干扰EC109细胞中p53的表达，NF-κB就不能诱导miR-34a表达，这提示NF-κB对miR-34a的调控需要野生型p53的参与。进一步的研究显示野生型p53对NF-κB诱导miR-34a的转录活性是必须的，但NF-κB与miR-34a的结合并不受p53突变的影响。以上结果提示NF-κB可以在转录水平调控食管鳞癌细胞中miR-34a的表达，但这种调控作用需要野生型p53的参与——p53可能协同NF-κB促进miR-34a的转录活性，但不影响NF-κB与miR-34a的结合。此外，研究还发现NF-κB可能不影响p53对食管鳞癌细胞中miR-34a的调控。3．NF-κB调控miR-330影响食管鳞癌细胞的生物学行为。采用CCK8、PI染色流式细胞仪检测和Edu掺入法检测miR-330对食管鳞癌细胞生长、周期及增殖的影响，进一步采用荧光定量PCR、CHIP和报告基因技术研究NF-κB对食管鳞癌细胞中miR-330的表达调控及相关机制。结果显示增加miR-330的表达，食管鳞癌细胞的生长加快，S期和G2期细胞增多，细胞增殖增加，提示miR-330可能对食管鳞癌的发生发展有促进作用。过表达NF-κB p65后EC109细胞中miR-330的表达明显下调，而KYSE450细胞中的miR-330表达则显著升高。报告基因实验显示在这两种细胞中，NF-κB p65都可以通过miR-330调控区-3117位的κB位点发挥促进转录活性的作用。CHIP实验也表明两株细胞中NF-κB均可以与miR-330调控区结合。研究结果提示miR-330对食管鳞癌细胞的生长有促进的作用。NF-κB可以在转录水平调控食管鳞癌细胞中miR-330的表达，但在不同的细胞中调控效应不一致，具体机制有待进一步研究。综上所述，本课题的研究结果提示NF-κB对不同背景的食管鳞癌细胞的生物学特性有不同的影响，p53状态的不同可能为其中的一个影响因素。MiR-34a和miR-330为食管鳞癌中NF-κB调控网络中的两个新成员，而在不同食管鳞癌细胞中NF-κB对它们的差异调控可能是导致NF-κB对不同食管鳞癌细胞生物学特性调控不同的原因之一。细胞中p53状态的不同是导致NF-κB差异调控miR-34a的原因，而NF-κB对miR-330不同调控的机制还有待进一步的研究。本课题初步揭示了NF-κB在食管鳞癌细胞生物学行为调控中的复杂作用及通过对下游miRNA分子的差异调控产生不同的作用效应。本研究有助于深入认识食管鳞癌发生发展的机制并为食管鳞癌的诊治提供依据。更多还原

【Abstract】 Esophageal cancer is one of the most mortality malignancies in the world, includingtwo major histological forms-esophageal squamous cell carcinoma (ESCC) andesophageal adenocarcinoma, and more than90%of esophageal cancer are ESCC.Epidemiology studies indicate that any factor that causes chronic irritation andinflammation of the esophageal mucosa appears to increase the incidence of ESCC. Theincidence of esophageal cancer is increasing recently, however the overall5-year survivalrate is only20%-30%. It is urgent and important to investigate the mechanism for theinitiation and development of ESCC and find novel target for disease diagnosis and therapy.NF-κB pathway plays a key role in linking inflammation to caner development andprogression. It can be triggered by a variety of stimuli, such as pro-inflammatory cytokines,components of bacterial and virus, genotoxic stress and so on. Activated NF-κB affects notonly tumor cells but also inflammatory cells, and involves in the regulation of cell cycle,apoptosis, angiogenesis, metastasis and inflammation. NF-κB plays a complex role in thedevelopment of epithelial cancer; sometimes functions as tumor-promoter, and sometimestumor-suppressor, which may be related to different genes regulated by NF-κB in differenttissues and cells. There are numerous studies reporting overexpression or abnormalactivation of NF-κB and its target genes in ESCC tissues and their association withtherapeutic responses and prognosis of esophageal cancer, indicating NF-κB plays a role inthe development of esophageal squamous cell cancer.MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate geneexpression at the posttranscriptional level. Emerging evidence suggests that miRNAs playimportant roles in regulation of tumor biology and inflammation. Altered expression ofmiRNA has been reported in a variety of cancers, and their expression profiles can be usedas hallmarks for diagnosis, classification and prognosis of human malignancies. Their targetgenes involve in cell proliferation, apoptosis, differentiation, and metastasis and so on.Recent data suggests that miRNAs also play causal roles in esophageal cancer development. Microarray analyses revealed dysregulated miRNA expressions in ESCC. Several miRNAshave been found in participate with esophageal cancer development too.This project is aimed to investigate the role of NF-κB in ESCC development and findfunctional miRNAs involved in NF-κB network in ESCC. So, in the first section, effects ofNF-κB on ESCC cell growth, apoptosis, cell cycle and proliferation were studied. Then,according to bioinformatic analysis and functional studies of miRNAs in recent papers, wescreened two miRNAs, miR-34a and miR-330, as potential targets for NF-κB regulation inESCC. Roles of miR-34a and miR-330in ESCC cell biology and their relationship withNF-κB were investigated.The major results are presented below:1．NF-κB plays a different role in controlling different ESCC cell growth.CCK8, flow cytometry and Edu cell imaging assay were used to detect the effect ofNF-κB on ESCC cell growth, apoptosis, cell cycle and proliferation. The results showedthat overexpression of p65in EC109cells inhibited cell growth, probably through inducingS phase cell cycle arrest and inhibiting cell proliferation. Also overexpression of p65induced apoptosis of EC109cells. While transfection of p65expression vectors promotecell growth in KYSE450cells, which might be caused by enhanced cell proliferation. Andectopic expression of p65in KYSE450cells seemed to have no effect on apoptosis. Geneexpression studies showed that several CDKI expressions, such as p21and p27, wereincreased by overexpressing p65in EC109cells. p53protein levels were also enhanced byp65overexpression, while expressions of the inhibitor of apoptosis protein, survivin weredownregulated. Gene expression studies in transfected KYSE450cells showed thatincreasing p65levels had no impact on all the above gene levels except p21whose proteinlevels were upregulated. These results indicate that NF-κB might have different roles indistinct ESCC cell.2．MiR-34a is a novel target for NF-κB in ESCC.Effects of miR-34a on ESCC cell growth, apoptosis, cell cycle, migration and invasionwere studied by CCK8, flow cytometry and transwell assays. Results demonstrated thatoverexpression of miR-34a inhibited cell growth, induced G1cell cycle arrest, impairedcell motility and invasiveness. However, ectopic expression of miR-34a did not induce apoptosis. These indicated that miR-34a might function as a tumor suppressor in ESCC.Also the regulationship between NF-κB and miR-34a was investigated.Overexpression of NF-κB p65subunit could increase miR-34a levels in EC109which wasowing to enhanced miR-34a transcriptional activity by p65. Mutation of the κB sites locatedin miR-34a promoter impaired p65induced transcriptional activity. And chromatinimmunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA) bothshowed that NF-κB could specifically bind to the third κB site located in miR-34a promoter.In addition, overexpression of NF-κB p65could not successfully induce miR-34aexpression in esophageal cancer cell lines with mutant p53(KYSE450) or decreased p53.Reporter assay further showed that NF-κB-induced miR-34a transcriptional activity wasreduced by p53impairment. Nevertheless, binding of NF-κB to miR-34a promoter was notaffected in cells with mutant p53. The above results demonstrated that NF-κB could inducemiR-34a expression at the transcriptional level and induction of miR-34a need wildtype p53function.3．NF-κB regulates miR-330contributing to ESCC cell growth regulation.Effects of miR-330on ESCC cell growth, cell cycle and proliferation were studied byCCK8, flow cytometry and Edu cell imaging assays. Results showed that overexpression ofmiR-330promoted cell growth. And cell cycle analysis indicated that cells withoverexpressed miR-330had increased S, G2/M phase cells. Edu cell imaging analysis alsoshowed that increasing miR-330enhanced ESCC cell growth. These data indicated thatmiR-330might have a tumor-promoting role in the development of ESCC.QRT-PCR, reporter assay and CHIP analysis were used to study the regulationshipbetween NF-κB and miR-330. Transfection with p65downregulated miR-330expressionsin EC109cells and upregulated miR-330levels in KYSE450cells. Reporter assay showedNF-κB p65could promote transcriptional activity in both ESCC cells. CHIP analysis alsoshowed that NF-κB could bind to the κB site in the regulatory region of miR-330gene.These results indicated that NF-κB has the ability of regulating miR-330expression at thetranscriptional levels, but has different impact on the miR-330expression in differentESCC cells.In conclusion, this study indicates that NF-κB plays different regulatory roles ondifferent ESCC cells. MiR-34a and miR-330are novel targets for NF-κB and contributing to ESCC carcinogenesis. Distinct regulation of these two miRNAs in different cells mightcause the divergent role of NF-κB on ESCC cell growth. And distinct regulation of miR-34aby NF-κB might owe different p53status, while the mechanism of different miR-330regulation by NF-κB needs further study. Our research is helpful for understanding thecomplex role of NF-κB and miRNAs in the carcinogenesis of esophageal squamous cellcancer and provides new evidence for screening targets for ESCC diagnosis and therapy.更多还原