【作者】 查郎；

【导师】 王子卫；

【作者基本信息】 重庆医科大学 ， 外科学， 2013， 博士

【摘要】 背景和目的：胃癌是世界上最常见的恶性肿瘤之一，在过去的20年中其死亡率虽然明显降低，但是在我国其发病率和死亡率仍然位于恶性肿瘤的前列，而肿瘤的转移是治疗失败的主要原因。迁移和侵袭是胃癌细胞转移的关键步骤。近年来，上皮细胞间质转化(epithelial-mesenchymal transitions, EMT)成为肿瘤转移的研究热点。EMT是一种常见的生理病理现象，与胚胎发育、伤口愈合及肿瘤侵袭转移等过程密切相关，主要表现为上皮细胞失去极性，细胞之间紧密连接和黏附连接下降，而获得了浸润性和游走迁移能力，演变为具有间质细胞形态和特性的细胞。因此，通过对胃癌细胞EMT的分子机制的研究，不仅能对胃癌转移的生物学行为提供新的研究方向，甚至能为胃癌的治疗提供潜在的策略。HMGA2（High mobility group protein A2）是一种非组蛋白染色体蛋白，本身没有转录活性，但是可以通过改变染色质结构调节其他基因的转录。HMGA2在正常组织中低表达或不表达，而在在胚胎形成时和恶性肿瘤组织中表达明显上调。在一些恶性肿瘤中HMGA2可以作为诊断的分子标志物或判断预后的独立因素。此外，HMGA2能促进上皮细胞间质化及维持肿瘤干细胞分化潜能和自我更新的能力，在肿瘤的形成、增殖及转移方面可能发挥重要作用。本研究首先在胃癌组织中检测HMGA2的表达并分析它与对应的胃癌病人临床病理参数之间的关系；其次，构建HMGA2过表达的慢病毒载体，转染内源性HMGA2低表达的胃癌细胞株，建立稳定过表达HMGA2的胃癌细胞株；合成HMGA2的siRNA的干扰片段，瞬时转染内源性高表达HMGA2的胃癌细胞株；分别检测干扰或过表达前后细胞生物学和分子生物学指标的改变。最后，筛选HMGA2下游可能的靶基因，并在胃癌细胞及动物模型上进行验证。探讨HMGA2促进EMT可能的分子机制。方法：（1）采用免疫组化SP法检测71例胃癌组织中HMGA2的表达，分析其与临床病理参数之间的关系。（2）Western-blot检测不同胃癌细胞株中HMGA2表达情况，构建过表达HMGA2的慢病毒载体，转染内源性低表达的胃癌细胞株并建立稳定株，使用MTT，侵袭小室及软琼脂克隆的方法比较过表达HMGA2后细胞在生长，侵袭转移性，成瘤性等细胞生物学指标的改变。合成HMGA2-siRNA，干扰内源性高表达HMGA2的胃癌细胞，分别检测过表达HMGA2前后与干扰HMGA2前后EMT标志物E-cadherin, N-cadherin和Vimentin的表达水平。建立裸鼠移植瘤模型，利用免疫组化的方法检测低表达HMGA2和过表达HMGA2的移植瘤EMT标志物的表达之间的差别。（3）使用染色质共沉淀加启动子芯片分析（ChIP on chip）鉴定HMGA2下游的直接作用靶点，使用KEGG_PATHWAY分析芯片结果，选取与肿瘤EMT相关的基因或信号通路进行分析，使用ChIP-qPCR（染色质共沉淀加荧光定量PCR），western-blot，qPCR及免疫荧光的方法进行验证。结果：（1）在胃癌组织中HMGA2的表达和胃癌患者淋巴结转移及临床TNM分期相关。（2）成功构建HMGA2过表达的慢病毒载体，外源性的HMGA2蛋白主要表达于细胞核内；转染内源性低表达的胃癌细胞株MKN28和GES-1后增强其迁移，侵袭和体外成瘤能力，但降低其生长率。过表达HMGA2后，胃癌细胞上皮标志物E-cadherin表达降低，间质标志物N-cadherin和Vimentin表达升高，细胞呈现出EMT状态；使用HMGA2-SiRNA干扰内源性高表达的MKN45细胞后，EMT状态出现一定程度的逆转。动物试验中发现，过表达HMGA2的MKN28细胞成瘤能力更强，但是移植瘤的生长速度减慢。（3）ChIP on chip分析发现1366个HMGA2可能的下游靶点，分析芯片结果后选出可能与EMT有关的两个基因TWIST1和AXIN1验证，发现过表达HMGA2可以促进TWIST1的表达，降低AXIN1的表达，使β-catenin进入胞核，进而激活Wnt/β-catenin信号通路。结论：HMGA2在胃癌组织中高表达，且与胃癌的侵润转移相关；胃癌细胞中稳定表达HMGA2可以促进胃癌细胞EMT的发生，增强胃癌细胞的侵袭转移和成瘤能力；胃癌细胞中HMGA2表达的降低可以逆转EMT状态；HMGA2通过上调TWIST1的表达，降低AXIN1的表达，促进使β-catenin进入细胞核，进而激活Wnt/β-catenin信号通路，诱发EMT。更多还原

【Abstract】 BACKGROUND AND OBJECTIVES: Gastric cancer mortalityhas declined markedly around the world over the last20years. Nonetheless,it still estimated to one of the most dangerous disease in our country andprobably is the second leading cause of cancer death. The death of gastriccancer is mainly caused by invasiveness and metastasis. Over the last fewyears, more and more evidence has indicated that EMT plays an importantrole in cancer progression and metastasis. In EMT process, cancer cells lostits epithelial properties and gain mesenchymal properties therebyenhancing their abilities of migration and invasiveness. However, theunderlying molecular mechanisms of these conditions are poorlyunderstood. Therefore, exploring the underlying molecular mechanisms ofEMT may provide a new treatment strategy for gastric cancer.High mobility group protein A2(HMGA2) is an architecturaltranscription factor that plays an important role in development andprogression of malignant neoplasias. Recently, some studies reported thatHMGA2is also implicated in epithelial-mesenchymal transitions (EMT)and cancer stem cells. In this study, at first, we investigate the relationship between theexpression of HMGA2and clinical parameter in gastric cancer. Secondly,we construct HMGA2overexpression lentiviral vectors and HMGA2siRNA. Then, we examined the changes of molecular and cellular biologyin gastric cancer cells with or without HMGA2overexpression. At last, wefind out target genes of HMGA2and validate them in vitro thereby exploremolecular mechanism of EMT elicited by HMGA2in gastric cancer.METHODS:(1) Immunohistochemistry was performed to examinethe HMGA2expression and investigated the relationship between theexpression of HMGA2and clinical parameter in gastric cancer.(2) Comparison of the expression of HMGA2in different gastricepithelial origin cell lines by western blot. Via homologus recombinationbetween pIRES2-EGFP and pLV-UbC-IRES2-EGFP, obtained lentiviralvectors plasmids for overexpressing HMGA2and then packaged intolentivirus particles. Inducted HMGA2gene in MKN28and GES-1celllines by above lentivirus particles and then analyses growth rates, migration,invasion and anchorage-independent growth by MTT, millicell and softagar assays after MKN28and GES-1cell lines were infected. Transienttransfected of HMGA2targeting siRNA into MKN45cells. Western blotwas performed to detect expression of E-cadherin, N-cadherin, Vimentin ingastric cancer cells with or without HMGA2overexpression.Immunohistochemistry were performed to detect expression of E-cadherin, N-cadherin, Vimentin in subcutaneous xenograft model of nude mouse.(3) Global mapping of HMGA2potential transcription factor bindingsites was identified by promoter microarray in these cells and the dateobtained from the microarrays were validated via chromatinimmunoprecipitation-PCR (ChIP-PCR) and qPCR. HMGA2potentialtarget genes were classified in KEGG database analyses. ChIP-qPCR，Western-blot，qPCR and immunofluorescence were performed to validateabove results from microarray.RESULTS:(1) The expression of HMGA2was correlated with TNMstage and lymph node metastasis.(2) Lentiviral vectors plasmids for overexpressing HMGA2wasconstructed successfully. Exogenous HMGA2expression was observed tolocate in the nucleus by immunofluorescence analysis. Overexpression ofHMGA2enhances oncogenic properties and improves migration andinvasion in MKN28and GES-1cell lines but reduced their growth rates.Furthermore, overexpression of HMGA2increased N-cadherin andVimentin expression and decreased E-cadherin expression in MKN28andGES-1cells. This condition could be reversed by transient transfected ofHMGA2targeting siRNA in MKN45cells. Overexpression of HMGA2inMKN28cells had a higher tumor formation rates but a smaller tumor sizein subcutaneous xenograft model of nude mouse.(3)1366genes were identified to be potential targets of HMGA2by ChIP on chip and then the target genes were analyzed for their roles inbiological functions and pathways with the KEGG databases.144of themwere clustered to35pathways potential in involved in cancer. TWIST1andAXIN1were singled out because they are related to EMT. HMGA2overexpression increased TWIST1expression and decreased AXIN1expression. β-catenin was observed to translocate from the usualmembrane/cytoplasm-bound site observed in MKN28without HMGA2expression to the nucleus in MKN28with HMGA2expression byimmunofluorescence.CONCLUSIONS: The expression of HMGA2was correlated withprogression of gastric cancer. HMGA2is an important contributor to EMTin gastric epithelial origin cell lines. HMGA2expression enhancedoncogenic properties of gastric cancer in vivo and in vitro. The molecularmechanism of EMT elicited by HMGA2is that HMGA2could activateWnt/β-catenin pathway via regulating expression of TWIST1and AXIN1.更多还原