【作者】 蔡洪科；

【导师】 邓甬川；

【作者基本信息】 浙江大学 ， 肿瘤外科学（专业学位）， 2013， 博士

【摘要】 研究背景结直肠癌是严重威胁人类健康的常见消化道恶性肿瘤。目前以手术为基础的综合治疗在大肠癌中发展迅速,尤其是化疗及分子靶向药物的发展及多学科诊疗模式(MDT)的建立,有效提高了大肠癌患者的生存,但统计结果显示其发病率及疾病相关死亡率仍上升非常明显。其中肿瘤转移是影响患者预后及致死的关键因素。大肠癌转移是一个十分复杂的过程,涉及多因素、多步骤,多条信号转导通路交联,相互影响。而上皮间质转化(Epithelial-mesenchymal transition, EMT)在大肠癌侵袭及转移中的作用越来越受到关注。在分子水平上对结直肠癌相关基因的研究已经较为深入,近些年越来越多的临床及基础科研工作者开始关注microRNAs (miRNAs, miRs)在大肠癌发生发展中的作用。miRNAs是在真核生物中发现的一类内源性的具有调控功能的非编码RNA,通过和靶基因1mRNA碱基配对引导沉默复合体(RISC)降解mRNA或阻碍其翻译过程。目前已有研究证实miRNAs在大肠癌早期诊断,预后判断及疗效预测中发挥作用。另外miRNAs在大肠癌细胞增殖及凋亡,新生血管生成等基础研究领域亦不断有数据更新。尽管miRNAs参与肿瘤发生及细胞凋亡的相关研究较多,但有关miRNAs介导EMT的研究仍较少。课题组之前曾关注miRNAs与肿瘤耐药的相关研究,发现1niR-200c能够抑制多药耐药基因及P-糖蛋白从而逆转乳腺癌对表柔比星的耐药。本课题依托前期miRNAs研究基础,以细胞粘附、肿瘤微环境及EMT为切入点,研究miR-194参与大肠癌EMT过程及侵袭、转移中的作用。实验方法及结果首先课题组结合前期实验基础及miRNAs芯片筛选结果,收集临床结直肠癌标本40例,利用stem-loop RT-qPCR法检测标本中：miR-194的相对表达并统计其与临床病理特征的相关性。结果显示在淋巴结阳性的原发肿瘤中miR-194的表达明显强于淋巴结阴性组,而患者不同性别、年龄、肿瘤部位、肿瘤大小、分化程度、浸润深度与1niR-194的表达无明显相关性。这些结果提示miR-194可能在结直肠癌侵袭及转移中发挥作用。其次课题组寻找研究miR-194在大肠癌中侵袭转移中作用的细胞模型,发现在同源性较好的大肠癌细胞系SW480与SW620中,SW620细胞miR-194相对表达明显高于SW480细胞,细胞的划痕及Transwell迁移及侵袭实验均证实了SW620细胞的迁移、侵袭能力明显强于SW480细胞。进一步研究发现在SW620细胞系中MMP-2在1mRNA及蛋白质水平上表达均明显高于SW480细胞系,但MMP-9mRNA表达两株细胞无明显差别。上皮标志物E-cadherin在SW480表达明显强于SW620,司质标志物Vimentin则呈相反的变化。因此证实MMP-2, E-cadherin, Vimentin在大肠癌EMT转化中发挥重要作用,SW480与SW620是miR-194功能研究的良好细胞模型。之后课题组将miR-194构建质粒载体稳定转染入相对低表达的SW480细胞中,筛选高表达miR-194的细胞克隆,成功建立稳定转染细胞系niR-194-480。细胞形态学观察发现稳定转染后的miR-194-480细胞由转染前的扁圆形转变为梭形,发生明显的间质性改变。细胞增殖MTT实验显示miR-194转染SW480后,细胞增殖能力并未见明显变化。而迁移、侵袭实验结果则表明稳转细胞系miR-194-480在细胞迁移、侵袭能力方面大大增强。因此,我们认为1niR-194可能对大肠癌细胞增殖影响不大,却明显增加了大肠癌细胞的迁移、侵袭能力。之后RT-qPCR及Western Blot以及明胶酶谱实验证实,高表达miR-194能够增加MMP-2蛋白表达及活性,降解细胞外基质,同时E-cadherin表达下降,而Vimentin则明显表达增强。因此证实miR-194通过增加MMP-2, Vimentin表达,降低E-cadherin表达,在大肠癌EMT改变中发挥重要作用。同样,细胞骨架染色实验证实miR-194-480细胞系在细胞微丝数量上明显多于未转染细胞系。这说明miR-194在大肠癌细胞中能够影响细胞微丝,改变细胞骨架,增加大肠癌细胞的迁移、侵袭能力。接下来课题组对miR-194可能参与的细胞信号转导通路进行了预测。经生物信息学筛选,TargetScan, miRanda, RNA22等miRNA靶基因预测软件预测,PKC有可能是miR-194的靶标,PKC对ERK通路的活化也是目前的研究热点。课题组预想miR-194是否通过PKC的活化,从而激活ERK通路,参与大肠癌EMT转化。但实验结果与预期差异较大,在阴性对照组及转染组均未检测到PKC-α的表达,分析原因可能是miR-194参与了其他通路而反馈抑制了PKC-α,当然不能排除自身实验技术的不够成熟。由于课题时间有限未能更进一步深入探究。结论miR-194在淋巴结阳性的结直肠癌患者中高表达,而与患者性别、年龄、肿瘤部位、肿瘤大小、分化程度、浸润深度无明显相关性；miR-194通过调节MMP-2, E-cadherin, Vimentin的表达,参与大肠癌细胞EMT,能够明显增加大肠癌细胞的迁移、侵袭能力；miR-194参与大肠癌EMT过程中伴随细胞骨架的改变；PKC通路可能在miR-194参与大肠癌EMT转化过程中不起关键作用。更多还原

【Abstract】 Background:Colorectal cancer is the most common gastrointestinal malignancy, which seriously threat to human health. Currently, multidisciplinary treatments based on surgical resection is rapid development in colorectal cancer. Especially chemotherapy, molecular targeted drugs are familiar in clinical application, which effectively improve the overall survival for the colorectal cancer patients. However, statistics showed that the incidence and disease-related mortality rate is still rising obviously. Diastant metastases is a key factor affecting prognosis and death for colorectal cancer patients.The formation of metastases is a multistep process, in which malignant cells disseminate from the primary tumour to colonize distant organs. This is a highly inefficient and complex process. It is now recognized that the epithelial mesenchymal transition (EMT) may represent a critical component permitting the progression of carcinomas towards invasive and metastatic disease. Colorectal cancer carcinogenesis has been extensively studied at a molecular level in recent years and has recently entered the era of microRNAs (miRNAs). MiRNAs are a family of small, highly conserved non-coding RNAs that post-transcriptionally regulate gene expression, which act as endogenous suppressors of gene expression through imperfect binding of RNA-induced silencing complex (RISC) to the target mRNAs. MiRNAs have been studied in colorectal cancer as potential biomarkers of diagnosis, prognosis or response to treatment as well as colon cancer cell proliferation, apoptosis and angiogenesis. However, researches on miRNAs mediated EMT are still rare. Our studay group previously concerned reaearch on the relationship between miRNAs and chemotherapy-resistance, which showed up-regulation of miR-200c with transfection of miR-200c mimics in breast cancer cells could enhance the chemosensitivity to epirubicin and reduce expression of multidrug resistance-1mRNA and P-glycoprotein. In this study, our group will focus on the effects and potential mechanisms of microRNA-194on EMT process of colorectal cancer.Methods and resultsFirstly, our research group collected40specimens of colorectal cancer, using stem-loop RT-qPCR technique to evaluate the relative expression of miR-194in different specimens and add up the correlation with clinicopathological features. The results showed that miR-194expression was significantly increased in lymph node-positive colorectal cancers. However, miR-194expression level was not significant correlation to gender, age, tumor location, tumor size, degree of differentiation and T stage. The results suggest that miR-194may play an important role in metastasis of colorectal cancer.Secondly, our team looking for appropriate cell model to the further study on the effects and potential mechanisms of microRNA-194in colorectal cancer invasion and metastasis. We found miR-194was significantly higher expression in SW620than SW480cell line. Subsequent wound healing and Transwell assay confirmed the ability of migration and invasion of SW620cells was significantly increased than SW480cells. Further studies showed that the expression of MMP-2not only in mRNA but protein level was significantly higher in SW620v.s SW480, however, MMP-9mRNA expression was no significant difference. The expression of epithelial cell marker E-cadherin was significantly higher in SW480, mesenchymal cell marker Vimentin was contrary. Therefore it confirmed that MMP-2, E-cadherin, Vimentin play an important role in colorectal cancer EMT process.Next, miR-194plasmid vector is constructed and stably transfected into the relatively low expression cell lin SW480. We successfully established a stably transfected cell line and named miR-194-480by screening high expression of miR-194cell clones. Morphological observation showed the shape of miR-194-480was significantly into fusiform compared with the non-transfected cell line SW480, which illustrate the visible interstitial changes. MTT assay showed overexpression of miR-194had no significant effect in cell proliferation, while, wound healing and Transwell assay confirmed the ability of migration and invasion of miR-194-480was significantly increased. Therefore, we believe that miR-194may have little effect in proliferation of colon cancer cells, but manifest increase the ability of migration and invasion. RT-qPCR, Western Blot and gelatin zymography assay demonstrated overexpression of miR-194can increase MMP-2protein expression and activity involved in degradation of the extracellular matrix, while decreased expression of E-cadherin, however Vimentin expression was significantly enhanced. Therefore, we confirmed that overexpression of miR-194play an important role in colorectal cancer EMT process by changing the expression of MMP-2, E-cadherin and Vimentin.Cytoskeletal staining experiment confirmed that overexpression miR-194can increase the number of cell microfilaments. The result showed that miR-194can affect cell microfilament, changing cytoskeleton, and ultimately increase the ability of migration and invasion in colon cancer cell.Our group next concern the cellular signal transduction pathways. By predicting of bioinformatics screening as TargetScan, miRanda, RNA22software, PKC may be a target of miR-194. More and more research concerned PKC activated the ERK pathway. We predict whether miR-194can increase PKC expression, thereby activating the ERK pathway, finally involved in colorectal cancer EMT transformation. But we got a negative result. Analyze the reasons may be involved in other pathways and miR-194feedback inhibition of PKC-alpha, of course, can not be ruled out experimental techniques are not mature enough. Analysis of the reasons, miR-194may be involved in other pathways then feedback inhibition of PKC-alpha, and of course can not rule out our immature experimental techniques. However, we are unable to further delve into the subject because of time constraints.Conclusions:MiR-194expression was significantly increased in lymph node-positive colorectal cancers, however, which was not significant correlation to gender, age, tumor location, tumor size, degree of differentiation and T stage. MiR-194play an important role in colorectal cancer EMT process by changing the expression of MMP-2, E-cadherin and Vimentin. Overexpression miR-194can increase the number of cell microfilaments. PKC-alpha pathway may not be the core signaling pathway in miR-194-mediated EMT process in colorectal cancer.更多还原