长链非编码RNA 00152在胃癌发生、发展中的机制研究

发布时间：2018-05-25 13:39

本文选题：胃癌 + 长链非编码RNA　；参考：《南京医科大学》2016年博士论文

【摘要】：胃癌作为全球第四大癌症,严重威胁到人类健康,特别是对于发展中国家。世界每年胃癌新发病例近百万,发展中国家占近2/3,其中42%发生在中国。胃癌的早期诊断与治疗对患者的病情及预后至关重要,也是国内胃癌领域有待突破的主要研究方向。胃癌的发生发展涉及多基因异常调控、多步骤参与,最终使细胞生物学行为由正常演变为异常。研究发现胃癌发生发展与基因有着密切的联系胃癌体内的某些特定基因与正常人的基因相关比发生了变化,有可能这些变异的基因是导致胃癌的关键。当细胞行为异常是基于基因水平发生变化时,便可引发细胞结构和功能的不可逆性改变,很大可能造成细胞癌变。胃癌的远处转移及复发也是胃癌患者死亡的最主要原因,是影响临床疗效和预后的关键因素。深入研究胃癌的发病机制以及引起胃癌发生转移的具体分子机制,可以为探索新的诊断方法及靶向治疗位点提供坚实的理论基础,也为胃癌的早期诊断和预后评价提供依据。既往针对于胃癌发生发展的机制研究多集中于传统的蛋白编码基因,主要涉及遗传学,表观遗传学以及相关信号通路的调控。随着高通量测序技术发展,研究者已经揭示在人类基因组中非编码RNA(Non-coding RNA, ncRNA)比例占据了95%以上,蛋白编码RNA以及功能性非编码RNA间复杂相互作用在胃癌的发生发展中具有重要的作用。长链非编码LNA(long non-coding RNA, IncRNA)作为ncRNA的重要组成成分之一,也越来越受到重视。目前已经揭示lncRNA可以通过多种途径参与表观遗传,基因转录及转录后水平的调控,并且具有作为早期疾病诊断标记物的潜力。近年来,非编码RNA尤其是长链非编码RNA(Long non-coding RNA, lncRNA)在肿瘤的发生机制研究中越来越受到研究者的关注。目前为止,已有众多IncRNA被报道与肿瘤细胞增殖,侵袭转移及肿瘤复发高度相关。同样,在胃癌研究中,已有报道发现在胃癌组织与癌旁组织中具有明显差异表达的IncRNA谱,并且某些IncRNA,如H19,HULC可通过不同机制调控下游靶基因影响胃癌细胞的增殖或侵袭能力进而参与胃癌的发生发展。但目前关于IncRNA在胃癌中的研究尚停留在起步阶段,探索和发现新的IncRNA及其具体调控机制,可以为胃癌的发病机制研究提供新的理论依据。长链非编码RNA是指一类转录本长度大于200个核苷酸的RNA,位于细胞核内或胞浆内,不参与或很少参与编码蛋白,主要以RNA的形式在表观遗传、转录及转录后等层面上调控基因表达水平。LncRNA主要参与了基因组印记,染色体沉默以及染色质修饰、ml RNA转录激活、转录干扰,及转录后调控、核内运输、调节原癌基因活化等多种重要的cis-或trans-调控过程,它不但可活化某些蛋白编码基因表达,亦可以抑制蛋白编码基因的表达。目前研究进展已经揭示lncRNA具有作为疾病早期诊断标记物的潜力。本研究中我们利用生物信息学分析、表达谱芯片结合经典分子生物学技术等,在分子水平、细胞水平、实验动物水平以及临床样本中探索异常表达的长链非编码RNALinc00152对胃癌生物学行为的影响及机制。通过Affymetrix lncRNA microarray基因芯片检测三例胃癌患者癌与癌旁组织中lncRNA的表达水平,芯片结果显示：与癌旁组织相比,有271条lncRNA呈现不同程度的差异表达,提示lncRNA可能参与胃癌的发生发展。进一步确定芯片结果的可靠性,研究组再将其他学者已经报道的胃癌lncRNA表达谱结果采用针对差异lncRNA联合比对分析发现,有3条lncRNA在两组芯片中均有显著差异表达。其中,长链非编码Linc00152在两组芯片结果中差异水平最大,且各组内表达一致性较好,结果较稳定。研究组再以72例胃癌患者的癌组织和癌旁组织为实验样本,采用qRT-PCR进行Linc00152表达验证。结果发现,Linc00152在癌组织中呈现与芯片结果一致的高表达状态。结合患者的临床信息发现,Linc00152的表达水平与胃癌患者的肿瘤大小呈明显正相关关系,而与TNM分期及淋巴结转移等相关性不明显。提示linc00O152可能参与胃癌细胞增殖有关,据此,研究组删选出linc00152高表达的胃癌细胞株(HGC-27, MGC803)建立体外细胞模型进行后续功能机制研究。为明确Linc00152的亚细胞定位,研究组采用胞浆-胞核分离RNA提取方法,分别提取两种细胞细胞的细胞浆及细胞核内RNA,通过特异性反转录方法检测两种细胞亚单位内LincOO152表达水平,结果显示,Linc00152主要存在与细胞浆中,仅有少许在细胞核中表达。为明确该段序列符合长链非编码RNA的非编码特性,研究组采用生物信息学软件预测方法,确定其非编码的生物学特性。采用目前为止公认的长链非编码RNAMEG3作为阳性对照,结果显示,Linc00152呈现明显非编码特性。为探索Linc00152对细胞功能的影响,我们用shRNA研究Linc00152的敲除效果,用CCK8检测、EDU实验研究对细胞增殖的影响。再用体外实验验证Linc00152表达与肿瘤生长的相关性。用RNA拉下实验预测探索潜在的结合蛋白,并与反义Linc00152比较,发现位于约140 kDa附近的蛋白质丰富。进一步质谱鉴定表明,表皮生长因子受体是由Linc00152所捕获的蛋白质。为进一步探索Linc00152对EGFR具体调控机制,我们首先利用RNA免疫共沉淀技术(RNA Immunoprecipitation,RIP)探索Linc00152是否可以通过直接绑定EGFR蛋白进而影响其功能及相关效应产生。RIP研究显示Linc00152直接与EGF R蛋白结合。最终研究表明Linc00152可能通过EGFR依耐性信号通路促进胃癌细胞生长以及侵袭-转移级联反应,证明Linc00152在胃癌的发生发展中发挥着重要作用。综上所述,我们的研究对胃癌的发生发展,特别是肿瘤生长及转移中,功能性非编码RNA参与调控的机制进行了深入而广泛的探讨,为进一步理解该过程中纷繁复杂的信号通路提供了新的思路和视角,同时,为将来胃癌的早期诊断及靶向治疗提供坚实的理论基础。
[Abstract]:As the fourth largest cancer in the world, gastric cancer is a serious threat to human health, especially in developing countries. There are nearly a million new cases of gastric cancer in the world every year, and the developing countries account for nearly 2/3. 42% of them occur in China. Early diagnosis and treatment of gastric cancer are important to the patients' condition and prognosis, and are also the main breakthroughs in the field of domestic gastric cancer. The occurrence and development of gastric cancer involves the regulation of polygenic abnormalities, multi step participation, and the eventual evolution of cell biological behavior from normal to abnormal. The key to gastric cancer is the cause of gastric cancer. When the abnormal cell behavior is based on the change of gene level, it can cause irreversible changes in cell structure and function, which may lead to cell carcinogenesis. The distant metastasis and recurrence of gastric cancer is the most important cause of death of gastric cancer patients. It is the key factor affecting the clinical efficacy and prognosis. The study of the pathogenesis of gastric cancer and the specific molecular mechanisms that cause the metastasis of gastric cancer can provide a solid theoretical basis for exploring new diagnostic methods and target therapy sites, and also provide the basis for early diagnosis and prognosis evaluation of gastric cancer. Genes, mainly involved in genetics, epigenetics and regulation of related signaling pathways. With the development of high throughput sequencing technology, researchers have revealed that the proportion of non coded RNA (Non-coding RNA, ncRNA) in the human genome occupies more than 95%, the complex interaction between protein encoded RNA and functional non coded RNA is occurring in the occurrence of gastric cancer. The long chain non coding LNA (long non-coding RNA, IncRNA), as one of the important components of ncRNA, is also becoming more and more important. It has been revealed that lncRNA can be involved in epigenetic, gene transcription and post transcriptional regulation in a variety of ways, and it has the potential as a marker for early diagnosis of disease. In recent years, non coded RNA, especially long chain non coded RNA (Long non-coding RNA, lncRNA), has attracted more and more attention in the study of the pathogenesis of tumor. So far, many IncRNA have been reported to be related to tumor cell proliferation, invasion and metastasis and tumor recurrence. Also, there have been reports in the research of gastric cancer. There is a distinct differential expression of IncRNA spectrum in gastric cancer tissues and adjacent tissues, and some IncRNA, such as H19, HULC can regulate the proliferation or invasion of gastric cancer cells by different mechanisms by different mechanisms, and then participate in the development of gastric cancer. However, the research on IncRNA in gastric cancer is still in the initial stage. The new IncRNA and its specific regulatory mechanism can provide a new theoretical basis for the study of the pathogenesis of gastric cancer. Long chain non coding RNA refers to a class of RNA, which is more than 200 nucleotides in length, is located in the nucleus or in the cytoplasm, and does not participate in or rarely participate in the encoding protein, mainly in the form of epigenetic, transcriptional and post transcriptional in the form of RNA. The level of gene expression level.LncRNA is mainly involved in genomic imprinting, chromosome silence and chromatin modification, ML RNA transcription activation, transcriptional interference, post transcriptional regulation, nuclear transport, and regulating the activation of proto oncogene activation in many important cis- or trans- processes. It can not only activate the expression of some protein encoding genes, but also can be used for gene expression. The current research progress has revealed the potential of lncRNA as a marker for early diagnosis of disease. In this study, we use bioinformatics analysis, expression spectrum chips and classical molecular biology techniques to explore abnormal molecular level, cell level, experimental animal level and clinical samples. The effect of long chain non coding RNALinc00152 on biological behavior of gastric cancer and its mechanism. The expression level of lncRNA in cancer and para cancerous tissues of three patients with gastric cancer was detected by Affymetrix lncRNA microarray gene chip. The results showed that 271 lncRNA showed different degrees of differential expression compared with para cancerous tissue, suggesting lncRNA Can participate in the development of gastric cancer. Further determine the reliability of the results of the chip. The research group then found that 3 lncRNA were significantly different in the two groups of microarrays. The results of the lncRNA expression profile of gastric cancer have been reported by other scholars. Among them, the long chain non coded Linc00152 is in two groups of chip results. In the study group, the cancer tissue and the paracancerous tissue in 72 cases of gastric cancer were used as the experimental samples, and the Linc00152 expression was verified by qRT-PCR. The results showed that Linc00152 showed high expression in the cancer tissue in accordance with the results of the chip. It was found that the expression level of Linc00152 was positively correlated with the tumor size of gastric cancer patients, but the correlation with TNM staging and lymph node metastasis was not obvious. It suggested that linc00O152 might be involved in the proliferation of gastric cancer cells. Accordingly, the study group selected the linc00152 high expression of gastric cancer cell line (HGC-27, MGC803) to establish the cell model in vitro. In order to identify the subcellular location of Linc00152, the study group took the cytoplasm and nucleus separation RNA extraction method to extract the cytoplasm and the RNA in the nucleus of two cell cells, and detected the level of LincOO152 in the subunits of the two cells by the specific reverse transcription method. The results showed that the main presence of Linc00152 was fine and fine. In the cytoplasm, only a few are expressed in the nucleus. In order to clarify the non coding characteristics of this segment consistent with the long chain non coded RNA, the research group uses the bioinformatics software prediction method to determine its non coding biological characteristics. The long chain non coded RNAMEG3 is used as a positive control, and the results show that Linc00152 is obviously not present. In order to explore the effect of Linc00152 on cell function, we used shRNA to study the knockout effect of Linc00152, the effect of CCK8 test on the proliferation of cells, and the correlation between the expression of Linc00152 and the growth of the tumor in vitro. The binding protein of the detects was predicted by the RNA pull down experiment, and the ratio of the antisense Linc00152 to the antisense Linc00152 ratio was predicted. More protein was found near about 140 kDa. Further mass spectrometry identification showed that the epidermal growth factor receptor was a protein captured by Linc00152. In order to further explore the specific regulation mechanism of Linc00152 on EGFR, we first explored whether Linc00152 can be passed by RNA immunoprecipitation (RIP) technique. Direct binding of EGFR protein then affects its function and related effects resulting in.RIP studies showing that Linc00152 is directly associated with EGF R protein. The final study shows that Linc00152 may promote the growth of gastric cancer cells and the invasion and metastasis cascade reaction through the EGFR dependent signaling pathway, which proves that Linc00152 plays an important role in the development of gastric cancer. To sum up, our research has made a thorough and extensive study of the mechanism of the involvement of functional non coded RNA in the development of gastric cancer, especially in tumor growth and metastasis. It provides new ideas and perspectives for further understanding of the complicated and complex signaling pathways in the process, at the same time, for the early diagnosis and targeting of gastric cancer. Treatment provides a solid theoretical basis.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

【相似文献】