本文選題：胃癌 + 長鏈非編碼RNA　； 參考：《南京醫(yī)科大學》2016年博士論文

【摘要】：胃癌作為全球第四大癌癥,嚴重威脅到人類健康,特別是對于發(fā)展中國家。世界每年胃癌新發(fā)病例近百萬,發(fā)展中國家占近2/3,其中42%發(fā)生在中國。胃癌的早期診斷與治療對患者的病情及預后至關(guān)重要,也是國內(nèi)胃癌領(lǐng)域有待突破的主要研究方向。胃癌的發(fā)生發(fā)展涉及多基因異常調(diào)控、多步驟參與,最終使細胞生物學行為由正常演變?yōu)楫惓！Ｑ芯堪l(fā)現(xiàn)胃癌發(fā)生發(fā)展與基因有著密切的聯(lián)系胃癌體內(nèi)的某些特定基因與正常人的基因相關(guān)比發(fā)生了變化,有可能這些變異的基因是導致胃癌的關(guān)鍵。當細胞行為異常是基于基因水平發(fā)生變化時,便可引發(fā)細胞結(jié)構(gòu)和功能的不可逆性改變,很大可能造成細胞癌變。胃癌的遠處轉(zhuǎn)移及復發(fā)也是胃癌患者死亡的最主要原因,是影響臨床療效和預后的關(guān)鍵因素。深入研究胃癌的發(fā)病機制以及引起胃癌發(fā)生轉(zhuǎn)移的具體分子機制,可以為探索新的診斷方法及靶向治療位點提供堅實的理論基礎(chǔ),也為胃癌的早期診斷和預后評價提供依據(jù)。既往針對于胃癌發(fā)生發(fā)展的機制研究多集中于傳統(tǒng)的蛋白編碼基因,主要涉及遺傳學,表觀遺傳學以及相關(guān)信號通路的調(diào)控。隨著高通量測序技術(shù)發(fā)展,研究者已經(jīng)揭示在人類基因組中非編碼RNA(Non-coding RNA, ncRNA)比例占據(jù)了95%以上,蛋白編碼RNA以及功能性非編碼RNA間復雜相互作用在胃癌的發(fā)生發(fā)展中具有重要的作用。長鏈非編碼LNA(long non-coding RNA, IncRNA)作為ncRNA的重要組成成分之一,也越來越受到重視。目前已經(jīng)揭示lncRNA可以通過多種途徑參與表觀遺傳,基因轉(zhuǎn)錄及轉(zhuǎn)錄后水平的調(diào)控,并且具有作為早期疾病診斷標記物的潛力。近年來,非編碼RNA尤其是長鏈非編碼RNA(Long non-coding RNA, lncRNA)在腫瘤的發(fā)生機制研究中越來越受到研究者的關(guān)注。目前為止,已有眾多IncRNA被報道與腫瘤細胞增殖,侵襲轉(zhuǎn)移及腫瘤復發(fā)高度相關(guān)。同樣,在胃癌研究中,已有報道發(fā)現(xiàn)在胃癌組織與癌旁組織中具有明顯差異表達的IncRNA譜,并且某些IncRNA,如H19,HULC可通過不同機制調(diào)控下游靶基因影響胃癌細胞的增殖或侵襲能力進而參與胃癌的發(fā)生發(fā)展。但目前關(guān)于IncRNA在胃癌中的研究尚停留在起步階段,探索和發(fā)現(xiàn)新的IncRNA及其具體調(diào)控機制,可以為胃癌的發(fā)病機制研究提供新的理論依據(jù)。長鏈非編碼RNA是指一類轉(zhuǎn)錄本長度大于200個核苷酸的RNA,位于細胞核內(nèi)或胞漿內(nèi),不參與或很少參與編碼蛋白,主要以RNA的形式在表觀遺傳、轉(zhuǎn)錄及轉(zhuǎn)錄后等層面上調(diào)控基因表達水平。LncRNA主要參與了基因組印記,染色體沉默以及染色質(zhì)修飾、ml RNA轉(zhuǎn)錄激活、轉(zhuǎn)錄干擾,及轉(zhuǎn)錄后調(diào)控、核內(nèi)運輸、調(diào)節(jié)原癌基因活化等多種重要的cis-或trans-調(diào)控過程,它不但可活化某些蛋白編碼基因表達,亦可以抑制蛋白編碼基因的表達。目前研究進展已經(jīng)揭示lncRNA具有作為疾病早期診斷標記物的潛力。本研究中我們利用生物信息學分析、表達譜芯片結(jié)合經(jīng)典分子生物學技術(shù)等,在分子水平、細胞水平、實驗動物水平以及臨床樣本中探索異常表達的長鏈非編碼RNALinc00152對胃癌生物學行為的影響及機制。通過Affymetrix lncRNA microarray基因芯片檢測三例胃癌患者癌與癌旁組織中l(wèi)ncRNA的表達水平,芯片結(jié)果顯示：與癌旁組織相比,有271條lncRNA呈現(xiàn)不同程度的差異表達,提示lncRNA可能參與胃癌的發(fā)生發(fā)展。進一步確定芯片結(jié)果的可靠性,研究組再將其他學者已經(jīng)報道的胃癌lncRNA表達譜結(jié)果采用針對差異lncRNA聯(lián)合比對分析發(fā)現(xiàn),有3條lncRNA在兩組芯片中均有顯著差異表達。其中,長鏈非編碼Linc00152在兩組芯片結(jié)果中差異水平最大,且各組內(nèi)表達一致性較好,結(jié)果較穩(wěn)定。研究組再以72例胃癌患者的癌組織和癌旁組織為實驗樣本,采用qRT-PCR進行Linc00152表達驗證。結(jié)果發(fā)現(xiàn),Linc00152在癌組織中呈現(xiàn)與芯片結(jié)果一致的高表達狀態(tài)。結(jié)合患者的臨床信息發(fā)現(xiàn),Linc00152的表達水平與胃癌患者的腫瘤大小呈明顯正相關(guān)關(guān)系,而與TNM分期及淋巴結(jié)轉(zhuǎn)移等相關(guān)性不明顯。提示linc00O152可能參與胃癌細胞增殖有關(guān),據(jù)此,研究組刪選出linc00152高表達的胃癌細胞株(HGC-27, MGC803)建立體外細胞模型進行后續(xù)功能機制研究。為明確Linc00152的亞細胞定位,研究組采用胞漿-胞核分離RNA提取方法,分別提取兩種細胞細胞的細胞漿及細胞核內(nèi)RNA,通過特異性反轉(zhuǎn)錄方法檢測兩種細胞亞單位內(nèi)LincOO152表達水平,結(jié)果顯示,Linc00152主要存在與細胞漿中,僅有少許在細胞核中表達。為明確該段序列符合長鏈非編碼RNA的非編碼特性,研究組采用生物信息學軟件預測方法,確定其非編碼的生物學特性。采用目前為止公認的長鏈非編碼RNAMEG3作為陽性對照,結(jié)果顯示,Linc00152呈現(xiàn)明顯非編碼特性。為探索Linc00152對細胞功能的影響,我們用shRNA研究Linc00152的敲除效果,用CCK8檢測、EDU實驗研究對細胞增殖的影響。再用體外實驗驗證Linc00152表達與腫瘤生長的相關(guān)性。用RNA拉下實驗預測探索潛在的結(jié)合蛋白,并與反義Linc00152比較,發(fā)現(xiàn)位于約140 kDa附近的蛋白質(zhì)豐富。進一步質(zhì)譜鑒定表明,表皮生長因子受體是由Linc00152所捕獲的蛋白質(zhì)。為進一步探索Linc00152對EGFR具體調(diào)控機制,我們首先利用RNA免疫共沉淀技術(shù)(RNA Immunoprecipitation,RIP)探索Linc00152是否可以通過直接綁定EGFR蛋白進而影響其功能及相關(guān)效應(yīng)產(chǎn)生。RIP研究顯示Linc00152直接與EGF R蛋白結(jié)合。最終研究表明Linc00152可能通過EGFR依耐性信號通路促進胃癌細胞生長以及侵襲-轉(zhuǎn)移級聯(lián)反應(yīng),證明Linc00152在胃癌的發(fā)生發(fā)展中發(fā)揮著重要作用。綜上所述,我們的研究對胃癌的發(fā)生發(fā)展,特別是腫瘤生長及轉(zhuǎn)移中,功能性非編碼RNA參與調(diào)控的機制進行了深入而廣泛的探討,為進一步理解該過程中紛繁復雜的信號通路提供了新的思路和視角,同時,為將來胃癌的早期診斷及靶向治療提供堅實的理論基礎(chǔ)。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

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