本文選題：NPC　切入點：miR-155　出處：《南方醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：MicroRNAs (miRNAs)是真核生物中發(fā)現(xiàn)的一類內(nèi)源性的具有調(diào)控功能的非編碼小、RNA分子,大約由21—25個核苷酸組成,其本身不具有開放閱讀框架(ORF)及蛋白質(zhì)編碼基因,有獨特的特征序列,主要在轉(zhuǎn)錄后水平調(diào)控基因的表達。Lee等在研究線蟲C.elegans遺傳發(fā)育篩選中首次發(fā)現(xiàn)miRNA lin-4; Reinhart等發(fā)現(xiàn)第二個非編碼rniRNA let-7。miRNAs在生物體的物質(zhì)代謝、細胞周期、細胞分化、凋亡、個體形態(tài)的形成和發(fā)育等一系列重要生命活動的調(diào)控過程中扮演著重要角色。迄今,已發(fā)現(xiàn)多個發(fā)揮原癌基因或抑瘤基因作用的miRNAs,女let-7、miR-21、miR-17、miR-143和miR-145、miR-372和miR-373以及miR-26a等,它們通過調(diào)控下游靶基因的轉(zhuǎn)錄和翻譯參與腫瘤的演進過程。研究表明,50%以上的miRNAs定位在腫瘤相關(guān)的基因組區(qū)域(cancer associated genomic regions, CAGR),包括LOH區(qū)、染色體擴增區(qū)及脆性位點等,其表達水平在許多腫瘤中發(fā)生改變,可能起到原癌基因或腫瘤抑制基因的作用,因此也將此類miRNA稱作oncomirs,在腫瘤發(fā)生、發(fā)展及轉(zhuǎn)移等過程中發(fā)揮重要作用。 miR-155位于人類21號染色體上,B-cell integration cluster (Bic)基因的第三個外顯子內(nèi),此基因不含開放閱讀框,過表達可促進細胞異常增殖；miR-155表達受Bic的轉(zhuǎn)錄水平和miRNA加工等調(diào)控。研究表明,miR-155在造血細胞生成、免疫反應(yīng)和炎癥反應(yīng)中發(fā)揮效應(yīng)：miR-155在許多常見腫瘤(如淋巴瘤、白血病、乳腺癌、肺癌、結(jié)腸癌、甲狀腺癌、宮頸癌、胰腺癌等)中表達上調(diào)。一些研究顯示,miR-155與腫瘤發(fā)生、轉(zhuǎn)移或預(yù)后等相關(guān),被認為是癌性微小RNA(oncomiR);比如在小鼠體內(nèi)轉(zhuǎn)基因過表達miR-155可誘發(fā)淋巴瘤、白血病和骨髓瘤；miR-155可促進乳腺癌、肺癌和肝癌細胞的增殖；此外,miR-155可增強乳腺癌細胞的抗凋亡能力和化療耐藥性。 鼻咽癌(Nasopharyngeal Carcinoma, NPC)是指發(fā)生于鼻咽粘膜的惡性腫瘤。發(fā)病年齡大多為中年人,亦有青少年患病者。中國的廣東、廣西、福建、湖南等地為高發(fā)區(qū)。NPC惡性程度較高,早期即可出現(xiàn)頸部淋巴結(jié)轉(zhuǎn)移。研究表明,環(huán)境因素和EB病毒(Epstein-Barr virus, EBV)感染在致癌過程中均可能起十分重要的作用；最近,Sengupta等利用高度精確和敏感的基因表達譜發(fā)現(xiàn)了8個在激光捕獲顯微切割(1aser-capture microdissection, LCM)后的NPC組織和正常鼻咽部上皮組織中具有顯著性表達差異的miRNA,即表達下調(diào)的miR-29c、 miR-34b、miR-34c、miR-212、miR-216和miR-217以及表達上調(diào)的miR-151和miR-192。此外,他們還通過生物信息學(xué)預(yù)測和實驗驗證,確定了miR-29c的靶基因為編碼細胞外基質(zhì)蛋白(extraceilular matrix proteins),包括多種膠原蛋白(collagens)(?)laminin y1,其與NPC侵襲轉(zhuǎn)移密切相關(guān)。miR-155在NPC組織中表達上調(diào),那么miR-155是否與NPC發(fā)生發(fā)展及轉(zhuǎn)移等相關(guān),目前尚未見報道。因此,miR-155表達失調(diào)在NPC發(fā)生發(fā)展及轉(zhuǎn)移中扮演何種角色,miR-155是否作為NPC一個oncomir等一系列問題都值得我們深入探討。 基于miR-155研究現(xiàn)狀及其在明確NPC癌變早期的分子機制中的潛在意義,本課題的研究目的為：明確miR-155在NPC細胞株和組織中的表達譜及其對NPC細胞生物學(xué)行為的影響,包括增殖、遷移和EMT等。我們通過上調(diào)和下調(diào)niR-155的表達水平明確其對NPC細胞生物學(xué)行為的影響,隨后進行下游靶基因的篩選和驗證,初步探討miR-155在NPC如上生物學(xué)行為中所扮演的角色及其機制,這將為深入理解NPC發(fā)病的分子機制和尋找新的分子靶向治療靶點奠定理論基礎(chǔ),為此本課題進行了如下研究： 方法： 1.NPC細胞株和組織標本中miR-155的表達譜 采用qRT-PCR檢測CNE1、CNE2、5-8F、6-10B、C666-1、HNE1、SUNE1和HONE18種NPC細胞株miR-155表達譜(以永生化鼻咽上皮細胞株NP69作為對照)。收集NPC組織標本15例和慢性鼻咽炎癥組織標本10例,應(yīng)用qRT-PCR檢測miR-155的表達譜,結(jié)果分析運用2-△△Ct方法進行比較。 2.建立穩(wěn)定過表達miR-155的NPC細胞株 利用慢病毒表達載體pLenti-miR-155生產(chǎn)攜帶miR-155的慢病毒,收集病毒上清分別感染NPC細胞株CNE1、CNE2、HONE1和SUNE1,48-72h后于倒置熒光顯微鏡下檢測EGFP表達以觀察感染情況,若感染效率低于90%,應(yīng)用流式細胞儀分選EGFP+細胞,大量擴增細胞并保種；提取RNA,應(yīng)用qRT-PCR檢測攜帶有miR-155轉(zhuǎn)基因的NPC細胞株中miR-155表達水平。 3.過表達miR-155對NPC細胞增殖、遷移及EMT的影響 CCK8實驗、平板克隆和細胞周期檢測細胞增殖能力改變；Transwell小室檢測細胞遷移能力改變,qRT-PCR、Western blot及免疫熒光(ICC)檢測EMT相關(guān)基因E-cadherin、α-catenin、Fibronectin、N-caherin及vimentin表達水平的變化。 4.利用miR155阻遏物下調(diào)NPC細胞中內(nèi)源性niR-155表達水平以進一步明確miR-155的如上功能 利用脂質(zhì)體介導(dǎo)瞬時轉(zhuǎn)染技術(shù)將化學(xué)合成的miR155阻遏物(miR155inhibitors)轉(zhuǎn)染至HONE1和SUNE1中,48h收集細胞并提取RNA, qRT-PCR檢測niR-155表達以評價抑制效果,CCK8和Transwell小室分別評價下調(diào)內(nèi)源性miR-155表達水平對細胞增殖和遷移能力的影響,Western blot檢測EMT相關(guān)基因表達。 結(jié)果： 1.NPC細胞株和組織標本中miR-155的表達譜 采用qRT-PCR檢測NPC細胞株CNE1、CNE2、5-8F、6-10B、C666-1、HNE1、 SUNE1和HONE1中miR-155表達情況(以NP69為對照)。結(jié)果顯示,miR-155在以上8株NPC細胞株中的表達水平均顯著高于NP69(圖1-1A),其中以CNE1最高,HONE1次之。qRT-PCR檢測15例NPC組織和10例鼻咽炎癥組織中miR-155表達,結(jié)果顯示兩者間無顯著差異(圖1-1B),這可能是因為我們所選用的正常對照為慢性炎組織標本,而miR-155已被證實在炎性淋巴細胞中高表達,因而干擾了miR-155的檢測。 2.建立穩(wěn)定過表達miR-155的NPC細胞株 慢病毒載體pLenti-miR-155經(jīng)測序鑒定,明確序列正確(圖2-1)；接著利用攜帶miR-155的慢病毒感染NPC細胞株,48-72h后于倒置熒光顯微鏡下通過檢測EGFP表達確認感染成功,隨后提取RNA, qRT-PCR檢測證實攜帶有miR-155轉(zhuǎn)基因的NPC細胞株中miR-155表達正常(表2-1和圖2-4),差異均具有統(tǒng)計學(xué)意義(P0.001),預(yù)示成功構(gòu)建穩(wěn)定過表達miR-155的NPC細胞株。 3.過表達miR-155對NPC細胞增殖、遷移及EMT的影響。 3.1增殖能力 CCK8實驗、平板克隆和細胞周期等實驗結(jié)果均表明過表達miR-155可促進NPC細胞增殖(圖2-5、圖2-6和圖2-7)。 3.2遷移能力及EMT相關(guān)基因的變化 Transwell實驗結(jié)果顯示：過表達miR-155促進了HONE1和CNE2體外遷移(表2-2和圖2-8)。 EMT相關(guān)基因的變化：qRT-PCR結(jié)果顯示,在CNE2和HONE1中過表達miR-155后,上皮細胞標志物E-cadherin和a-catenin表達水平下降(圖2-9),差異具有統(tǒng)計學(xué)意義(P0.05),而間質(zhì)細胞標志物N-cadherin和vimentin表達水平升高(圖2-9),前者差異有統(tǒng)計學(xué)意義(P0.05),后者差異無統(tǒng)計學(xué)意義(P=0.212)。 Western blot結(jié)果：CNE2和HONE1過表達miR-155后E-cadherin和α-catenin表達水平下降,而間質(zhì)細胞標志物Fibronectin口vimentin表達水平升高(圖2-10)。 ICC結(jié)果：CNE2、HONE1過表達miR-155后,E-cadherin(?)α-catenin表達下降,Fibronectin、N-caherin及vimentin表達水平升高,與Western blot結(jié)果一致,(圖2-11) 上皮-間充質(zhì)轉(zhuǎn)化(EMT)是指上皮細胞通過特定程序轉(zhuǎn)化為具有間質(zhì)表型細胞的生物學(xué)過程,EMT是上皮細胞來源的惡性腫瘤細胞獲得遷移和侵襲能力的重要生物學(xué)過程。在EMT發(fā)生過程中常會導(dǎo)致上皮細胞標志物E-cadherin、 α-catenin等表達水平下調(diào),造成細胞間粘附能力降低,而間質(zhì)細胞標記物Fibronectin、N-caherin(?)vimentin表達水平升高,則導(dǎo)致細胞遷移能力增強。 4.利用miR155阻遏物下調(diào)NPC細胞中內(nèi)源性niR-155表達對細胞增殖、遷移和EMT相關(guān)基因表達的影響 4.1miR155inhibitors下調(diào)NPC細胞中內(nèi)源性miR-155表達 利用脂質(zhì)體介導(dǎo)瞬時轉(zhuǎn)染技術(shù)將化學(xué)合成的miR155阻遏物(miRl55inhibitors)導(dǎo)入HONE1和SUNE1中,48h收集細胞并提取RNA, qRT-PCR檢測miR-155表達,結(jié)果顯示,內(nèi)源性miR-155的抑制效率達80%左右(圖2-12)。 4.2細胞增殖能力變化 CCK8生長曲線結(jié)果顯示,HONE1和SUNE1中內(nèi)源性miR-155表達下調(diào)后,細胞增殖能力下降(圖2-13)。 4.3細胞遷移能力及EMT相關(guān)基因水平的變化 Transwell小室實驗：HONE1和SUNE1中內(nèi)源性miR-155表達下調(diào)后,細胞遷移能力減弱(表2-8和圖2-14)。 Western blot結(jié)果：下調(diào)HONE1和SUNE1中miR-155表達,導(dǎo)致E-cadherin表達上調(diào)和vimentin表達下調(diào)(圖2-15)。 結(jié)論： miR-155可促進NPC細胞體外增殖和遷移能力,且能促進EMT發(fā)生。
[Abstract]:MicroRNAs (miRNAs) is a kind of endogenous found in eukaryotes with the regulatory function of non encoding small RNA molecules by about 21 - 25 nucleotides, which itself does not have the open reading frame (ORF) and protein encoding gene, has the unique feature of sequence, mainly in the post transcriptional regulation of gene the expression of.Lee in the study of nematode C.elegans genetic development for the first time that miRNA Lin-4 screening; Reinhart found second non rniRNA encoding let-7.miRNAs in organism metabolism, cell cycle, cell differentiation, apoptosis, plays an important role in regulation of a form of the formation and development of a series of important life activities. So far. Play has been found that a number of oncogenes or tumor suppressor genes of miRNAs, miR-21, miR-17, let-7, miR-143 and miR-145, miR-372 and miR-373 and miR-26a, which through the regulation of downstream target genes The evolution of transcription and translation in tumors. The study shows that the genomic regions in tumor associated miRNAs more than 50% (cancer associated genomic regions, CAGR), including LOH, and the amplified region of chromosome fragile sites, its expression level changed in many cancers, may play oncogenes or tumor suppressors the role of genes, so it will be this kind of miRNA called oncomirs, play an important role in tumorigenesis, development and metastasis process.
MiR-155 is located on human chromosome 21, B-cell integration cluster (Bic) gene exon third, this gene does not contain an open reading frame, expression can promote cell proliferation; the expression of miR-155 Bic transcription and miRNA processing control. The research results show that the formation of miR-155 in hematopoietic cells, immune effect reaction and inflammation: miR-155 in many common tumors (such as lymphoma, leukemia, breast cancer, lung cancer, colorectal cancer, thyroid cancer, cervical cancer, pancreatic cancer, etc.) in expression. Some studies showed that miR-155 and tumor, metastasis or prognosis, considered to be cancer micro RNA (oncomiR) for example, in mice; transgenic overexpression of miR-155 induced lymphoma, leukemia and myeloma; miR-155 can promote breast cancer, lung cancer and liver cancer cell proliferation; in addition, miR-155 can enhance the anti apoptosis of breast cancer cells Ability and chemotherapeutic resistance.
Nasopharyngeal carcinoma (Nasopharyngeal Carcinoma NPC) refers to the occurrence of malignant tumor in nasopharyngeal mucosa. The age of onset is mostly middle-aged, are teenagers. Chinese in Guangdong, Guangxi, Fujian, Hunan and other places for higher.NPC in high incidence area of malignancy, early can lead to neck lymph node metastasis. The study shows that the environmental factors and EB (Epstein-Barr virus EBV) virus infection in carcinogenesis may play an important role; recently, Sengupta using a highly accurate and sensitive gene expression was found in 8 laser capture microdissection (1aser-capture microdissection, LCM) with significant differential expression after miRNA NPC tissues and normal nasopharynx in the epithelial tissue, which regulated the expression of miR-29c, miR-34b, miR-34c, miR-212, miR-216 and miR-217 and up-regulated miR-151 and miR-192. in addition, he also through bioinformatics pre Measurement and experimental verification, identified miR-29c target genes for encoding the extracellular matrix protein (Extraceilular matrix proteins), including a variety of collagen (collagens) (?) laminin Y1, NPC and the invasion and metastasis of.MiR-155 in NPC tissue expression, whether miR-155 and NPC development and metastasis, it is have not been reported. Therefore, the expression of miR-155 disorders play a role in development and metastasis occurred in NPC, miR-155 or NPC as an oncomir and a series of problems are worthy of our in-depth study.
Based on the significance of the current research situation of miR-155 and its potential molecular mechanism in the carcinogenesis of NPC early in the clear, the purpose of this study is to clarify the expression of miR-155 in NPC cell lines and tissues of the spectrum and its influence on the biological behavior of NPC cells including proliferation, migration and EMT. The up-regulated and down regulated the expression level of niR-155 clear the influence on the biological behavior of NPC cells, followed by screening and identification of downstream target genes, preliminary study of role and mechanism of miR-155 play in the biological behavior of NPC in the above, which will deeply understand the molecular mechanism of NPC pathogenesis and find new molecular targeted therapeutic target lays the theoretical foundation for the paper the following research:
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