鼻咽癌中miR-155功能的初探

发布时间：2018-03-17 21:01

  本文选题：NPC　切入点：miR-155　出处：《南方医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：MicroRNAs (miRNAs)是真核生物中发现的一类内源性的具有调控功能的非编码小、RNA分子,大约由21—25个核苷酸组成,其本身不具有开放阅读框架(ORF)及蛋白质编码基因,有独特的特征序列,主要在转录后水平调控基因的表达。Lee等在研究线虫C.elegans遗传发育筛选中首次发现miRNA lin-4; Reinhart等发现第二个非编码rniRNA let-7。miRNAs在生物体的物质代谢、细胞周期、细胞分化、凋亡、个体形态的形成和发育等一系列重要生命活动的调控过程中扮演着重要角色。迄今,已发现多个发挥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,它们通过调控下游靶基因的转录和翻译参与肿瘤的演进过程。研究表明,50%以上的miRNAs定位在肿瘤相关的基因组区域(cancer associated genomic regions, CAGR),包括LOH区、染色体扩增区及脆性位点等,其表达水平在许多肿瘤中发生改变,可能起到原癌基因或肿瘤抑制基因的作用,因此也将此类miRNA称作oncomirs,在肿瘤发生、发展及转移等过程中发挥重要作用。 miR-155位于人类21号染色体上,B-cell integration cluster (Bic)基因的第三个外显子内,此基因不含开放阅读框,过表达可促进细胞异常增殖；miR-155表达受Bic的转录水平和miRNA加工等调控。研究表明,miR-155在造血细胞生成、免疫反应和炎症反应中发挥效应：miR-155在许多常见肿瘤(如淋巴瘤、白血病、乳腺癌、肺癌、结肠癌、甲状腺癌、宫颈癌、胰腺癌等)中表达上调。一些研究显示,miR-155与肿瘤发生、转移或预后等相关,被认为是癌性微小RNA(oncomiR);比如在小鼠体内转基因过表达miR-155可诱发淋巴瘤、白血病和骨髓瘤；miR-155可促进乳腺癌、肺癌和肝癌细胞的增殖；此外,miR-155可增强乳腺癌细胞的抗凋亡能力和化疗耐药性。 鼻咽癌(Nasopharyngeal Carcinoma, NPC)是指发生于鼻咽粘膜的恶性肿瘤。发病年龄大多为中年人,亦有青少年患病者。中国的广东、广西、福建、湖南等地为高发区。NPC恶性程度较高,早期即可出现颈部淋巴结转移。研究表明,环境因素和EB病毒(Epstein-Barr virus, EBV)感染在致癌过程中均可能起十分重要的作用；最近,Sengupta等利用高度精确和敏感的基因表达谱发现了8个在激光捕获显微切割(1aser-capture microdissection, LCM)后的NPC组织和正常鼻咽部上皮组织中具有显著性表达差异的miRNA,即表达下调的miR-29c、 miR-34b、miR-34c、miR-212、miR-216和miR-217以及表达上调的miR-151和miR-192。此外,他们还通过生物信息学预测和实验验证,确定了miR-29c的靶基因为编码细胞外基质蛋白(extraceilular matrix proteins),包括多种胶原蛋白(collagens)(?)laminin y1,其与NPC侵袭转移密切相关。miR-155在NPC组织中表达上调,那么miR-155是否与NPC发生发展及转移等相关,目前尚未见报道。因此,miR-155表达失调在NPC发生发展及转移中扮演何种角色,miR-155是否作为NPC一个oncomir等一系列问题都值得我们深入探讨。 基于miR-155研究现状及其在明确NPC癌变早期的分子机制中的潜在意义,本课题的研究目的为：明确miR-155在NPC细胞株和组织中的表达谱及其对NPC细胞生物学行为的影响,包括增殖、迁移和EMT等。我们通过上调和下调niR-155的表达水平明确其对NPC细胞生物学行为的影响,随后进行下游靶基因的筛选和验证,初步探讨miR-155在NPC如上生物学行为中所扮演的角色及其机制,这将为深入理解NPC发病的分子机制和寻找新的分子靶向治疗靶点奠定理论基础,为此本课题进行了如下研究： 方法： 1.NPC细胞株和组织标本中miR-155的表达谱 采用qRT-PCR检测CNE1、CNE2、5-8F、6-10B、C666-1、HNE1、SUNE1和HONE18种NPC细胞株miR-155表达谱(以永生化鼻咽上皮细胞株NP69作为对照)。收集NPC组织标本15例和慢性鼻咽炎症组织标本10例,应用qRT-PCR检测miR-155的表达谱,结果分析运用2-△△Ct方法进行比较。 2.建立稳定过表达miR-155的NPC细胞株 利用慢病毒表达载体pLenti-miR-155生产携带miR-155的慢病毒,收集病毒上清分别感染NPC细胞株CNE1、CNE2、HONE1和SUNE1,48-72h后于倒置荧光显微镜下检测EGFP表达以观察感染情况,若感染效率低于90%,应用流式细胞仪分选EGFP+细胞,大量扩增细胞并保种；提取RNA,应用qRT-PCR检测携带有miR-155转基因的NPC细胞株中miR-155表达水平。 3.过表达miR-155对NPC细胞增殖、迁移及EMT的影响 CCK8实验、平板克隆和细胞周期检测细胞增殖能力改变；Transwell小室检测细胞迁移能力改变,qRT-PCR、Western blot及免疫荧光(ICC)检测EMT相关基因E-cadherin、α-catenin、Fibronectin、N-caherin及vimentin表达水平的变化。 4.利用miR155阻遏物下调NPC细胞中内源性niR-155表达水平以进一步明确miR-155的如上功能 利用脂质体介导瞬时转染技术将化学合成的miR155阻遏物(miR155inhibitors)转染至HONE1和SUNE1中,48h收集细胞并提取RNA, qRT-PCR检测niR-155表达以评价抑制效果,CCK8和Transwell小室分别评价下调内源性miR-155表达水平对细胞增殖和迁移能力的影响,Western blot检测EMT相关基因表达。 结果： 1.NPC细胞株和组织标本中miR-155的表达谱 采用qRT-PCR检测NPC细胞株CNE1、CNE2、5-8F、6-10B、C666-1、HNE1、 SUNE1和HONE1中miR-155表达情况(以NP69为对照)。结果显示,miR-155在以上8株NPC细胞株中的表达水平均显著高于NP69(图1-1A),其中以CNE1最高,HONE1次之。qRT-PCR检测15例NPC组织和10例鼻咽炎症组织中miR-155表达,结果显示两者间无显著差异(图1-1B),这可能是因为我们所选用的正常对照为慢性炎组织标本,而miR-155已被证实在炎性淋巴细胞中高表达,因而干扰了miR-155的检测。 2.建立稳定过表达miR-155的NPC细胞株 慢病毒载体pLenti-miR-155经测序鉴定,明确序列正确(图2-1)；接着利用携带miR-155的慢病毒感染NPC细胞株,48-72h后于倒置荧光显微镜下通过检测EGFP表达确认感染成功,随后提取RNA, qRT-PCR检测证实携带有miR-155转基因的NPC细胞株中miR-155表达正常(表2-1和图2-4),差异均具有统计学意义(P0.001),预示成功构建稳定过表达miR-155的NPC细胞株。 3.过表达miR-155对NPC细胞增殖、迁移及EMT的影响。 3.1增殖能力 CCK8实验、平板克隆和细胞周期等实验结果均表明过表达miR-155可促进NPC细胞增殖(图2-5、图2-6和图2-7)。 3.2迁移能力及EMT相关基因的变化 Transwell实验结果显示：过表达miR-155促进了HONE1和CNE2体外迁移(表2-2和图2-8)。 EMT相关基因的变化：qRT-PCR结果显示,在CNE2和HONE1中过表达miR-155后,上皮细胞标志物E-cadherin和a-catenin表达水平下降(图2-9),差异具有统计学意义(P0.05),而间质细胞标志物N-cadherin和vimentin表达水平升高(图2-9),前者差异有统计学意义(P0.05),后者差异无统计学意义(P=0.212)。 Western blot结果：CNE2和HONE1过表达miR-155后E-cadherin和α-catenin表达水平下降,而间质细胞标志物Fibronectin口vimentin表达水平升高(图2-10)。 ICC结果：CNE2、HONE1过表达miR-155后,E-cadherin(?)α-catenin表达下降,Fibronectin、N-caherin及vimentin表达水平升高,与Western blot结果一致,(图2-11) 上皮-间充质转化(EMT)是指上皮细胞通过特定程序转化为具有间质表型细胞的生物学过程,EMT是上皮细胞来源的恶性肿瘤细胞获得迁移和侵袭能力的重要生物学过程。在EMT发生过程中常会导致上皮细胞标志物E-cadherin、 α-catenin等表达水平下调,造成细胞间粘附能力降低,而间质细胞标记物Fibronectin、N-caherin(?)vimentin表达水平升高,则导致细胞迁移能力增强。 4.利用miR155阻遏物下调NPC细胞中内源性niR-155表达对细胞增殖、迁移和EMT相关基因表达的影响 4.1miR155inhibitors下调NPC细胞中内源性miR-155表达 利用脂质体介导瞬时转染技术将化学合成的miR155阻遏物(miRl55inhibitors)导入HONE1和SUNE1中,48h收集细胞并提取RNA, qRT-PCR检测miR-155表达,结果显示,内源性miR-155的抑制效率达80%左右(图2-12)。 4.2细胞增殖能力变化 CCK8生长曲线结果显示,HONE1和SUNE1中内源性miR-155表达下调后,细胞增殖能力下降(图2-13)。 4.3细胞迁移能力及EMT相关基因水平的变化 Transwell小室实验：HONE1和SUNE1中内源性miR-155表达下调后,细胞迁移能力减弱(表2-8和图2-14)。 Western blot结果：下调HONE1和SUNE1中miR-155表达,导致E-cadherin表达上调和vimentin表达下调(图2-15)。 结论： miR-155可促进NPC细胞体外增殖和迁移能力,且能促进EMT发生。
[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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