【作者】 肖胜军；

【导师】 韦敏怡；

【作者基本信息】 广西医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 背景与目的鼻咽癌是中国南方和东南亚地区最常见的恶性肿瘤之一,具有明显的区域分布特征、与EB病毒的密切关系。LMP1是EBVⅡ型潜伏感染的鼻咽癌细胞表达的一个病毒癌基因,他能促进上皮细胞转化,能通过NF-κB、p38、JNK等信号通路激活下游基因的转录,影响细胞的生长与增殖、凋亡以及侵蚀和转移能力。同时,它在EBVⅢ型潜伏感染的淋巴瘤细胞中能调节miRNAs的表达,并通过miRNAs的靶基因调控作用保持病毒的潜伏感染状态、影响肿瘤的生物学行为等等。miRNAs一种新型的、内源性的、非蛋白编码小RNA分子,长约22个核苷酸,发挥转录后基因表达调控作用。miRNAs通过miRISC抑制靶mRNA的翻译或促使其降解,从而调节细胞的发育、分化、增殖、凋亡、代谢等生命活动。其中部分miRNAs具有癌基因和肿瘤抑制基因的功能,称为癌基因miRNAs (oncomiRs),对肿瘤的生物学行为起重要作用。通过miRNA芯片进行高通量筛选,找出差异表达的miRNAs分子,特别是oncomiRs分子,能全面了解肿瘤发生和发展的分子机制、更为精确地对肿瘤的进行分型和预后。找出差异表达：miRNA分子后,可以通过多个靶基因预测软件对靶基因进行预测,对于了解其生物学功能具有重要意义。最近有学者通过对鼻咽癌组织与癌旁正常组织的差异表达miRNA的靶基因参与的信号通路进行计算机预测,来了解差异表达miRNA的生物学功能,并发现它们能通过靶基因干预多条与肿瘤生物学行为相关的信号通路,对肿瘤的生长与增殖、凋亡、转移及血管生成产生重要作用。miRNA的功能研究及生物信息学预测结果可以通过转染miRNA mimics、antagomiRs或miRNA抑制剂,正向或反向诱导miRNA的功能,来研究或验证细胞中该miRNA分子的功能。该转染的有效性可以通过靶基因的表达水平的改变获得证实。同时可以通过检测相关靶基因的表达来探讨其功能发生的具体机制。本实验旨在探讨LMP1对鼻咽癌细胞miRNA表达的调控作用,并探讨其对鼻咽癌生物学行为的影响。通过比较鼻咽癌细胞系CNE1与其EB病毒的潜伏膜蛋白1(latent membrane protein 1, LMP1)稳定转染细胞系CNE1-LMP1的癌基因微小RNA (oncogenic microRNAs, oncomiRs)表达谱的差异,探讨LMP1对EBVⅡ型潜伏感染的鼻咽癌细胞系CNE1oncomiRs表达的影响。并通过生物信息学分析,从中找出对细胞生物学功能影响较关键的分子；通过antagomiRs阻断后探讨其对鼻咽癌细胞生物学行为的影响,并在鼻咽癌组织中检测其表达及与LMP1的相关性及与临床病理特征的关系,探讨其在鼻咽癌组织中表达的意义。方法1.采用包含有132个oncomiRs分子的膜基microRNA芯片,检测鼻咽癌细胞系CNE1及其EBV LMP1的稳定转染细胞系oncomiRs的表达谱及差异表达谱。采用实时定量PCR检测验证表达差异较大的(达2倍)miRNAs分子,并对两者结果进行相关分析,验证芯片检测结果的可靠性。2.采用miRNA分子功能预测在线工具DIANA-mirPath,分析差异表达miRNA参与的已知信号通路——京都基因及基因组百科全书(KEGG)信号通路,来阐明它们参与的生物学功能。3.通过hsa-miR-19b knockdown探针阻断鼻咽癌细胞CNE1-LMP1中的hsa-miR-19b,然后分析其对细胞周期、增殖、凋亡和细胞迁移、侵袭的影响；及其对靶基因蛋白SOCS1及STAT3 (Signal transducer and activator of transcription 3)信号通路的影响,以初步探讨鼻咽癌细胞中hsa-miR-19b的功能和作用机制。4.选择芯片筛选及qRT-PCR验证获得的表达差异较为明显的hsa-miR-19b分子,采用实时定量PCR检测46例鼻咽癌组织中差异表达最大的miRNA分子hsa-miR-19b表达,并采用免疫组化技术原位检测46例鼻咽癌组织中LMP1表达,并探讨其与LMP1表达的相关性。并探讨两者表达与临床病理特征之间的关系,探讨其表达作为诊断分子标记的意义。结果1.CNE1的oncomiRs表达谱在芯片包含的132个oncomiRs中,CNE1中检出oncomiRs分子21个；其中高表达的(表达量与内参RUN48比大于1)有4个,低表达(表达量与内参RUN48比小于1)的有17个。2. CNE1-LMP1的oncomiRs表达谱在芯片包含的132个oncomiRs中,NE1-LMP1中检出30个；其中高表达的(表达量与内参RUN48比大于1)有7个,低表达(表达量与内参RUN48比小于1)的有23个。3.CNE1和CNE1-LMP1的oncomiRs差异表达谱通过比较较鼻咽癌细胞系CNE1与其LMP1稳定转染细胞系CNE1-LMP1的肿瘤相关miRNAs (oncomiRs)表达谱的差异,其中CNE1检出oncomiRs分子21个,而CNE1-LMP1中检出30个,显示LMP1能调节miRNAs的表达。与CNE1比较,CNE1-LMP1中miRNAs分子表达总量增加,有9个在CNE1-LMP1中特异性表达。分析共同表达的21个miRNA分子发现,CNE1-LMP1中有8个轻度降低,13个表达升高,其中表达升高达两倍以上的miRNA分子有7个:hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149>、hsa-miR-150、hsa-miR-188和hsa-miR-205。9个在CNE1-LMP1中特异性表达的miRNA中,hsa-miR-122a呈高水平表达,其表达量相对值大于1。综上所述,CNE1-LMP1中miRNA表达的总体水平升高。4. qRT-PCR验证两细胞系中差异表达较大的miRNA分子通过荧光定量RT-RCR检测,对芯片检测发现的7个(hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149、hsa-miR-150、hsa-miR-188和hsa-miR-205)表达差异较大的miRNA进行验证,芯片检测发现结果差异与qRT-RCR检测发现的结果差异相关(r=0.970,P=0.000)。两种方法检测结果一致。5. DIANA-mirPath对差异表达miRNA参与的信号通路及功能预测通过软件预测到与细胞信号通路相关的靶基因数目总共有95个；在差异表达的8个miRNA分子中,miR-17有预测参与信号通路靶基因44个,miR-19b有53个,其余6个共计4个。由此可见miR-17和miR-19b对信号通路的贡献占了绝大部分。两者参与了多条信号通路,与肿瘤密切相关的有环境信息的信号转导,细胞的运动性、生长与死亡及细胞通讯,多种肿瘤相关通路等。同时前期发现miR-19b的改变倍数及表达量显著高于miR-17,因此将miR-19b挑出进行后续功能研究。6.鼻咽癌细胞CNE1-LMP1中hsa-miR-19b功能初探hsa-miR-19b阻断后,细胞增殖下降(5pmol组抑制率为20.45%,11pmol组为44.50%)；细胞周期阻滞于G1,hsa-miR-19b阻断后G1期细胞百分比明显增加,而细胞凋亡明显增加；hsa-miR-19b阻断后细胞的迁移、侵袭能力下降。同时,其靶基因SOCS1蛋白表达明显升高,使STAT3磷酸化水平下降。7.鼻咽癌组织中LMP1与hsa-miR-19b的表达及与临床病理特征的关系NPC组织中LMP1蛋白阳性表达率为60.95%(28/46), hsa-miR-19b的表达量为68.27±69.00；LMP1蛋白表达及hsa-miR-19b表达均与肿瘤分期和淋巴结转移相关(P<0.05)。同时LMP1蛋白表达与hsa-miR-19b表达相关(r=0.390,P<0.05)。结论1.鼻咽癌细胞系CNE1与其LMP1稳定转染细胞系CNE1-LMP1的oncomiRs表达谱存在差异。2.LMP1能调节鼻咽癌细胞系oncomiRs的表达,使miRNAs的总体表达水平升高,其可能机制为通过其转录激活功能促进miRNAs的编码基因表达miRNAs。3.LMP1可能调节鼻咽癌细胞系oncomiRs的表达,可以成为其发挥病毒癌基因作用的另一重要的通路。4.在8个差异表达较大的miRNA分子中,hsa-miR-17和hsa-miR-19b的靶基因对细胞信号通路的贡献最大,其参与的信号转导、细胞的运动、生长与死亡、细胞通讯的信号通路、肿瘤相关信号通路与肿瘤的发生发展密切相关。5. hsa-miR-19b能促进鼻咽癌细胞生长与增殖、抑制细胞凋亡；其作用的可能途径有：通过抑制其靶基因SOCS1而抑制STAT3信号通路。hsa-miR-19b还能促进细胞迁移和侵袭。6.鼻咽部低分化鳞状细胞癌组织中存在LMP1和hsa-miR-19b的表达。7.鼻咽部低分化鳞状细胞癌组织中,LMP1可能参与了hsa-miR-19b的表达调控。8.LMP1和hsa-miR-19b可以作为鼻咽部低分化鳞状细胞癌的分期及转移的诊断分子标记。更多还原

【Abstract】 Background and objective:The incidence of nasopharyngeal carcinoma (NPC) is most prevalent in Southeast Asia, particularly in Southern China. NPC is characterized by regional distribution and EBV-associated. LMP1 is a viral oncogene expressed in nasopharyngeal carcinoma cells, which exhibite latency type II of EBV infection. LMP1 can promote epithelial cells transformation through hijacking cell signaling pathway such as NF-κB, p38/MAPK and JNK signaling to activate the transcription of downstream genes. The activated protein expression profile are widely involed in cell growth, proliferation, apoptosis, invasion and metastasis. Meanwhile, LMP1 can regulate the expression of miRNAs in lymphoma that exhibte latency typeⅢof EBV infection.This regulation can keep the latent infection of EBV and change the biological characters of tumor cells.miRNAs is a novel class of small(-22 nucleotide) non-protein-coding RNA molecular, which function as post-transcriptional gene regulator. miRNAs recruit other factors to assemble a complex named with miRISC(miRNA induced silencing complex), which suppress translation or promote degration of target mRNAs. miRNAs regulate diverse biological process such as development, differetiation, proliferation, apoptosis and metabolism. Differential expression profile study by highthrough screening through miRNA microarray to find differentially expressed miRNA, especially oncomiRs, is a good way to elucidate the globle molecular mechanism of development and progression of tumor, and also a good index in typing and prognosis of cancer. The targets of differentially expressed miRNA can be predicted by various predicting software to comprehend their biological function in cells. Recently, one group found the differentially expressed miRNAs between nasopharyngeal carcinoma tissue and adjacent normal tissue. Then the cell signal pathway mediated by their targets were predicted in silicon to elucidate their biological effects. Various signal pathway, which includes several pathway associated with tumoric biological characters, were interferred by their targets and were involved in cell growth, proliferation, apoptosis, metastasis and angiogenesis. Function of miRNA can be studied in model cells by transfecting with miRNA mimics and antagomiRs (miRNA inhibitors) to induced the function of miRNA positively or inversely. The results from bioinformatics can also be verified by transfecting assay. The efficacy of transfection can be validation by targets detection. Simultaneously, the detailed mechanism of miRNA functioning can be indicated by targets assay.In order to explore the regulating effection of LMP1 on oncomiRs expression in CNE1 cell line and ultimate effection on biological characters in nasopharyngeal carcinoma. We investigate the differential oncomiRs expression profile between nasopharyngeal carcinoma cell line CNE1 and it’s steady EBV-LMP1-transfected cell line CNE1-LMP1. Among the differentially expressed oncomiRs, the miRNAs play a key role in cellular biological function were picked out by bioinformatical assay. AntagomiRs were used to block the selected miRNAs to investigate it’s role in biological characters of nasopharyngeal cells. It’s expression in nasophayngeal carcinoma tissue were detected and the association with expression of LMP1 were assessed. Both of them were evaluated their association with clinopathological characters of nasopharyngeal carcinoma to explore the significance of their expression in nasopharyngeal carcinoma tissue.Then detect the most differentially expressed miRNA in tissues of nasopharyngeal carcinoma, evaluate the association between it’s expression value and LMP1 expression and their expression with clincopathological characters. And transfection knockdown probe into model cell to block the miRNA to elucidate its prelimilary function.Methods:1. A membrane-based microRNA array that targets 132 of the most well studied oncomiRs were used to detect the expression profile of CNE1 and CNE1-LMP1. And then real time qRT-PCR assay verified the expression data of the most differentiatedly expressed(changed over 2 folds) miRNAs. The data of miRNA array and qRT-PCR were analysised by correlation analysis to verify the reliability of miRNA array assay.2. The KEGG (kyoto encyclopedia of genes and genomes) pathway employed by the differentially expressed oncomiRs to execute their biological function were predicted by DIANA-mirPath, a online functional prediction tool of miRNA.3. Cell cycle, proliferation, apoptosis, cell migration and invasion in CNE1-LMP1 cells were evaluated after knockdown of hsa-miR-19b. STAT3 signaling pathway and SOCS1 (a target gene of hsa-miR-19b, also suppressor of STAT3 signaling) were also detected to elucidate the preliminary function and fundamental mechanism of hsa-miR-19b.4.46 cases of nasopharyngeal carcinoma were enrolled in the study before radiotherapy. LMP1 protein detected by immunohistochemisty and hsa-miR-19b by real time qRT-PCR. The correlation between these two index were assessed. hsa-miR-19b detected by real time qRT-PCR. The correlation between LMP1 and hsa-miR-19b expression were assessed. Meanwhile, evaluate the correlation between LMP1 or hsa-miR-19b and clinicopathological characters.Results:1. The oncomiRs expression profile of CNE1 cells shows as below. Among the restricted 132 miRNAs,21 were detectable in CNE1. There is 4 miRNAs with higher expression levels(expression levels higher than internal reference RUN48); And 17 with lower expression levels(expression levels lower than internal reference RUN48).2. The oncomiRs expression profile of CNE1-LMP1 cells shows as below. Among the restricted 132 miRNAs,30 were detectable in CNE1. There is 7 miRNAs with higher expression levels(expression levels higher than internal reference RUN48). And 23 with lower expression levels(expression levels lower than internal reference RUN48).3. The differential expression profile between CNE1 and CNE1-LMP1. Among the restricted 132 miRNAs,30 were detectable in CNE1-LMP1 and 21 in CNE1. There is 21 shared miRNAs, among which 7 miRNAs’expression level (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150, hsa-miR-188 and hsa-miR-205) elevated over two folds. Among the 9 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a has a highest expression level surpass the internal control sample. Altogether, CNE1-LMP1 showed higher total miRNAs levels.4. qRT-PCR verify the expression detected by miRNA array. The expression differentiation of 7 most differentiatedly expressed miRNAs confirmed by the real qRT-PCR. The results show that they are closely correlated(r=0.970, P=0.000).5. DIANA-miPath predict the pathway and function of differentially expressed miRNAsThe software predicts that the total targets involved in cell signal pathway is 95. Among 8 differentially expressed miRNAs, miR-17 has 44 targets, miR-19b has 53 and the other 6 miRNAs has only 4. From this, we included that miR-17 and miR-19b contribute to most of the biological function through signal pathway. The two major miRNAs involved in various pathway. Among them, the signal pathway which closely associated with tumor includes enviromental signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway. Taking the previous expression data together(miR-19b shared the higher expression level and changed times), we pick out miR-19b for further functional assay.6. The preliminary function of hsa-miR-19b in CNE1-LMP1After knockdown the hsa-miR-19b, cell proliferation decreased (5pmol group cell proliferation inhibit rate is 20.45%, llpmol group is 44.50%); the percentage of cells in G1 stage increased, cell cycle is blocked in G1 stage; And the apoptosis increased. Meanwhile, the expression of it’s target protein, SOCS1 increased; lead to lower phosphorylation level of STAT3.7. The in situ expression of LMP1 and hsa-miR-19b and their correlation with clinopathological charactersThe positive expression rate of LMP1 protein is 60.95%(28/46). The relative expression value of hsa-miR-19b is 68.27±69.00. Both LMP1 and hsa-miR-19b were associated with stage and lymphatic metastasis(P<0.05).The expression of LMP1 were associated with the expression of hsa-miR-19b (r=0.390, P<0.05).Conclusions:1. oncomiRs are differentially expressed between CNE1 cells and CNE1-LMP1 cells.2. LMP1 can regulate the expression of oncomiRs in nasopharyngeal carcinoma cell line and elevate the total miRNAs level through it’s transcription-activating function.3. Regulating the expression of miRNAs would be another important pathway employed by LMP1 to function as a viral oncogene.4. Among 8 differentially expressed miRNAs, hsa-miR-17 and has-miR-17 contribute most to the cell signal pathway, among which signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway were closely associated with the development and progression of tumor.5. Hsa-miR-19b can promote cell cycle and proliferation, inhibit apoptosis. The possible mechanism employed in these process may be constructively activate the STAT3 signaling. In which hsa-miR-19b inhibit the translation of it’s target gene SOCS1, and ultimately abolished the blockage of the phosphorylation of STAT3. Hsa-miR-19b can also promote cell migration and ivasion in vitro.6. LMP1 and hsa-miR-19b showed expression in low differentiated squamous cell carcinoma of nsaopharynx epithelia.7. LMP1 would be involved in expression regulation of hsa-miR-19b in low differentiated squamous cell carcinoma of nsaopharynx epithelia.8. LMP1 and hsa-miR-19b can functionate as a diagnosis molecular marker in low differentiated squamous cell carcinoma of nsaopharynx epithelia.更多还原