LMP1对人鼻咽癌细胞系CNE1癌基因微小RNA表达谱的影响

发布时间：2018-06-28 21:48

本文选题：微小RNA + 癌基因微小RNA　；参考：《广西医科大学》2010年博士论文

【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(P0.05)。同时LMP1蛋白表达与hsa-miR-19b表达相关(r=0.390,P0.05)。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。
[Abstract]:Background and purpose
Nasopharyngeal carcinoma is one of the most common malignant tumors in southern and Southeast Asia, and has obvious regional distribution characteristics. The close relationship with EB virus.LMP1 is a viral oncogene expressed in nasopharyngeal carcinoma cells with EBV II latent infection. He can promote the transformation of epithelial cells and can activate the downstream genes through the signal pathways such as NF- kappa B, p38, JNK and so on. At the same time, it can regulate the expression of miRNAs in the lymphoma cells of EBV type III latent infection, and maintain the latent infection state of the virus through the regulation of the target gene of miRNAs, and affect the biological behavior of the tumor and so on.
MiRNAs a new, endogenous, non protein encoded small RNA molecule, with about 22 nucleotides long, exerting the regulation of gene expression after transcriptional gene expression,.MiRNAs, which inhibits the translation or degradation of target mRNA through miRISC, and regulates cell development, differentiation, proliferation, apoptosis, and metabolites. Some of these miRNAs have oncogenes and tumor suppressor. The function of the gene, known as the oncogene miRNAs (oncomiRs), plays an important role in the biological behavior of the tumor.
Through high throughput screening by miRNA chip, identifying differentially expressed miRNAs molecules, especially oncomiRs molecules, can fully understand the molecular mechanism of tumor development and development, and more accurately classify and prognosis the tumor. Find out the differential expression: after miRNA, multiple target gene prediction software can be used to predict the target gene. It is of great significance to understand its biological function. Recently, some scholars have predicted the biological function of differential expression of miRNA by means of the signal pathway involved in the expression of the target gene of miRNA in the differential expression of the nasopharyngeal carcinoma tissue from the normal tissue adjacent to the cancer, and found that they can interfere with the target gene to interfere with the biological behavior of the tumor. Related signaling pathways play an important role in tumor growth and proliferation, apoptosis, metastasis and angiogenesis.
The functional study of miRNA and the results of bioinformatics prediction can be carried out by transfection of miRNA mimics, antagomiRs or miRNA inhibitor to induce or reverse the function of miRNA, to study or verify the function of the miRNA molecule in the cell. The effectiveness of the transfection can be confirmed by the change of the expression level of the target gene. The related target genes are expressed to explore the specific mechanism of its function.
The purpose of this study was to investigate the effect of LMP1 on the expression of miRNA in nasopharyngeal carcinoma cells and to explore its effect on the biological behavior of nasopharyngeal carcinoma. By comparing the latent membrane protein 1 (latent membrane protein 1, LMP1) of the nasopharyngeal carcinoma cell line CNE1 and its EB virus (latent membrane protein 1, LMP1), the oncogene micro RNA (oncogenic microRNAs) was steadily transfected into the cell line CNE1-LMP1. Rs) the difference of expression profiles and the effect of LMP1 on the expression of CNE1oncomiRs in nasopharyngeal carcinoma cell line of EBV type II latent infection. And through bioinformatics analysis, the key molecules of cell biological function were found, and the effects on the biological behavior of nasopharyngeal carcinoma were investigated by antagomiRs blocking and in nasopharyngeal carcinoma tissue. The expression of LMP1 and its correlation with clinicopathological characteristics were detected, and its significance in nasopharyngeal carcinoma tissues was discussed.
Method
1. the expression and differential expression profiles of the stable transfected cell line oncomiRs of nasopharyngeal carcinoma cell line CNE1 and its EBV LMP1 were detected by a membrane based microRNA chip containing 132 oncomiRs molecules. Real-time quantitative PCR detection was used to verify the expression of miRNAs molecules with a large difference (up to 2 times), and the results were analyzed to verify the chip detection. The reliability of the result.
2. the miRNA molecular function was used to predict the online tool DIANA-mirPath to analyze the known signal pathways involved in the differential expression of miRNA, the Kyoto gene and the genome Encyclopedia (KEGG) signal pathway, to clarify the biological functions of their participation.
3. the hsa-miR-19b in nasopharyngeal carcinoma cell CNE1-LMP1 was blocked by hsa-miR-19b knockdown probe, and the effects on cell cycle, proliferation, apoptosis, cell migration and invasion were analyzed, and the effect on the target gene protein SOCS1 and STAT3 (Signal transducer and activator of transcription 3) signal pathway to explore nasopharyngeal carcinoma preliminarily. The function and mechanism of hsa-miR-19b in cells.
4. the hsa-miR-19b molecules with more distinct differences in expression were obtained by selection of chip and qRT-PCR. Real-time quantitative PCR was used to detect the most differentially expressed hsa-miR-19b expression in 46 cases of nasopharyngeal carcinoma, and the expression of LMP1 in 46 nasopharyngeal carcinoma tissues was detected by immunohistochemistry in situ, and the correlation between the expression of LMP1 and the expression of LMP1 in 46 cases of nasopharyngeal carcinoma was investigated. The relationship between the two expressions and clinicopathological features was discussed, and the significance of its expression as diagnostic molecular marker was discussed.
Result
OncomiRs expression profile of 1.CNE1
Of the 132 oncomiRs contained in the chip, 21 of the oncomiRs molecules were detected in CNE1, of which 4 were highly expressed (the ratio of expression to the internal reference RUN48 was greater than 1), and 17 of the low expression (the expression and the RUN48 ratio of the internal reference were less than 1).
OncomiRs expression profiles of 2. CNE1-LMP1
Of the 132 oncomiRs contained in the chip, 30 were detected in NE1-LMP1, of which 7 were highly expressed (the ratio of expression and the RUN48 ratio of the internal reference was greater than 1), and 23 of the low expression (the expression and the RUN48 ratio of the internal reference were less than 1).
OncomiRs differential expression profiles of 3.CNE1 and CNE1-LMP1
By comparing the difference of miRNAs (oncomiRs) expression profiles between CNE1 and LMP1 stable transfected cell line CNE1-LMP1, CNE1 detected 21 oncomiRs molecules, and 30 in CNE1-LMP1, indicating that LMP1 could regulate the expression of miRNAs. Compared with CNE1, the total expression of the molecule increased in CNE1-LMP1, and 9 in the CNE1-LMP1. Specific expression in MP1. Analysis of 21 miRNA molecules expressed by co expression found that 8 slightly decreased in CNE1-LMP1 and 13 expressions rising, of which 7 miRNA molecules raised up to two times higher than 7: hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150, hsa-miR-188 and hsa-miR-205.9 in CNE1-LMP1. In A, hsa-miR-122a was highly expressed, and its relative expression was greater than 1.. In summary, the overall level of miRNA expression in CNE1-LMP1 increased.
4. qRT-PCR validation of differentially expressed miRNA molecules in two cell lines
Through the fluorescence quantitative RT-RCR detection, 7 (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150, hsa-miR-188 and hsa-miR-205) were detected by the fluorescence quantitative detection. The difference of the difference between the detection results of the chip detection was related to the difference of the results of qRT-RCR detection (r=0.970, P=0.000). The two methods of detection were detected. The fruit is the same.
5. DIANA-mirPath signaling pathways and functional prediction of differential expression of miRNA
The number of target genes associated with the cell signaling pathway was predicted by software, and 95 of the 8 miRNA molecules expressed differently were predicted to participate in 44 of the signaling pathway targets, 53 miR-19b and 4 of the other 6. This shows that the contribution of miR-17 and miR-19b to the signaling pathway is most. Signal transduction is closely related to the tumor, including signal transduction of environmental information, cell motility, growth and death, cell communication, and multiple tumor related pathways. At the same time, the change multiple and expression of miR-19b were found to be significantly higher than that of miR-17, so miR-19b was selected for follow-up study.
6. hsa-miR-19b function in nasopharyngeal carcinoma cell CNE1-LMP1
After hsa-miR-19b blockage, cell proliferation decreased (the inhibition rate of group 5pmol was 20.45%, group 11pmol was 44.50%), cell cycle was blocked in G1, the percentage of cells in G1 phase increased obviously after hsa-miR-19b blocking, and cell apoptosis increased obviously; cell migration and invasion energy decreased after hsa-miR-19b blocking. Meanwhile, the expression of SOCS1 protein in target gene increased obviously. The phosphorylation level of STAT3 is reduced.
7. expression of LMP1 and hsa-miR-19b in nasopharyngeal carcinoma and its relationship with clinicopathological characteristics
The positive expression rate of LMP1 protein in NPC tissue was 60.95% (28/46), and the expression of hsa-miR-19b was 68.27 + 69. The expression of LMP1 protein and hsa-miR-19b expression were related to the tumor stage and lymph node metastasis (P0.05), and the expression of LMP1 protein was associated with the expression of hsa-miR-19b (r=0.390, P0.05).
conclusion
1. oncomiRs expression profiles of nasopharyngeal carcinoma cell line CNE1 and its LMP1 transfected cell line CNE1-LMP1 were different.
2.LMP1 can regulate the expression of oncomiRs in nasopharyngeal carcinoma cell line and increase the overall expression level of miRNAs. The possible mechanism is to promote the expression of miRNAs. in the miRNAs gene through its transcriptional activation function.
3.LMP1 may regulate the expression of oncomiRs in nasopharyngeal carcinoma cell line, and it can become another important pathway to play the role of viral oncogene.
4. of the 8 highly differentially expressed miRNA molecules, the target genes of hsa-miR-17 and hsa-miR-19b have the greatest contribution to the cell signaling pathway, and their involvement in signal transduction, cell movement, growth and death, cell communication signaling pathways, and tumor related signaling pathways are closely related to the development of tumor development.
5. hsa-miR-19b can promote the growth and proliferation of nasopharyngeal carcinoma cells and inhibit cell apoptosis. The possible pathways are: inhibition of the target gene SOCS1 and the inhibition of the STAT3 signaling pathway.Hsa-miR-19b can also promote cell migration and invasion.
6. the expression of LMP1 and hsa-miR-19b in nasopharyngeal poorly differentiated squamous cell carcinoma.
7. in nasopharyngeal poorly differentiated squamous cell carcinoma, LMP1 may be involved in the regulation of hsa-miR-19b expression.
8.LMP1 and hsa-miR-19b can be used as diagnostic markers for staging and metastasis of poorly differentiated squamous cell carcinoma of the nasopharynx.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

【参考文献】