鼻咽癌相关基因ID2与EMT关系的初步探讨

发布时间：2018-07-08 08:44

本文选题：鼻咽癌 + EMT　；参考：《南方医科大学》2010年硕士论文

【摘要】： 研究背景和目的鼻咽癌(NasoPharyngeal Carcinoma,NPC)是一种主要发生在我国南方地区的鼻咽部恶性肿瘤,由于NPC发生的部位比较隐蔽,早期症状常不明显而容易被忽略,因此确诊的患者60%以上都是中、晚期病例,常伴有转移发生。寻找鼻咽癌发生发展及转移的机制,是我们当前研究的主要方向。肿瘤的形成和侵袭转移是一个多步骤、多阶段、多因子参与的复杂而又连续的过程,涉及了一些关键的瘤基因和抑瘤基因。在本研究中,我们从公共基因芯片数据库GEO(Gene expression omnibus)中寻找并下载鼻咽癌的相关基因芯片数据,并使用Genclip软件对其进行分析,找到鼻咽癌公共基因芯片数据中与上皮间充质转化(Epithelial——mesenchymal transition, EMT)相关的基因35个,其中包括本课题所研究的ID2,它在鼻咽癌组织和细胞中表达明显上调,提示该基因可能正向参与促进了鼻咽癌的发病过程。 ID蛋白家族成员共有四个ID1, ID2, ID3, ID4,它们最初被发现在负性调节细胞分化方面起了重要作用,后来越来越多的研究证明它们在调节谱系定型,基因表达,细胞命运,细胞增殖,以及在神经发生,淋巴组织形成,新生血管形成中细胞的分化时间也起了关键作用。ID蛋白家族成员被发现在很多肿瘤中过表达,比如大肠癌,胶质母细胞瘤,髓母细胞瘤,神经细胞瘤,胰腺癌,甲状腺癌,鳞状细胞癌,前列腺癌,乳腺癌,子宫内膜癌,宫颈癌和黑色素瘤等。为了阐明ID2基因在鼻咽癌发病中的作用,我们利用RNAi技术高效、特异的靶向沉默机制干扰鼻咽癌细胞SUNE1中ID2表达,建立稳定靶向干扰ID2表达的siRNA鼻咽癌细胞,以观察干扰ID2后鼻咽癌细胞生物学功能改变,并探寻其与EMT的关系,为鼻咽癌的发病机制和治疗提供新的思路。方法 1.鼻咽癌EMT相关基因的筛选从公共基因芯片数据库GEO (Gene expression omnibus)中寻找并下载鼻咽癌的相关基因芯片数据,并使用Genclip软件对下载的数据进行分析,寻找鼻咽癌公共基因芯片数据中与上皮间充质转化(Epithelial——mesenchymal transition, EMT)相关的基因；并用生物信息学方法对筛选出来的基因进一步分析。 2. ID2在鼻咽癌中的表达特性鉴定免疫组化SP法从蛋白水平检测ID2在非癌鼻咽组织和鼻咽癌中的表达水平,初步探讨ID2在组织水平与鼻咽癌发生发展的关系。荧光定量PCR法从mRNA水平检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1中的表达水平,初步探讨ID2在永生化鼻咽上皮细胞和鼻咽癌细胞中的表达差异,并找到高表达ID2的鼻咽癌细胞作为下一步干扰对象。 3. ID2表达沉默对鼻咽癌细胞生物学影响及部分与EMT相关基因表达水平的检测构建ID2慢病毒干扰载体,包装成成熟的慢病毒颗粒感染高表达ID2的SUNE1细胞,有限稀释法筛选出稳定的阳性和空载克隆,荧光定量PCR检测各干扰克隆细胞中ID2干扰效率,筛选干扰效率最高的细胞克隆并将该细胞克隆作为实验对象被进一步研究。MTT法、平板克隆形成实验检测ID2沉默后对细胞体外增殖的影响；Transwell检测ID2沉默后细胞迁移运动能力的改变。荧光定量PCR法检测与ID2相关的TGF-beta以及与EMT相关的转录因子Slug, SIP1, Twist, Snail,上皮标志物E-cadherin,间质标志物Vimentin在四株细胞株SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv, SUNE1中mRNA的表达水平。 4.统计学方法采用SPSS 13.0统计软件包进行统计学处理,第二章中因免疫组化各指标均为等级资料,故其组间的比较采用非参数秩和检验,ID2的表达在非癌鼻咽组织和鼻咽癌比较采用Mann-Whitney U检验,ID2的表达与鼻咽癌临床分期的关系检验用Kruskal Wallis Test,检验水准均取双侧α=0.05；荧光定量PCR法检测鼻咽癌细胞株ID2表达特性结果比较采用单因素方差分析,多重比较采用SNK检验。第三章中荧光定量PCR、运动、平板克隆形成实验采用单因素方差分析；MTT采用析因设计的方差分析；多重比较采用SNK或LSD检验。结果 1.鼻咽癌EMT相关基因的筛选在公共的鼻咽癌芯片数据中找到35个与EMT相关的差异表达基因,而且这些基因功能大致与细胞组分、细胞粘附、通信、信号转导、分化、运动、迁移以及细胞表面受体相关的信号转导等有关,这些功能往往都被认为与肿瘤的侵袭和转移有关。 2.ID2在鼻咽癌中的表达特性鉴定 1、共收集鼻咽炎组18例、鼻咽癌组52例临床标本。SP免疫组化结果表明,ID2表达主要定位在细胞核和细胞浆。ID2在鼻咽癌和非癌鼻咽组织中的表达具有统计学差异(Z=-5.553, P=0.000), ID2的表达情况与临床分期没有统计学差异(χ2=0.963,P=0.810) 2、荧光定量PCR法检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1 mRNA水平表达具有统计学差异(F=2.980, P=0.002), SUNE1表达最高,表达量为NP69 ID2表达量的78倍,作为下一步干扰对象。 3.ID2表达沉默对鼻咽癌细胞生物学影响及部分与EMT相关基因表达水平的检测 1、构建ID2慢病毒干扰载体,包装慢病毒后感染SUNE1细胞,有限稀释法筛选GFP+单克隆,荧光定量PCR检测各克隆的ID2表达情况,筛选出干扰效率最高的2个单克隆为下一步实验的研究对象。阳性克隆的干扰效率与SUNE1和阴性对照细胞表达具有统计学差异(F=48.693,P=0.000),同时检测ID2在阴性对照中的表达相对于SUNE1强度较一致(0.933±0.006)。阳性干扰克隆命名为SUNE1/plv-ID2-1和SUNE1/plv-ID2-2,阴性对照克隆为SUNE1/plv。 2、MTT法检测细胞增殖情况,与阴性对照SUNE1/Plv和SUNE1细胞相比,干扰克隆细胞SUNE1/plv-ID2-1和SUNE1/plv-ID2-2的增殖速度明显减慢并且呈时间依赖关系,具有统计学差异(F=3.301,P=0.000)；平板克隆形成实验显示四组细胞的克隆形成率具有统计学差异(F=253.434,P=0.000), SUNE1/plv-ID2细胞克隆形成减少,SUNE1和阴性对照组不具有统计学差异(P=0.866)。综合表明ID2表达干扰后,SUNE1细胞体外生长被显著抑制。 3、Transwell小室检测结果显示,与SUNE1和SUNE1/plv细胞相比,SUNE1 /plv-ID2细胞穿过聚碳酸酯膜的细胞数没有明显变化,差异不具有显著性(F=0.624,P=0.610)。 4、荧光定量PCR法检测结果显示TGF-beta, E-cadherin, Slug, SIP1, Vimentin在4组细胞间的mRNA表达不具有统计学差异(F=0.405,P=0.754；F=0.571,P=0.650; F=0.541,P=0.668; F=0.456,P=0.721; F=1.058,P=0.419),而Twist, Snail在4组细胞间的表达具有统计学差异(F=94.627,P=0.000;F=155.295,P=0.000)。结论 1、生物信息学方法筛选出35个与鼻咽癌EMT相关的基因 2、免疫组化SP法证实ID2的表达与鼻咽癌的发生发展有关系。 3、荧光定量PCR法检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1中的表达具有统计学差异,SUNE1表达最高。 4、ID2干扰后部分细胞体外生物学功能发生改变。 5、ID2干扰后部分与EMT相关的基因1nRNA表达水平出现改变。
[Abstract]:Background and purpose of research
NasoPharyngeal Carcinoma (NPC) is a malignant tumor of nasopharynx, which mainly occurs in the southern part of China. Because the location of NPC is more concealed, the early symptoms are often not obvious and easy to be ignored. Therefore, more than 60% of the confirmed patients are in the middle and late cases, often accompanied by metastasis. The mechanism is the main direction of our current research.
The formation and invasion of tumor is a complex and continuous process involving multiple steps, multistages and multiple factors. It involves some key tumor genes and tumor suppressor genes. In this study, we find and download the related gene chip data of nasopharyngeal carcinoma from the public gene chip database GEO (Gene Expression Omnibus) and use G The enclip software analyzed it to find 35 genes related to Epithelial - mesenchymal transition (EMT) in the common gene chip data of nasopharyngeal carcinoma, including ID2, which was studied in this subject. It was expressed clearly in the tissues and cells of nasopharyngeal carcinoma, suggesting that the gene may be positively involved in promoting the nose. The process of the cancer of the pharynx.
The ID family members have four ID1, ID2, ID3, ID4. They were initially found to play an important role in the differentiation of negative regulatory cells. More and more studies have shown that they are regulating lineage stereotypes, gene expression, cell fate, cell proliferation, and cell differentiation in the generation of the divine, lymphatic tissue, and neovascularization. Time also plays a key role in the.ID protein family that has been found to be overexpressed in many tumors, such as colorectal cancer, glioblastoma, medulloblastoma, neurocytoma, pancreatic cancer, thyroid cancer, squamous cell carcinoma, prostate cancer, breast cancer, endometrial cancer, cervical cancer and melanoma.
In order to elucidate the role of ID2 gene in the pathogenesis of nasopharyngeal carcinoma, we use RNAi technology to interfere with the expression of ID2 in nasopharyngeal carcinoma cell SUNE1 by efficient and specific targeting silencing mechanism, to establish a siRNA nasopharyngeal carcinoma cell with stable target interference ID2 expression, in order to observe the biological function changes of the nasopharyngeal carcinoma cells after interference and explore the relationship between the nasopharyngeal carcinoma cells and the nasopharyngeal carcinoma cells, and to make the nasopharynx to be nasopharynx. The pathogenesis and treatment of cancer provide a new way of thinking.
Method
Screening of EMT related genes in 1. nasopharyngeal carcinoma
The related gene chip data of nasopharyngeal carcinoma were searched and downloaded from the public gene chip database GEO (Gene Expression Omnibus), and the data were analyzed by Genclip software to find the genes associated with the epithelial mesenchymal transition (Epithelial -- mesenchymal transition, EMT) in the common gene chip data of nasopharyngeal carcinoma. The selected genes were further analyzed by bioinformatics.
Identification of expression characteristics of 2. ID2 in nasopharyngeal carcinoma
The expression level of ID2 in noncancerous nasopharyngeal tissue and nasopharyngeal carcinoma was detected by immunohistochemical SP method, and the relationship between the level of ID2 and the development of nasopharyngeal carcinoma was preliminarily discussed.
The expression level of ID2 in the immortalized nasopharyngeal epithelial cells NP69 and nasopharyngeal carcinoma cells SUNE1,5-8F, 6-10B, CNE2, HNE1, CNE1, C666-1, HONE1 in the immortalized nasopharyngeal epithelial cells, NP69 and nasopharyngeal carcinoma cells was detected by fluorescence quantitative PCR, and the difference in the expression of ID2 in the immortalized nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells was preliminarily discussed, and the nasopharyngeal carcinoma cells with high expression were found as the next interference. Object.
3. the effect of ID2 silencing on the biological effects of nasopharyngeal carcinoma cells and the detection of some EMT related gene expression levels
The ID2 lentivirus interference carrier was constructed and packaged into mature lentivirus particles infected with SUNE1 cells with high expression of ID2. The stable positive and unloaded clones were screened by the finite dilution method. The ID2 interference efficiency in the interference cloned cells was detected by fluorescence quantitative PCR, and the cell clones with the highest interference efficiency were screened and the cell clone was used as the experimental object. One step was to study the.MTT method. The effect of ID2 silencing on the proliferation of cells in vitro was detected by a flat clone formation test, and Transwell was used to detect the change of cell migration and movement after ID2 silencing.
ID2 related TGF-beta and EMT related transcription factors Slug, SIP1, Twist, Snail, epithelial markers E-cadherin, and interstitial marker Vimentin were detected in the expression level of four cell lines in four cell lines.
4. statistical method
SPSS 13 statistical software package was used for statistical processing, and the second chapters were classified as grade data due to immunohistochemistry, so the comparison between the groups was compared with the non parametric rank sum test. The expression of ID2 was compared with Mann-Whitney U test in non cancer nasopharyngeal tissue and nasopharyngeal carcinoma. The relationship between the expression of ID2 and the clinical stage of nasopharyngeal carcinoma was tested by Kruskal Wal. Lis Test, the test level was both bilateral alpha =0.05; fluorescence quantitative PCR method was used to detect the expression characteristics of ID2 in nasopharyngeal carcinoma cell lines by single factor variance analysis and multiple comparison using SNK test. The third chapter fluorescence quantitative PCR, exercise, flat clone formation experiment using single factor difference analysis; MTT using analysis of variance analysis of factorial design; The SNK or LSD test is used for the re comparison.
Result
Screening of EMT related genes in 1. nasopharyngeal carcinoma
35 differentially expressed genes associated with EMT are found in the common nasopharyngeal carcinoma chip data, and the functions of these genes are related to cell components, cell adhesion, communication, signal transduction, differentiation, movement, migration, and cell surface receptor related signal transduction. These functions are often considered to be associated with tumor invasion and metastasis.
Identification of 2.ID2 expression in nasopharyngeal carcinoma
1, a total of 18 cases of nasopharyngitis group were collected. The results of.SP immunohistochemical staining in 52 cases of nasopharyngeal carcinoma group showed that the expression of ID2 expression in the nucleus and cytoplasm.ID2 in nasopharyngeal carcinoma and non cancer nasopharynx tissues was statistically different (Z=-5.553, P=0.000), and there was no statistical difference between the expression of ID2 and the clinical stage (x 2=0.963, P=0.810).
2, the fluorescence quantitative PCR method was used to detect ID2 in the immortalized nasopharyngeal epithelial cells NP69 and nasopharyngeal carcinoma cells SUNE1,5-8F, 6-10B, CNE2, HNE1, CNE1, C666-1, HONE1 mRNA, and the expression was the highest, and the expression amount was 78 times as the next step.
Biological effects of 3.ID2 silencing on nasopharyngeal carcinoma cells and detection of partial EMT related gene expression
1, the ID2 lentivirus interference carrier was constructed, SUNE1 cells were infected after the lentivirus was packaged, the GFP+ monoclonal was screened by the finite dilution method, and the ID2 expression of each clone was detected by the fluorescence quantitative PCR, and the 2 monoclonal antibodies with the highest interference efficiency were selected for the next experiment. The interference efficiency of the positive clones was expressed with the SUNE1 and negative control cells. The statistical difference (F=48.693, P=0.000), and the expression of ID2 in negative control was the same as that of SUNE1 (0.933 + 0.006). The positive clones were named SUNE1/plv-ID2-1 and SUNE1/plv-ID2-2, and the negative control clones were SUNE1/plv.
2, MTT assay was used to detect cell proliferation. Compared with negative control SUNE1/Plv and SUNE1 cells, the proliferation rate of interference cloned cells, SUNE1/plv-ID2-1 and SUNE1/plv-ID2-2, was significantly slower and time dependent, with statistical differences (F=3.301, P=0.000). The clone formation rate of the four groups was statistically significant. F=253.434 (P=0.000), SUNE1/plv-ID2 cell clone formation decreased, SUNE1 and negative control group did not have statistical difference (P=0.866). The growth of SUNE1 cells in vitro was significantly inhibited after ID2 expression interference.
3, the results of Transwell cell detection showed that compared with SUNE1 and SUNE1/plv cells, the number of cells passing through the polycarbonate membrane of SUNE1 /plv-ID2 cells did not change significantly, and the difference was not significant (F=0.624, P=0.610).
4, the results of fluorescence quantitative PCR assay showed that the expression of TGF-beta, E-cadherin, Slug, SIP1, Vimentin did not have statistical differences between the 4 groups of cells (F=0.405, P=0.754; F=0.571, P=0.650), but there were statistical differences between the 4 groups of cells. F=155.295, P=0.000).
conclusion
1, bioinformatics methods screened 35 genes related to nasopharyngeal carcinoma (EMT).
2, immunohistochemical SP method confirmed that the expression of ID2 was related to the occurrence and development of nasopharyngeal carcinoma.
3, the expression of ID2 in the immortalized nasopharyngeal epithelial cells NP69 and nasopharyngeal carcinoma cells SUNE1,5-8F, 6-10B, CNE2, HNE1, CNE1, C666-1, HONE1 in the immortalized nasopharyngeal epithelial cells NP69 and nasopharyngeal carcinoma cells were statistically different, and the SUNE1 expression was the highest.
4, the biological function of some cells changed after ID2 interference.
5, the expression level of 1nRNA related to EMT after ID2 interference was changed.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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