鼻咽癌临床进展过程中转录因子活性谱动态变化规律研究
发布时间：2018-07-28 07:59
【摘要】： [研究背景] 鼻咽癌是一种多步骤进展的多基因遗传性恶性肿瘤。目前,已发现鼻咽癌中越来越多的遗传学和表观遗传学分子改变,并且癌基因过表达或活性过高以及抑癌基因的表达缺失或失活可发生在不同临床／病理进展阶段。我们实验室经过多年的研究,发现数个候选抑瘤／易感基因参与多个细胞信号通路,并与鼻咽癌细胞增殖、侵袭和转移相关基因的表达有关。重要的是,这些基因表达缺失或失活同样也发生在鼻咽癌发生发展的不同阶段。上述研究表明,许多参与信号通路的遗传分子(尤其是组织特异性)相继激活或失活导致转录因子的活性以及所调控的靶基因表达异常,从而促进了鼻咽癌发生和发展。因此,基因异常表达谱与异常的转录调控模式密切相关。 转录因子是基因调控网络中的重要分子。它接受上游信号通路异常信号,引起基因的表达紊乱,与肿瘤的发生发展密切相关。了解转录因子活性在肿瘤进展中的动态变化规律有利于阐明基因调控和表达异常的分子机制。尽管已有文献报道了某些转录因子在鼻咽癌组织或细胞中存在异常表达或活性改变,但是系统阐述转录因子活性在鼻咽癌不同临床进展阶段动态变化规律的研究尚无人问津。从而,本研究采用转录因子芯片分别检测和分析鼻咽癌不同临床进展阶段转录因子活性动态变化规律以及鼻咽癌细胞系与正常鼻咽上皮细胞系活性差异转录因子。 [鼻咽癌不同临床进展阶段动态变化的转录因子活性谱的构建] 对鼻咽癌不同临床进展阶段的组织样本进行检测,分析转录因子活性在临床进展中的动态变化。组织样本分为两组：1.独立样本组(12例),每例样本单独提取核蛋白,分别进行芯片检测；2.混合样本组(13例),同一临床阶段的样本混合,提取核蛋白后进行芯片检测。抽提临床Ⅰ-Ⅳ期组织核蛋白后,用Combo Protein/DNA芯片(含有345个检测位点)检测转录因子活性。采用One-way ANOVA和studentt检验方法获得55个差异转录因子。通过聚类分析发现26个转录因子在独立样本组和混合样本组中的活性变化趋势基本一致。这些转录因子在鼻咽癌临床进展阶段中活性增高并且呈动态性的变化。 [AP2和ATF家族分子在鼻咽癌不同临床进展阶段的表达分析] 通过线性回归分析,发现在26个活性增高的转录因子中,16个转录因子与鼻咽癌临床进展呈正相关性。这些转录因子分别是GAS/ISRE, CdxA/NKX2, HOXD8, PPUR, NFκB, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF/CREB, RFX, C/EBP, Snail, PRDI-BFc和Stat5b。许多文献提示,AP2和ATF家族分子与多种肿瘤的发生有关。因此,我们选择这两个转录因子进行验证和分析。首先用EMSA证实了转录因子AP2和ATF的活性变化规律,进而扩大样本进一步对这两个转录因子家族的主要分子AP2α、AP2β、AP2γ、ATF1和ATF2以及靶基因EGFR和MMP-2进行免疫组化分析。采用Spearman's rank test和Fisher's exact test分析转录因子表达与鼻咽癌临床进展阶段和靶基因表达的相关性。结果显示,AP2α、AP2β、AP2γ、ATF1和ATF2在肿瘤组织中的表达明显高于正常鼻咽上皮,且与临床进展相关。AP2a与靶基因EGFR、ATF1和ATF2与靶基因MMP-2之间的表达具有相关性。Western blot和RT-PCR进一步验证了以上结果。 [鼻咽癌细胞系活性差异转录因子分析] 采用博奥生物技术有限公司的转录因子芯片(含有270个检测位点、)检测正常鼻咽上皮细胞系(NP69)、非转移性(6-10B)和转移性(5-8F)鼻咽癌细胞系中的转录因子活性。通过比较信号值,获得差异转录因子。鼻咽癌细胞中有10个转录因子上调,8个转录因子下调。值得注意的是,在上调的10个转录因子中,AP2,ATF/CREB, C/EBP和RFX与鼻咽癌组织的分析结果一致。与正常鼻咽上皮比较,它们在鼻咽癌组织中表达上调且与临床阶段的进展相关。与NP69相比,AP2, ATF/CREB和Spl的活性在两株鼻咽癌细胞中明显升高,EMSA分析进一步证实了芯片结果。RT-PCR和Western blot分析结果表明,AP2α、AP2β、AP2γ、ATF1、ATF2、Sp1和Sp3 mRNA表达水平在鼻咽癌细胞明显上调,其中AP2a、AP2γ、ATF1、ATF2、Sp1和Sp3蛋白水平在5-8F细胞高于6-10B细胞。此外,我们发现Spl、Sp3以及Spl靶基因VEGF和MMP-9在鼻咽癌组织高表达。 [Spl对5-8F细胞侵袭能力的影响] 为了明确Spl对5-8F细胞侵袭能力的影响,使用Sp1 siRNA转染细胞以干预Spl的表达。Sp1 siRNA能显著下调5-8F细胞中Spl的蛋白水平以及VEGF表达和MMP-9分泌。同时,用光辉霉素(一种Spl特异性的抑制剂)处理5-8F细胞后,发现光辉霉素能够呈剂量依赖性地抑制Sp1、VEGF和MMP-9的表达,同时5-8F细胞迁移和侵袭能力降低。这表明Spl表达和活性增高而引起MMP-9和VEGF的过表达,可能是5-8F细胞侵袭和转移能力增强的重要原因之一。 综上所述,鼻咽癌不同临床阶段进展过程中转录因子活性动态变化规律的研究为我们进一步探索鼻咽癌相关基因转录调控机制提供了有价值的理论和实验基础。尽管目前我们得到了一些与临床进展相关的转录因子及其活性变化模式,但这并不代表我们揭示了所有与鼻咽癌发生发展密切相关的转录因子。本实验通过高通量的Protein/DNA芯片分析的确揭示了部分差异转录因子的活性变化规律,而这些转录因子在鼻咽癌进展过程中的动态变化规律可能与异常的基因表达谱密切相关。随着系统生物学和整合组学研究的进展,通过将不同临床进展阶段动态变化的转录因子差异活性谱与基因差异表达谱进行整合,将有利于阐明这些临床进展相关的差异转录因子对基因转录的调控机制,为鼻咽癌的诊断、预后提供检测分子标记以及临床治疗靶点。
[Abstract]:[research background]
Nasopharyngeal carcinoma is a multistep genetic malignant tumor progressed. At present, more and more genetic and epigenetic changes have been found in nasopharyngeal carcinoma, and the overexpression or exorbitant activity of the oncogene and the deletion or inactivation of the tumor suppressor gene can occur at different stages of clinical / pathological progress. Several years of research have found that several candidate tumor suppressor / susceptibility genes participate in multiple cell signaling pathways and are related to the expression of genes related to proliferation, invasion and metastasis of nasopharyngeal carcinoma cells. The genetic molecules (especially tissue specificity) activation or inactivation of the pathway lead to the activity of the transcription factors and the abnormal expression of the regulated target genes, thus promoting the occurrence and development of nasopharyngeal carcinoma. Therefore, the abnormal expression profile of the gene is closely related to the abnormal transcription regulation mode.
The transcription factor is an important molecule in the gene regulatory network. It accepts the abnormal signal of the upstream signal pathway and causes the disorder of gene expression. It is closely related to the development of tumor. Understanding the dynamic changes of the activity of the transcription factor in the progression of the tumor is beneficial to elucidate the molecular mechanism of gene regulation and abnormal expression. Although the literature has been reported in the literature There are some transcriptional factors that have abnormal expression or activity in nasopharyngeal carcinoma tissues or cells. However, the study of the dynamic changes of transcription factor activity in different clinical stages of nasopharyngeal carcinoma is still unknown. Therefore, the transcriptional factor chip is used to detect and analyze the different clinical stages of nasopharyngeal carcinoma. The dynamic change of transcription factor activity and transcription factor of activity difference between nasopharyngeal carcinoma cell line and normal nasopharyngeal epithelial cell line.
[construction of dynamic transcription factor activity spectrum for nasopharyngeal carcinoma at different clinical stages]
Detection of tissue samples in different clinical stages of nasopharyngeal carcinoma and analysis of the dynamic changes in the activity of transcription factors in clinical progress. The tissue samples were divided into two groups: 1. independent sample groups (12 cases), each sample was separately extracted and detected by chip, 2. mixed sample group (13 cases), mixed samples at the same clinical stage, and proposed After the nucleoprotein was detected, the activity of transcription factor was detected by Combo Protein/DNA chip (containing 345 detection loci). 55 differential transcription factors were obtained by One-way ANOVA and studentt test. 26 transcription factors were found in independent sample group and mixed sample by cluster analysis. The activity trends of these groups were basically consistent. These transcription factors increased and showed dynamic changes in the nasopharyngeal carcinoma clinical stage.
Expression analysis of [AP2 and ATF family molecules in nasopharyngeal carcinoma at different clinical stages
By linear regression analysis, it was found that 16 transcriptional factors were positively related to the clinical progress of nasopharyngeal carcinoma in 26 highly active transcription factors. These transcription factors were GAS/ISRE, CdxA/NKX2, HOXD8, PPUR, NF kappa B, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF. And ATF family molecules are associated with a variety of tumors. Therefore, we select these two transcription factors for validation and analysis. First, EMSA has been used to confirm the changes in the activity of transcription factors AP2 and ATF, and then further expand the sample to further study the main molecules of these two transcription factor families, AP2 a, AP2 beta, AP2 gamma, ATF1 and ATF2, and target gene EGFR and EGFR. MMP-2 rank test and Fisher's exact test were used to analyze the correlation between the expression of transcription factors and the expression of target genes in the clinical progression of nasopharyngeal carcinoma. The results showed that the expression of AP2 a, AP2 beta, AP2 gamma, ATF1 and ATF2 in the tumor tissues was significantly higher than that of the normal nasopharyngeal epithelium. Gene EGFR, ATF1 and ATF2 were correlated with the expression of target gene MMP-2..Western blot and RT-PCR further verified the above results.
[differential transcription factor analysis of nasopharyngeal carcinoma cell line activity]
The transcriptional factor chip of BOO biotechnology Limited (containing 270 detection sites) was used to detect the transcriptional factor activity of normal nasopharyngeal epithelial cell line (NP69), non metastatic (6-10B) and metastatic (5-8F) nasopharyngeal carcinoma cell lines. The differential transcription factors were obtained by comparing the signal values. 10 transcription factors were up regulated in nasopharyngeal carcinoma cells, 8 It is worth noting that AP2, ATF/CREB, C/EBP, and RFX are consistent with the analysis of nasopharyngeal carcinoma in the 10 transcription factors up to up. Compared with normal nasopharyngeal epithelium, they are up-regulated in nasopharyngeal carcinoma and are related to the progress of the clinical stage. Compared with NP69, the activity of AP2, ATF/CREB and Spl is in two nasopharynx. The EMSA analysis further confirmed that the results of.RT-PCR and Western blot analysis showed that AP2 alpha, AP2 beta, AP2 gamma, ATF1, ATF2, Sp1 and Sp3 mRNA cells were obviously up-regulated in nasopharyngeal carcinoma cells. The expression of VEGF and MMP-9 in nasopharyngeal carcinoma was high.
The effect of [Spl on the invasiveness of 5-8F cells]
In order to determine the effect of Spl on the invasive ability of 5-8F cells, the use of Sp1 siRNA transfected cells to interfere with Spl expression.Sp1 siRNA can significantly reduce the protein level of Spl in 5-8F cells and VEGF expression and MMP-9 secretion. Meanwhile, after treating the cells with the splendorycin (a kind of Spl specific inhibitor), it is found that the radiomycin can be dose-dependent. The expression of Sp1, VEGF and MMP-9 is inhibited and the migration and invasion of 5-8F cells are reduced. This indicates that the increased expression and activity of Spl cause the overexpression of MMP-9 and VEGF, which may be one of the important reasons for the enhancement of the invasion and metastasis ability of 5-8F cells.
To sum up, the study of the dynamic changes in the activity of transcription factors during the progression of nasopharyngeal carcinoma at different stages has provided a valuable theoretical and experimental basis for our further exploration of the transcriptional regulation mechanism of nasopharyngeal cancer related genes. But this does not mean that we reveal all the transcriptional factors that are closely related to the development of nasopharyngeal carcinoma. This experiment did reveal the changes in the activity of some differential transcription factors by high throughput Protein/DNA chip analysis, and the dynamic changes of these transcription factors in the progression of nasopharyngeal carcinoma may be related to the abnormal gene table. With the progress of systematic biology and integrated omics, the integration of the differential activity spectrum of transcription factors and gene differential expression profiles that vary dynamically in different stages of clinical progress will be beneficial to elucidate the regulatory mechanism of these differential transcription factors related to gene transcription in the diagnosis of nasopharyngeal carcinoma, Prognosis provides detection of molecular markers and clinical therapeutic targets.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63
本文编号：2149430
[Abstract]:[research background]
Nasopharyngeal carcinoma is a multistep genetic malignant tumor progressed. At present, more and more genetic and epigenetic changes have been found in nasopharyngeal carcinoma, and the overexpression or exorbitant activity of the oncogene and the deletion or inactivation of the tumor suppressor gene can occur at different stages of clinical / pathological progress. Several years of research have found that several candidate tumor suppressor / susceptibility genes participate in multiple cell signaling pathways and are related to the expression of genes related to proliferation, invasion and metastasis of nasopharyngeal carcinoma cells. The genetic molecules (especially tissue specificity) activation or inactivation of the pathway lead to the activity of the transcription factors and the abnormal expression of the regulated target genes, thus promoting the occurrence and development of nasopharyngeal carcinoma. Therefore, the abnormal expression profile of the gene is closely related to the abnormal transcription regulation mode.
The transcription factor is an important molecule in the gene regulatory network. It accepts the abnormal signal of the upstream signal pathway and causes the disorder of gene expression. It is closely related to the development of tumor. Understanding the dynamic changes of the activity of the transcription factor in the progression of the tumor is beneficial to elucidate the molecular mechanism of gene regulation and abnormal expression. Although the literature has been reported in the literature There are some transcriptional factors that have abnormal expression or activity in nasopharyngeal carcinoma tissues or cells. However, the study of the dynamic changes of transcription factor activity in different clinical stages of nasopharyngeal carcinoma is still unknown. Therefore, the transcriptional factor chip is used to detect and analyze the different clinical stages of nasopharyngeal carcinoma. The dynamic change of transcription factor activity and transcription factor of activity difference between nasopharyngeal carcinoma cell line and normal nasopharyngeal epithelial cell line.
[construction of dynamic transcription factor activity spectrum for nasopharyngeal carcinoma at different clinical stages]
Detection of tissue samples in different clinical stages of nasopharyngeal carcinoma and analysis of the dynamic changes in the activity of transcription factors in clinical progress. The tissue samples were divided into two groups: 1. independent sample groups (12 cases), each sample was separately extracted and detected by chip, 2. mixed sample group (13 cases), mixed samples at the same clinical stage, and proposed After the nucleoprotein was detected, the activity of transcription factor was detected by Combo Protein/DNA chip (containing 345 detection loci). 55 differential transcription factors were obtained by One-way ANOVA and studentt test. 26 transcription factors were found in independent sample group and mixed sample by cluster analysis. The activity trends of these groups were basically consistent. These transcription factors increased and showed dynamic changes in the nasopharyngeal carcinoma clinical stage.
Expression analysis of [AP2 and ATF family molecules in nasopharyngeal carcinoma at different clinical stages
By linear regression analysis, it was found that 16 transcriptional factors were positively related to the clinical progress of nasopharyngeal carcinoma in 26 highly active transcription factors. These transcription factors were GAS/ISRE, CdxA/NKX2, HOXD8, PPUR, NF kappa B, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF. And ATF family molecules are associated with a variety of tumors. Therefore, we select these two transcription factors for validation and analysis. First, EMSA has been used to confirm the changes in the activity of transcription factors AP2 and ATF, and then further expand the sample to further study the main molecules of these two transcription factor families, AP2 a, AP2 beta, AP2 gamma, ATF1 and ATF2, and target gene EGFR and EGFR. MMP-2 rank test and Fisher's exact test were used to analyze the correlation between the expression of transcription factors and the expression of target genes in the clinical progression of nasopharyngeal carcinoma. The results showed that the expression of AP2 a, AP2 beta, AP2 gamma, ATF1 and ATF2 in the tumor tissues was significantly higher than that of the normal nasopharyngeal epithelium. Gene EGFR, ATF1 and ATF2 were correlated with the expression of target gene MMP-2..Western blot and RT-PCR further verified the above results.
[differential transcription factor analysis of nasopharyngeal carcinoma cell line activity]
The transcriptional factor chip of BOO biotechnology Limited (containing 270 detection sites) was used to detect the transcriptional factor activity of normal nasopharyngeal epithelial cell line (NP69), non metastatic (6-10B) and metastatic (5-8F) nasopharyngeal carcinoma cell lines. The differential transcription factors were obtained by comparing the signal values. 10 transcription factors were up regulated in nasopharyngeal carcinoma cells, 8 It is worth noting that AP2, ATF/CREB, C/EBP, and RFX are consistent with the analysis of nasopharyngeal carcinoma in the 10 transcription factors up to up. Compared with normal nasopharyngeal epithelium, they are up-regulated in nasopharyngeal carcinoma and are related to the progress of the clinical stage. Compared with NP69, the activity of AP2, ATF/CREB and Spl is in two nasopharynx. The EMSA analysis further confirmed that the results of.RT-PCR and Western blot analysis showed that AP2 alpha, AP2 beta, AP2 gamma, ATF1, ATF2, Sp1 and Sp3 mRNA cells were obviously up-regulated in nasopharyngeal carcinoma cells. The expression of VEGF and MMP-9 in nasopharyngeal carcinoma was high.
The effect of [Spl on the invasiveness of 5-8F cells]
In order to determine the effect of Spl on the invasive ability of 5-8F cells, the use of Sp1 siRNA transfected cells to interfere with Spl expression.Sp1 siRNA can significantly reduce the protein level of Spl in 5-8F cells and VEGF expression and MMP-9 secretion. Meanwhile, after treating the cells with the splendorycin (a kind of Spl specific inhibitor), it is found that the radiomycin can be dose-dependent. The expression of Sp1, VEGF and MMP-9 is inhibited and the migration and invasion of 5-8F cells are reduced. This indicates that the increased expression and activity of Spl cause the overexpression of MMP-9 and VEGF, which may be one of the important reasons for the enhancement of the invasion and metastasis ability of 5-8F cells.
To sum up, the study of the dynamic changes in the activity of transcription factors during the progression of nasopharyngeal carcinoma at different stages has provided a valuable theoretical and experimental basis for our further exploration of the transcriptional regulation mechanism of nasopharyngeal cancer related genes. But this does not mean that we reveal all the transcriptional factors that are closely related to the development of nasopharyngeal carcinoma. This experiment did reveal the changes in the activity of some differential transcription factors by high throughput Protein/DNA chip analysis, and the dynamic changes of these transcription factors in the progression of nasopharyngeal carcinoma may be related to the abnormal gene table. With the progress of systematic biology and integrated omics, the integration of the differential activity spectrum of transcription factors and gene differential expression profiles that vary dynamically in different stages of clinical progress will be beneficial to elucidate the regulatory mechanism of these differential transcription factors related to gene transcription in the diagnosis of nasopharyngeal carcinoma, Prognosis provides detection of molecular markers and clinical therapeutic targets.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63
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