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Dynamic Activity Profile Study of Transcription Factors Activated in Nasopharyngeal Carcinoma Progression

Author: SuBo
Tutor: LiGuiYuan
School: Central South University
Course: Pathology and Pathophysiology
Keywords: Nasopharyngeal Transcription factor Spectrum of activity Protein / DNA chip
CLC: R739.63
Type: PhD thesis
Year: 2010
Downloads: 111
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Abstract

[Background] The NPC is a multi-step progress of multi-gene genetic malignancy. Has found more and more NPC genetics and epigenetics molecules of change, and oncogene overexpression or activity is too high, and the expression of the tumor suppressor gene deletion or inactivation can occur in different clinical / pathological stage of progress . Our laboratory after years of research, found a number of candidate tumor suppressor / susceptibility genes involved in multiple cell signaling pathways, and nasopharyngeal carcinoma cell proliferation, invasion and metastasis related gene expression. Importantly, the expression of these genes also occur at different stages of development of the NPC deletion or inactivation. These studies have shown that many genetic molecules involved in the signaling pathway (tissue-specific) succession activation or inactivation causes abnormal activity of transcription factors as well as the regulation of target gene expression, thus contributing to nasopharyngeal carcinoma and development. Therefore, closely related to abnormal gene expression of the transcriptional regulation of the spectrum with abnormal mode. Transcription factors are important molecules in the gene regulatory network. It accepts the upstream signaling pathways abnormal signal, causing disorder of gene expression is closely related to tumor development. Understanding the dynamic variation of the transcription factor activity in tumor progression conducive to elucidate the molecular mechanisms of gene regulation and expression of abnormal. Despite It has been reported that some transcription factors in NPC tissues or cells in the presence of abnormal expression or activity change, but the system expounded transcription factor activity in nasopharyngeal different clinical stages of progress dynamic variation no one cares. Thus, the transcription factor activity differences transcription factor chip detection and analysis of dynamic variation as well as nasopharyngeal carcinoma cell line of nasopharyngeal different stages of clinical progress transcription factor activity and normal nasopharyngeal epithelial cell line. [The nasopharyngeal different clinical stages of progress Construction of dynamic changes in transcription factor activity spectrum] the different clinical stages of progress in nasopharyngeal tissue samples for testing, analysis of the dynamic changes of the activity of transcription factors in the clinical progression. Tissue samples were divided into two groups: (1) an independent sample group (12 cases), each patient sample individually extracted nucleoprotein, respectively microarray; 2. Mixed sample group (13 cases), the samples of the same clinical stage mixed, extracted nucleoprotein after chip detection. Extraction clinical stage Ⅰ - Ⅳ of Organizational nucleoprotein Combo Protein / DNA chips (containing 345 test sites) to detect the activity of transcription factors. One-way ANOVA and studentt inspection method to get 55 different transcription factors. By cluster analysis found that 26 transcription factor activity in an independent sample group and the mixed sample group trend is basically the same. The activity of these transcription factors in nasopharyngeal clinical progression stage increased and was dynamic changes. [AP2 and ATF family of molecular expression analysis in the nasopharyngeal different clinical stage of progress] by linear regression analysis, positive correlation was found in 26 increased the activity of the transcription factor, 16 transcription factors with the clinical progression of nasopharyngeal carcinoma. These transcription factors are GAS / ISRE, CdxA/NKX2, HOXD8, PPUR, NFκB, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF / CREB, RFX, C / EBP, Snail, PRDI-BFc and Stat5b . Many literature suggests, AP2 and ATF family molecules of various tumors. Therefore, we chose these two transcription factors verification and analysis. First EMSA confirmed the activity of transcription factor AP2 and ATF variation, thereby expanding the sample to further these two transcription factor family molecule AP2α AP2β AP2γ ATF1 and ATF2 target gene EGFR and MMP-2 immunohistochemistry Analysis. Spearman's rank test and Fisher's exact test to analyze the expression of transcription factors with nasopharyngeal carcinoma clinical stage of progress and target gene expression correlation. The results showed that, AP2α, AP2β AP2γ ATF1 and ATF2 expression in tumor tissue was significantly higher than the normal nasopharyngeal epithelium, and associated with clinical progression. AP2a target genes EGFR, ATF1 and ATF2 of the target gene in the expression of MMP-2 between relevant. Western blot and RT-PCR to further verify the above results. Nasopharyngeal carcinoma cell line the activity differences transcription factor analysis] Boao Biotech Co. transcription factor chip (containing 270 detection sites) to detect normal nasopharyngeal epithelial cell line (NP69), non-metastatic (6-10B ) and metastatic (5-8F) transcription factor activity in nasopharyngeal carcinoma cell line. Obtained by comparing the signal value, the differences in transcription factors. Nasopharyngeal carcinoma cells in 10 transcription factor upregulates down eight transcription factors. It is worth noting that the 10 up-regulated transcription factor AP2, ATF / CREB, C / EBP and RFX nasopharyngeal carcinoma tissue analysis results are consistent. Compared with normal nasopharyngeal epithelial, they raised and progress with clinical stage nasopharyngeal carcinoma. Compared with NP69, AP2, the ATF / CREB and Spl activity in two nasopharyngeal carcinoma cells was significantly higher, EMSA analysis further confirmed the microarray results. RT-PCR and Western blot analysis showed that the AP2α, AP2β AP2γ ATF1, ATF2 Sp1 and Sp3 mRNA expression levels in NPC cells were significantly increased, which AP2a, AP2γ, ATF1, ATF2, Sp1 and Sp3 protein levels in 5 -8F cells than 6-10B cells. In addition, we found that Spl, Sp3 Spl target gene VEGF and MMP-9 in nasopharyngeal carcinoma high expression. The the [Spl 5-8F cell invasion] in order to clear the Spl 5-8F cell invasion, Sp1 siRNA transfected cells to interfering with the expression of the Spl. Sp1 siRNA significantly down-regulated 5-8F the Spl protein levels in the cells and the expression of VEGF and MMP-9 secretion. Meanwhile, 5-8F cells after treatment with mithramycin (a Spl-specific inhibitor), found that mithramycin dose-dependently inhibited Sp1, VEGF and MMP-9 expression, while 5-8F cell migration reduce and invasion capacity. This indicates that the the Spl expression and activity increased due to the over-expression of MMP-9 and VEGF, may be one of the important reasons for 5-8F cell invasion and metastasis ability. In summary, the process of nasopharyngeal carcinoma with different clinical stages of progress transcription factor activity in the study of the dynamic changes in the law for us to further explore the NPC associated gene transcriptional regulation mechanism provides a valuable theoretical and experimental basis. Although we have been associated with clinical progression transcription factor activity mode, but that does not mean we reveal all NPC development is closely related to the transcription factor. The experimental high-throughput Protein / DNA microarray analysis indeed revealed the part of the difference in transcription factor activity variation, the dynamic variation of these transcription factors in nasopharyngeal carcinoma progression may be closely related to the abnormal gene expression profiles. With the progress of systems biology and integrated set of studies, through the different stages of clinical progression of the dynamic changes in the transcription factor difference spectrum of activity and differential gene expression spectrum integration will be beneficial to clarify these clinical progress related to differences in transcription factor gene transcription regulatory mechanism for nasopharyngeal cancer diagnosis, prognosis detect molecular markers, and clinical therapeutic targets.

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