鼻咽癌臨床進展過程中轉錄因子活性譜動態(tài)變化規(guī)律研究
發(fā)布時間：2018-07-28 07:59
【摘要】： [研究背景] 鼻咽癌是一種多步驟進展的多基因遺傳性惡性腫瘤。目前,已發(fā)現(xiàn)鼻咽癌中越來越多的遺傳學和表觀遺傳學分子改變,并且癌基因過表達或活性過高以及抑癌基因的表達缺失或失活可發(fā)生在不同臨床／病理進展階段。我們實驗室經(jīng)過多年的研究,發(fā)現(xiàn)數(shù)個候選抑瘤／易感基因參與多個細胞信號通路,并與鼻咽癌細胞增殖、侵襲和轉移相關基因的表達有關。重要的是,這些基因表達缺失或失活同樣也發(fā)生在鼻咽癌發(fā)生發(fā)展的不同階段。上述研究表明,許多參與信號通路的遺傳分子(尤其是組織特異性)相繼激活或失活導致轉錄因子的活性以及所調控的靶基因表達異常,從而促進了鼻咽癌發(fā)生和發(fā)展。因此,基因異常表達譜與異常的轉錄調控模式密切相關。 轉錄因子是基因調控網(wǎng)絡中的重要分子。它接受上游信號通路異常信號,引起基因的表達紊亂,與腫瘤的發(fā)生發(fā)展密切相關。了解轉錄因子活性在腫瘤進展中的動態(tài)變化規(guī)律有利于闡明基因調控和表達異常的分子機制。盡管已有文獻報道了某些轉錄因子在鼻咽癌組織或細胞中存在異常表達或活性改變,但是系統(tǒng)闡述轉錄因子活性在鼻咽癌不同臨床進展階段動態(tài)變化規(guī)律的研究尚無人問津。從而,本研究采用轉錄因子芯片分別檢測和分析鼻咽癌不同臨床進展階段轉錄因子活性動態(tài)變化規(guī)律以及鼻咽癌細胞系與正常鼻咽上皮細胞系活性差異轉錄因子。 [鼻咽癌不同臨床進展階段動態(tài)變化的轉錄因子活性譜的構建] 對鼻咽癌不同臨床進展階段的組織樣本進行檢測,分析轉錄因子活性在臨床進展中的動態(tài)變化。組織樣本分為兩組：1.獨立樣本組(12例),每例樣本單獨提取核蛋白,分別進行芯片檢測；2.混合樣本組(13例),同一臨床階段的樣本混合,提取核蛋白后進行芯片檢測。抽提臨床Ⅰ-Ⅳ期組織核蛋白后,用Combo Protein/DNA芯片(含有345個檢測位點)檢測轉錄因子活性。采用One-way ANOVA和studentt檢驗方法獲得55個差異轉錄因子。通過聚類分析發(fā)現(xiàn)26個轉錄因子在獨立樣本組和混合樣本組中的活性變化趨勢基本一致。這些轉錄因子在鼻咽癌臨床進展階段中活性增高并且呈動態(tài)性的變化。 [AP2和ATF家族分子在鼻咽癌不同臨床進展階段的表達分析] 通過線性回歸分析,發(fā)現(xiàn)在26個活性增高的轉錄因子中,16個轉錄因子與鼻咽癌臨床進展呈正相關性。這些轉錄因子分別是GAS/ISRE, CdxA/NKX2, HOXD8, PPUR, NFκB, AP2, Fra-1/JUN, AP3, PTF1, GKLF, ATF/CREB, RFX, C/EBP, Snail, PRDI-BFc和Stat5b。許多文獻提示,AP2和ATF家族分子與多種腫瘤的發(fā)生有關。因此,我們選擇這兩個轉錄因子進行驗證和分析。首先用EMSA證實了轉錄因子AP2和ATF的活性變化規(guī)律,進而擴大樣本進一步對這兩個轉錄因子家族的主要分子AP2α、AP2β、AP2γ、ATF1和ATF2以及靶基因EGFR和MMP-2進行免疫組化分析。采用Spearman's rank test和Fisher's exact test分析轉錄因子表達與鼻咽癌臨床進展階段和靶基因表達的相關性。結果顯示,AP2α、AP2β、AP2γ、ATF1和ATF2在腫瘤組織中的表達明顯高于正常鼻咽上皮,且與臨床進展相關。AP2a與靶基因EGFR、ATF1和ATF2與靶基因MMP-2之間的表達具有相關性。Western blot和RT-PCR進一步驗證了以上結果。 [鼻咽癌細胞系活性差異轉錄因子分析] 采用博奧生物技術有限公司的轉錄因子芯片(含有270個檢測位點、)檢測正常鼻咽上皮細胞系(NP69)、非轉移性(6-10B)和轉移性(5-8F)鼻咽癌細胞系中的轉錄因子活性。通過比較信號值,獲得差異轉錄因子。鼻咽癌細胞中有10個轉錄因子上調,8個轉錄因子下調。值得注意的是,在上調的10個轉錄因子中,AP2,ATF/CREB, C/EBP和RFX與鼻咽癌組織的分析結果一致。與正常鼻咽上皮比較,它們在鼻咽癌組織中表達上調且與臨床階段的進展相關。與NP69相比,AP2, ATF/CREB和Spl的活性在兩株鼻咽癌細胞中明顯升高,EMSA分析進一步證實了芯片結果。RT-PCR和Western blot分析結果表明,AP2α、AP2β、AP2γ、ATF1、ATF2、Sp1和Sp3 mRNA表達水平在鼻咽癌細胞明顯上調,其中AP2a、AP2γ、ATF1、ATF2、Sp1和Sp3蛋白水平在5-8F細胞高于6-10B細胞。此外,我們發(fā)現(xiàn)Spl、Sp3以及Spl靶基因VEGF和MMP-9在鼻咽癌組織高表達。 [Spl對5-8F細胞侵襲能力的影響] 為了明確Spl對5-8F細胞侵襲能力的影響,使用Sp1 siRNA轉染細胞以干預Spl的表達。Sp1 siRNA能顯著下調5-8F細胞中Spl的蛋白水平以及VEGF表達和MMP-9分泌。同時,用光輝霉素(一種Spl特異性的抑制劑)處理5-8F細胞后,發(fā)現(xiàn)光輝霉素能夠呈劑量依賴性地抑制Sp1、VEGF和MMP-9的表達,同時5-8F細胞遷移和侵襲能力降低。這表明Spl表達和活性增高而引起MMP-9和VEGF的過表達,可能是5-8F細胞侵襲和轉移能力增強的重要原因之一。 綜上所述,鼻咽癌不同臨床階段進展過程中轉錄因子活性動態(tài)變化規(guī)律的研究為我們進一步探索鼻咽癌相關基因轉錄調控機制提供了有價值的理論和實驗基礎。盡管目前我們得到了一些與臨床進展相關的轉錄因子及其活性變化模式,但這并不代表我們揭示了所有與鼻咽癌發(fā)生發(fā)展密切相關的轉錄因子。本實驗通過高通量的Protein/DNA芯片分析的確揭示了部分差異轉錄因子的活性變化規(guī)律,而這些轉錄因子在鼻咽癌進展過程中的動態(tài)變化規(guī)律可能與異常的基因表達譜密切相關。隨著系統(tǒng)生物學和整合組學研究的進展,通過將不同臨床進展階段動態(tài)變化的轉錄因子差異活性譜與基因差異表達譜進行整合,將有利于闡明這些臨床進展相關的差異轉錄因子對基因轉錄的調控機制,為鼻咽癌的診斷、預后提供檢測分子標記以及臨床治療靶點。
[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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.63
本文編號：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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.63
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，本文編號：2149430
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