SND1促進(jìn)TGFβ信號(hào)傳導(dǎo)及乳腺癌轉(zhuǎn)移的分子機(jī)制
發(fā)布時(shí)間：2019-01-12 12:25
【摘要】：目的:乳腺癌是危害世界女性健康的惡性疾病之一,90%以上乳腺癌患者的死亡原因與晚期癌細(xì)胞的轉(zhuǎn)移相關(guān)。乳腺癌的轉(zhuǎn)移是一個(gè)十分復(fù)雜的過(guò)程,從癌細(xì)胞開(kāi)始局部浸潤(rùn)直到遠(yuǎn)端組織形成轉(zhuǎn)移灶的過(guò)程中多種細(xì)胞因子、信號(hào)通路及腫瘤細(xì)胞本身轉(zhuǎn)移相關(guān)基因的表達(dá)相互作用,共同參與乳腺癌的轉(zhuǎn)移。在這其中TGFβ信號(hào)通路可促進(jìn)乳腺癌細(xì)胞發(fā)生上皮-間充質(zhì)轉(zhuǎn)化及與侵襲轉(zhuǎn)移相關(guān)基因的表達(dá)促進(jìn)乳腺癌的轉(zhuǎn)移,在乳腺癌轉(zhuǎn)移過(guò)程中發(fā)揮了重要的作用。SND1蛋白是一個(gè)保守的多功能蛋白,近年來(lái)研究發(fā)現(xiàn)SND1作為轉(zhuǎn)錄共激活因子參與基因的轉(zhuǎn)錄調(diào)控過(guò)程同時(shí)也參與乳腺癌的發(fā)生發(fā)展過(guò)程。本課題組前期發(fā)現(xiàn)SND1蛋白與TGFβ信號(hào)通路若干效應(yīng)因子的啟動(dòng)子區(qū)域有很強(qiáng)的結(jié)合能力,提示SND1可能參與TGFβ信號(hào)通路下游效應(yīng)基因的轉(zhuǎn)錄調(diào)控。但SND1、TGFβ信號(hào)通路與乳腺癌轉(zhuǎn)移三者之間是否存在相關(guān)性依然未知,因此本課題目的是探討SND1通過(guò)影響TGFβ信號(hào)通路參與乳腺癌轉(zhuǎn)移的分子機(jī)制。方法:本課題主要包括七部分:1.利用臨床的乳腺癌免疫組化樣本進(jìn)行SND1免疫組化染色及重癥聯(lián)合免疫缺陷(Severe Combined Immunodeficiency,SCID)小鼠進(jìn)行體內(nèi)活體成瘤實(shí)驗(yàn)分析SND1與乳腺癌轉(zhuǎn)移與預(yù)后的相關(guān)性。2.在乳腺癌細(xì)胞系通過(guò)全基因組Ch IP-chip預(yù)測(cè)可能受SND1表達(dá)調(diào)控的基因,在TCGA乳腺癌數(shù)據(jù)庫(kù)找到與SND1共表達(dá)的基因,將二者結(jié)合進(jìn)行分析預(yù)測(cè)受SND1表達(dá)調(diào)控的基因。3.在乳腺癌細(xì)胞系中利用免疫印跡實(shí)驗(yàn),聚合酶鏈反應(yīng),熒光素酶實(shí)驗(yàn)檢測(cè)SND1對(duì)SMAD2、SMAD3、SMAD4基因m RNA水平,蛋白水平及轉(zhuǎn)錄水平的影響。利用軟件分析潛在受SND1表達(dá)影響的Smads下游靶基因,并利用聚合酶鏈反應(yīng)檢測(cè)SND1表達(dá)變化對(duì)其m RNA水平的影響。4.在乳腺癌細(xì)胞系中利用染色質(zhì)免疫共沉淀及電泳遷移率實(shí)驗(yàn)檢測(cè)SND1與SMAD2、SMAD3、SMAD4基因啟動(dòng)子結(jié)合情況。5.在乳腺癌細(xì)胞系中用免疫共沉淀實(shí)驗(yàn)及染色質(zhì)免疫共沉淀實(shí)驗(yàn)分析SND1調(diào)控SMAD2、SMAD3、SMAD4基因轉(zhuǎn)錄的分子機(jī)制。6.利用GST-pulldown實(shí)驗(yàn)、聚合酶鏈反應(yīng)、染色質(zhì)免疫共沉淀實(shí)驗(yàn)、免疫印跡實(shí)驗(yàn)及雙熒光素酶實(shí)驗(yàn)檢測(cè)SND1不同結(jié)構(gòu)域?qū)MAD2、SMAD3、SMAD4基因的轉(zhuǎn)錄水平及蛋白表達(dá)的影響。7.在穩(wěn)定沉默SND1的乳腺癌細(xì)胞及其對(duì)照細(xì)胞中用TGFβ1處理細(xì)胞,通過(guò)免疫印跡實(shí)驗(yàn),免疫熒光,熒光素酶實(shí)驗(yàn)分析SND1表達(dá)變化對(duì)TGFβ信號(hào)傳導(dǎo)的影響;利用受體激活實(shí)驗(yàn)檢測(cè)TGFβ1處理下SND1表達(dá)變化對(duì)細(xì)胞TGFβ受體激活情況的影響;利用劃痕實(shí)驗(yàn),transwell實(shí)驗(yàn)檢測(cè)在TGFβ1處理?xiàng)l件下SND1表達(dá)變化會(huì)對(duì)乳腺癌侵襲轉(zhuǎn)移的影響。結(jié)果:1.免疫組化分析及小鼠成瘤實(shí)驗(yàn)中結(jié)果均提示SND1蛋白高表達(dá)與乳腺癌轉(zhuǎn)移相關(guān),通過(guò)Kaplan-Meier分別對(duì)臨床病人及小鼠的生存期進(jìn)行分析,得出結(jié)論SND1蛋白的表達(dá)與預(yù)后差相關(guān)。2.通過(guò)對(duì)Ch IP-chip和TCGA數(shù)據(jù)進(jìn)行分析,篩選SMAD2、SMAD3、SMAD4作為SND1蛋白的靶基因。3.乳腺癌細(xì)胞中SND1蛋白可以調(diào)控SMAD2、SMAD3、SMAD4基因轉(zhuǎn)錄水平進(jìn)一步調(diào)控細(xì)胞內(nèi)Smad2、Smad3和Smad4的蛋白表達(dá);SND1的表達(dá)變化也會(huì)引起Smad2、Smad3和Smad4蛋白下游部分靶基因m RNA水平的改變。4.SND1蛋白通過(guò)與SMAD2、SMAD3、SMAD4的基因啟動(dòng)子兩個(gè)保守模序結(jié)合促進(jìn)基因轉(zhuǎn)錄。5.SND1蛋白可以與GCN5結(jié)合,并募集GCN5結(jié)合到SMAD2、SMAD3、SMAD4基因啟動(dòng)子上,通過(guò)GCN5通過(guò)催化組蛋白H3K9乙�；龠M(jìn)基因轉(zhuǎn)錄。6.SND1的TD結(jié)構(gòu)域可與GCN5結(jié)合并在促進(jìn)SMAD2、SMAD3、SMAD4基因轉(zhuǎn)錄過(guò)程中發(fā)揮了主要作用。7.在乳腺癌中SND1蛋白表達(dá)降低不會(huì)影響TGFβ1處理后TGFβ受體激活情況但會(huì)降低TGFβ1處理后乳腺癌細(xì)胞內(nèi)源Smad2和Smad3的磷酸化水平及TGFβ信號(hào)傳導(dǎo),并抑制TGFβ1處理乳腺癌細(xì)胞后細(xì)胞的侵襲轉(zhuǎn)移能力增加。結(jié)論:1.SND1蛋白可以結(jié)合到SMAD2、SMAD3、SMAD4的基因啟動(dòng)子保守模序并招募GCN5到達(dá)該區(qū)域,通過(guò)GCN5催化該區(qū)域組蛋白H3K9乙�；龠M(jìn)基因轉(zhuǎn)錄,SND1的TD結(jié)構(gòu)域在此過(guò)程中發(fā)揮了重要作用。2.SND1蛋白可以通過(guò)調(diào)控TGFβ下游效應(yīng)分子的表達(dá)影響TGFβ信號(hào)通路的信號(hào)傳導(dǎo),并進(jìn)一步影響了TGFβ信號(hào)通路對(duì)乳腺癌轉(zhuǎn)移的調(diào)控。
[Abstract]:Objective: Breast cancer is one of the malignant diseases that are harmful to the health of women in the world. The cause of death in more than 90% of patients with breast cancer is related to the metastasis of advanced cancer cells. The transfer of breast cancer is a very complicated process, from the start of local infiltration of cancer cells to the expression of various cytokines, signaling pathways and the expression of the genes involved in the transfer of the tumor cells in the course of the formation of a metastasis in the distal tissue, which is commonly involved in the transfer of breast cancer. In this case, the TGF signal pathway can promote the epithelial-to-mesenchymal transition of the breast cancer cells and promote the metastasis of the breast cancer with the expression of the invasion and metastasis-related genes, and plays an important role in the breast cancer metastasis process. The SND1 protein is a conserved multi-function protein. In recent years, SND1 has been found to be involved in the transcription regulation of the transcription co-active factor in the development of breast cancer. In the early stage of the research group, the promoter region of several effector factors of the SND1 protein and the TGF signaling pathway has strong binding capacity, and the SND1 may be involved in the transcription regulation of the downstream effector gene of the TGF signaling pathway. However, there is still an unknown correlation between SND1, TGF signal pathway and breast cancer metastasis. Therefore, the purpose of this study is to investigate the molecular mechanism of SND1 to participate in the metastasis of breast cancer by influencing TGF signal pathway. Method: This topic mainly includes seven parts: 1. In order to study the correlation between SND1 and breast cancer metastasis and prognosis, SND1 and SCID mice were used to study the correlation between SND1 and breast cancer metastasis and prognosis. In the breast cancer cell line, the gene which may be regulated by the SND1 expression can be predicted by the full-genome Ch IP-chip, and a gene co-expressed with the SND1 is found in the TCGA breast cancer database, and the genes which are regulated by the SND1 expression are predicted in combination. The effects of SND1 on SMAD2, SMAD3, SMAD4 gene m RNA level, protein level and transcription level were detected by immunoblotting experiment, polymerase chain reaction and luciferase experiment in breast cancer cell line. Smads downstream target genes, which were potentially affected by SND1 expression, were analyzed by software, and the effect of SND1 expression on its m-RNA level was detected by polymerase chain reaction. In the breast cancer cell line, the combination of SND1 and SMAD2, SMAD3, and SMAD4 gene promoter was detected by using chromatin immunoprecipitation and electrophoretic mobility assay. The molecular mechanisms of the transcription of SMAD2, SMAD3 and SMAD4 genes were analyzed by immunoprecipitation and chromatin immunoprecipitation in breast cancer cell lines. The effects of SND1 different domains on the level of transcription and protein expression of SMAD2, SMAD3 and SMAD4 genes were detected by using the GST-pulpit experiment, the polymerase chain reaction, the chromatin immunoprecipitation experiment, the immunoblotting experiment and the double-luciferase experiment. The cells were treated with TGF-1 in the stable silent SND1 breast cancer cells and their control cells, and the effect of SND1 expression on the signal conduction of TGF was analyzed by immunoblotting experiment, immunofluorescence and luciferase experiment. The effect of the change of SND1 expression on the activation of TGF-1 receptor was detected by the activation of the receptor. The effect of the change of SND1 expression on the invasion and metastasis of breast cancer under the treatment of TGF-1 was detected by a scratch test. Results: 1. The results showed that the high expression of SND1 protein was related to the metastasis of breast cancer, and the survival time of the clinical patients and the mice was analyzed by the Kaplan-Meier method. The conclusion was that the expression of SND1 protein was related to the poor prognosis. SMAD2, SMAD3 and SMAD4 were selected as the target gene of SND1 protein by analyzing Ch IP-chip and TCGA data. The SND1 protein in breast cancer cells can regulate the expression of Smad2, Smad3 and Smad4 in the SMAD2, SMAD3 and SMAD4 gene, and the expression of SND1 can also cause the change of the mRNA level of the target gene m in the downstream part of Smad2, Smad3 and Smad4. The gene promoter of SMAD4 can be combined with two conservative modes to promote the gene transcription. 5. The SND1 protein can be combined with the GCN5, and the GCN5 is incorporated into the SMAD2, SMAD3, SMAD4 gene promoter, and the gene transcription is promoted by the GCN5 by catalyzing histone H3K9 to promote the gene transcription. The SMAD4 gene plays a major role in the transcription process. The decrease in the expression of SND1 protein in breast cancer did not affect the activation of TGF-1 receptor after TGF-1 treatment, but decreased the level of the phosphorylation of endogenous Smad2 and Smad3 in breast cancer cells after TGF-1 treatment and the TGF-1 signaling, and inhibited the increase of the invasion and metastasis ability of TGF-1 in the treatment of breast cancer cells. Conclusion: 1. The SND1 protein can bind to the gene promoter of SMAD2, SMAD3 and SMAD4 and recruit the GCN5 to reach the region. The TD domain of SND1 plays an important role in this process.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9
[Abstract]:Objective: Breast cancer is one of the malignant diseases that are harmful to the health of women in the world. The cause of death in more than 90% of patients with breast cancer is related to the metastasis of advanced cancer cells. The transfer of breast cancer is a very complicated process, from the start of local infiltration of cancer cells to the expression of various cytokines, signaling pathways and the expression of the genes involved in the transfer of the tumor cells in the course of the formation of a metastasis in the distal tissue, which is commonly involved in the transfer of breast cancer. In this case, the TGF signal pathway can promote the epithelial-to-mesenchymal transition of the breast cancer cells and promote the metastasis of the breast cancer with the expression of the invasion and metastasis-related genes, and plays an important role in the breast cancer metastasis process. The SND1 protein is a conserved multi-function protein. In recent years, SND1 has been found to be involved in the transcription regulation of the transcription co-active factor in the development of breast cancer. In the early stage of the research group, the promoter region of several effector factors of the SND1 protein and the TGF signaling pathway has strong binding capacity, and the SND1 may be involved in the transcription regulation of the downstream effector gene of the TGF signaling pathway. However, there is still an unknown correlation between SND1, TGF signal pathway and breast cancer metastasis. Therefore, the purpose of this study is to investigate the molecular mechanism of SND1 to participate in the metastasis of breast cancer by influencing TGF signal pathway. Method: This topic mainly includes seven parts: 1. In order to study the correlation between SND1 and breast cancer metastasis and prognosis, SND1 and SCID mice were used to study the correlation between SND1 and breast cancer metastasis and prognosis. In the breast cancer cell line, the gene which may be regulated by the SND1 expression can be predicted by the full-genome Ch IP-chip, and a gene co-expressed with the SND1 is found in the TCGA breast cancer database, and the genes which are regulated by the SND1 expression are predicted in combination. The effects of SND1 on SMAD2, SMAD3, SMAD4 gene m RNA level, protein level and transcription level were detected by immunoblotting experiment, polymerase chain reaction and luciferase experiment in breast cancer cell line. Smads downstream target genes, which were potentially affected by SND1 expression, were analyzed by software, and the effect of SND1 expression on its m-RNA level was detected by polymerase chain reaction. In the breast cancer cell line, the combination of SND1 and SMAD2, SMAD3, and SMAD4 gene promoter was detected by using chromatin immunoprecipitation and electrophoretic mobility assay. The molecular mechanisms of the transcription of SMAD2, SMAD3 and SMAD4 genes were analyzed by immunoprecipitation and chromatin immunoprecipitation in breast cancer cell lines. The effects of SND1 different domains on the level of transcription and protein expression of SMAD2, SMAD3 and SMAD4 genes were detected by using the GST-pulpit experiment, the polymerase chain reaction, the chromatin immunoprecipitation experiment, the immunoblotting experiment and the double-luciferase experiment. The cells were treated with TGF-1 in the stable silent SND1 breast cancer cells and their control cells, and the effect of SND1 expression on the signal conduction of TGF was analyzed by immunoblotting experiment, immunofluorescence and luciferase experiment. The effect of the change of SND1 expression on the activation of TGF-1 receptor was detected by the activation of the receptor. The effect of the change of SND1 expression on the invasion and metastasis of breast cancer under the treatment of TGF-1 was detected by a scratch test. Results: 1. The results showed that the high expression of SND1 protein was related to the metastasis of breast cancer, and the survival time of the clinical patients and the mice was analyzed by the Kaplan-Meier method. The conclusion was that the expression of SND1 protein was related to the poor prognosis. SMAD2, SMAD3 and SMAD4 were selected as the target gene of SND1 protein by analyzing Ch IP-chip and TCGA data. The SND1 protein in breast cancer cells can regulate the expression of Smad2, Smad3 and Smad4 in the SMAD2, SMAD3 and SMAD4 gene, and the expression of SND1 can also cause the change of the mRNA level of the target gene m in the downstream part of Smad2, Smad3 and Smad4. The gene promoter of SMAD4 can be combined with two conservative modes to promote the gene transcription. 5. The SND1 protein can be combined with the GCN5, and the GCN5 is incorporated into the SMAD2, SMAD3, SMAD4 gene promoter, and the gene transcription is promoted by the GCN5 by catalyzing histone H3K9 to promote the gene transcription. The SMAD4 gene plays a major role in the transcription process. The decrease in the expression of SND1 protein in breast cancer did not affect the activation of TGF-1 receptor after TGF-1 treatment, but decreased the level of the phosphorylation of endogenous Smad2 and Smad3 in breast cancer cells after TGF-1 treatment and the TGF-1 signaling, and inhibited the increase of the invasion and metastasis ability of TGF-1 in the treatment of breast cancer cells. Conclusion: 1. The SND1 protein can bind to the gene promoter of SMAD2, SMAD3 and SMAD4 and recruit the GCN5 to reach the region. The TD domain of SND1 plays an important role in this process.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9
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10 胡蘊(yùn)慧;李雙靜;Ernesto Yag，
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