SND1促進TGFβ信號傳導及乳腺癌轉移的分子機制
發(fā)布時間：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胞的轉移相關。乳腺癌的轉移是一個十分復雜的過程,從癌細胞開始局部浸潤直到遠端組織形成轉移灶的過程中多種細胞因子、信號通路及腫瘤細胞本身轉移相關基因的表達相互作用,共同參與乳腺癌的轉移。在這其中TGFβ信號通路可促進乳腺癌細胞發(fā)生上皮-間充質轉化及與侵襲轉移相關基因的表達促進乳腺癌的轉移,在乳腺癌轉移過程中發(fā)揮了重要的作用。SND1蛋白是一個保守的多功能蛋白,近年來研究發(fā)現(xiàn)SND1作為轉錄共激活因子參與基因的轉錄調控過程同時也參與乳腺癌的發(fā)生發(fā)展過程。本課題組前期發(fā)現(xiàn)SND1蛋白與TGFβ信號通路若干效應因子的啟動子區(qū)域有很強的結合能力,提示SND1可能參與TGFβ信號通路下游效應基因的轉錄調控。但SND1、TGFβ信號通路與乳腺癌轉移三者之間是否存在相關性依然未知,因此本課題目的是探討SND1通過影響TGFβ信號通路參與乳腺癌轉移的分子機制。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)小鼠進行體內活體成瘤實驗分析SND1與乳腺癌轉移與預后的相關性。2.在乳腺癌細胞系通過全基因組Ch IP-chip預測可能受SND1表達調控的基因,在TCGA乳腺癌數(shù)據庫找到與SND1共表達的基因,將二者結合進行分析預測受SND1表達調控的基因。3.在乳腺癌細胞系中利用免疫印跡實驗,聚合酶鏈反應,熒光素酶實驗檢測SND1對SMAD2、SMAD3、SMAD4基因m RNA水平,蛋白水平及轉錄水平的影響。利用軟件分析潛在受SND1表達影響的Smads下游靶基因,并利用聚合酶鏈反應檢測SND1表達變化對其m RNA水平的影響。4.在乳腺癌細胞系中利用染色質免疫共沉淀及電泳遷移率實驗檢測SND1與SMAD2、SMAD3、SMAD4基因啟動子結合情況。5.在乳腺癌細胞系中用免疫共沉淀實驗及染色質免疫共沉淀實驗分析SND1調控SMAD2、SMAD3、SMAD4基因轉錄的分子機制。6.利用GST-pulldown實驗、聚合酶鏈反應、染色質免疫共沉淀實驗、免疫印跡實驗及雙熒光素酶實驗檢測SND1不同結構域對SMAD2、SMAD3、SMAD4基因的轉錄水平及蛋白表達的影響。7.在穩(wěn)定沉默SND1的乳腺癌細胞及其對照細胞中用TGFβ1處理細胞,通過免疫印跡實驗,免疫熒光,熒光素酶實驗分析SND1表達變化對TGFβ信號傳導的影響;利用受體激活實驗檢測TGFβ1處理下SND1表達變化對細胞TGFβ受體激活情況的影響;利用劃痕實驗,transwell實驗檢測在TGFβ1處理條件下SND1表達變化會對乳腺癌侵襲轉移的影響。結果:1.免疫組化分析及小鼠成瘤實驗中結果均提示SND1蛋白高表達與乳腺癌轉移相關,通過Kaplan-Meier分別對臨床病人及小鼠的生存期進行分析,得出結論SND1蛋白的表達與預后差相關。2.通過對Ch IP-chip和TCGA數(shù)據進行分析,篩選SMAD2、SMAD3、SMAD4作為SND1蛋白的靶基因。3.乳腺癌細胞中SND1蛋白可以調控SMAD2、SMAD3、SMAD4基因轉錄水平進一步調控細胞內Smad2、Smad3和Smad4的蛋白表達;SND1的表達變化也會引起Smad2、Smad3和Smad4蛋白下游部分靶基因m RNA水平的改變。4.SND1蛋白通過與SMAD2、SMAD3、SMAD4的基因啟動子兩個保守模序結合促進基因轉錄。5.SND1蛋白可以與GCN5結合,并募集GCN5結合到SMAD2、SMAD3、SMAD4基因啟動子上,通過GCN5通過催化組蛋白H3K9乙酰化促進基因轉錄。6.SND1的TD結構域可與GCN5結合并在促進SMAD2、SMAD3、SMAD4基因轉錄過程中發(fā)揮了主要作用。7.在乳腺癌中SND1蛋白表達降低不會影響TGFβ1處理后TGFβ受體激活情況但會降低TGFβ1處理后乳腺癌細胞內源Smad2和Smad3的磷酸化水平及TGFβ信號傳導,并抑制TGFβ1處理乳腺癌細胞后細胞的侵襲轉移能力增加。結論:1.SND1蛋白可以結合到SMAD2、SMAD3、SMAD4的基因啟動子保守模序并招募GCN5到達該區(qū)域,通過GCN5催化該區(qū)域組蛋白H3K9乙酰化促進基因轉錄,SND1的TD結構域在此過程中發(fā)揮了重要作用。2.SND1蛋白可以通過調控TGFβ下游效應分子的表達影響TGFβ信號通路的信號傳導,并進一步影響了TGFβ信號通路對乳腺癌轉移的調控。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R737.9
【相似文獻】
相關期刊論文 前10條
1 ;可導致乳腺癌轉移的超級基因[J];現(xiàn)代醫(yī)學儀器與應用;2008年01期
2 ;日本科學家發(fā)現(xiàn)抑制乳腺癌轉移的蛋白質[J];中國新藥雜志;2009年04期
3 高峰;;預知乳腺癌轉移的新研究[J];醫(yī)學與哲學(臨床決策論壇版);2009年06期
4 ;基因影響乳腺癌轉移被查明[J];中華中醫(yī)藥學刊;2010年07期
5 高鳳霞;;乳腺癌轉移的相關因素研究進展綜述[J];醫(yī)學信息(中旬刊);2011年01期
6 寶魯日;師永紅;;乳腺癌轉移相關基因的研究進展[J];內蒙古醫(yī)學院學報;2011年04期
7 ;藥物抑制乳腺癌轉移[J];中國腫瘤;1996年06期
8 徐楊榮;梁慶模;;乳腺癌轉移基因的研究進展[J];醫(yī)學綜述;2007年02期
9 羅萬春;易東;劉恩;龔利紅;;整數(shù)非線性規(guī)劃模型在乳腺癌轉移相關基因表達調控網絡建立中的應用[J];北京生物醫(yī)學工程;2008年02期
10 王福玉;;血清TPS水平預測乳腺癌轉移或術后復發(fā)的臨床觀察[J];醫(yī)學信息;2010年07期
相關會議論文 前10條
1 孫寶存;;乳腺癌轉移機制的研究現(xiàn)狀與進展[A];第三屆中國腫瘤學術大會教育論文集[C];2004年
2 王培林;黃信孚;謝玉泉;;人乳腺癌轉移抑制的初步實驗研究[A];2000全國腫瘤學術大會論文集[C];2000年
3 劉勝;劉玲琳;;對“從化”理論的認識及其在乳腺癌轉移治療中的應用[A];第十一屆全國中醫(yī)及中西醫(yī)結合乳腺病學術會議論文集[C];2009年
4 鄒天寧;湯學良;聶建云;張季;楊莊青;;腫瘤標志物CA15-3對乳腺癌轉移診斷價值的探討[A];2000全國腫瘤學術大會論文集[C];2000年
5 喬宇峰;馮玉梅;李曉青;李希;;KNSL4基因單核苷酸多態(tài)性與乳腺癌轉移的關系[A];第四屆中國腫瘤學術大會暨第五屆海峽兩岸腫瘤學術會議論文集[C];2006年
6 郭倩倩;;Rankl-Rank信號與乳腺癌轉移的關系研究進展[A];第十二次全國中醫(yī)、中西醫(yī)結合乳房病學術會議論文集[C];2011年
7 黃佳yN;李紅麗;馮濤;;骨形成蛋白BMP4在乳腺癌轉移中的作用和機制探討[A];第七屆全國醫(yī)學生物化學與分子生物學和第四屆全國臨床應用生物化學與分子生物學聯(lián)合學術研討會暨醫(yī)學生化分會會員代表大會論文集[C];2011年
8 馮玉梅;孫保存;李曉青;牛昀;寧連勝;方志沂;肖春花;王玉麗;張麗娜;李希;郝希山;;利用高通量基因芯片篩選乳腺癌轉移預測基因群并用于乳腺癌轉移預后分子分型[A];第四屆中國腫瘤學術大會暨第五屆海峽兩岸腫瘤學術會議教育集[C];2006年
9 楊清玲;陳昌杰;楊志鋒;;vMIP-Ⅱ N端重組肽的原核表達、純化及活性鑒定[A];華東六省一市生物化學與分子生物學會2010年學術交流會論文集[C];2010年
10 胡蘊慧;李雙靜;Ernesto Yag，
本文編號：2407772
本文鏈接：http://www.sikaile.net/yixuelunwen/zlx/2407772.html
最近更新
教材專著
