SND1促进TGFβ信号传导及乳腺癌转移的分子机制

发布时间：2019-01-12 12:25

【摘要】：目的:乳腺癌是危害世界女性健康的恶性疾病之一,90%以上乳腺癌患者的死亡原因与晚期癌细胞的转移相关。乳腺癌的转移是一个十分复杂的过程,从癌细胞开始局部浸润直到远端组织形成转移灶的过程中多种细胞因子、信号通路及肿瘤细胞本身转移相关基因的表达相互作用,共同参与乳腺癌的转移。在这其中TGFβ信号通路可促进乳腺癌细胞发生上皮-间充质转化及与侵袭转移相关基因的表达促进乳腺癌的转移,在乳腺癌转移过程中发挥了重要的作用。SND1蛋白是一个保守的多功能蛋白,近年来研究发现SND1作为转录共激活因子参与基因的转录调控过程同时也参与乳腺癌的发生发展过程。本课题组前期发现SND1蛋白与TGFβ信号通路若干效应因子的启动子区域有很强的结合能力,提示SND1可能参与TGFβ信号通路下游效应基因的转录调控。但SND1、TGFβ信号通路与乳腺癌转移三者之间是否存在相关性依然未知,因此本课题目的是探讨SND1通过影响TGFβ信号通路参与乳腺癌转移的分子机制。方法:本课题主要包括七部分:1.利用临床的乳腺癌免疫组化样本进行SND1免疫组化染色及重症联合免疫缺陷(Severe Combined Immunodeficiency,SCID)小鼠进行体内活体成瘤实验分析SND1与乳腺癌转移与预后的相关性。2.在乳腺癌细胞系通过全基因组Ch IP-chip预测可能受SND1表达调控的基因,在TCGA乳腺癌数据库找到与SND1共表达的基因,将二者结合进行分析预测受SND1表达调控的基因。3.在乳腺癌细胞系中利用免疫印迹实验,聚合酶链反应,荧光素酶实验检测SND1对SMAD2、SMAD3、SMAD4基因m RNA水平,蛋白水平及转录水平的影响。利用软件分析潜在受SND1表达影响的Smads下游靶基因,并利用聚合酶链反应检测SND1表达变化对其m RNA水平的影响。4.在乳腺癌细胞系中利用染色质免疫共沉淀及电泳迁移率实验检测SND1与SMAD2、SMAD3、SMAD4基因启动子结合情况。5.在乳腺癌细胞系中用免疫共沉淀实验及染色质免疫共沉淀实验分析SND1调控SMAD2、SMAD3、SMAD4基因转录的分子机制。6.利用GST-pulldown实验、聚合酶链反应、染色质免疫共沉淀实验、免疫印迹实验及双荧光素酶实验检测SND1不同结构域对SMAD2、SMAD3、SMAD4基因的转录水平及蛋白表达的影响。7.在稳定沉默SND1的乳腺癌细胞及其对照细胞中用TGFβ1处理细胞,通过免疫印迹实验,免疫荧光,荧光素酶实验分析SND1表达变化对TGFβ信号传导的影响;利用受体激活实验检测TGFβ1处理下SND1表达变化对细胞TGFβ受体激活情况的影响;利用划痕实验,transwell实验检测在TGFβ1处理条件下SND1表达变化会对乳腺癌侵袭转移的影响。结果:1.免疫组化分析及小鼠成瘤实验中结果均提示SND1蛋白高表达与乳腺癌转移相关,通过Kaplan-Meier分别对临床病人及小鼠的生存期进行分析,得出结论SND1蛋白的表达与预后差相关。2.通过对Ch IP-chip和TCGA数据进行分析,筛选SMAD2、SMAD3、SMAD4作为SND1蛋白的靶基因。3.乳腺癌细胞中SND1蛋白可以调控SMAD2、SMAD3、SMAD4基因转录水平进一步调控细胞内Smad2、Smad3和Smad4的蛋白表达;SND1的表达变化也会引起Smad2、Smad3和Smad4蛋白下游部分靶基因m RNA水平的改变。4.SND1蛋白通过与SMAD2、SMAD3、SMAD4的基因启动子两个保守模序结合促进基因转录。5.SND1蛋白可以与GCN5结合,并募集GCN5结合到SMAD2、SMAD3、SMAD4基因启动子上,通过GCN5通过催化组蛋白H3K9乙酰化促进基因转录。6.SND1的TD结构域可与GCN5结合并在促进SMAD2、SMAD3、SMAD4基因转录过程中发挥了主要作用。7.在乳腺癌中SND1蛋白表达降低不会影响TGFβ1处理后TGFβ受体激活情况但会降低TGFβ1处理后乳腺癌细胞内源Smad2和Smad3的磷酸化水平及TGFβ信号传导,并抑制TGFβ1处理乳腺癌细胞后细胞的侵袭转移能力增加。结论:1.SND1蛋白可以结合到SMAD2、SMAD3、SMAD4的基因启动子保守模序并招募GCN5到达该区域,通过GCN5催化该区域组蛋白H3K9乙酰化促进基因转录,SND1的TD结构域在此过程中发挥了重要作用。2.SND1蛋白可以通过调控TGFβ下游效应分子的表达影响TGFβ信号通路的信号传导,并进一步影响了TGFβ信号通路对乳腺癌转移的调控。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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