Coronin 1a对乳腺癌细胞增殖转移的影响

发布时间：2018-03-28 20:32

本文选题：Coronin　切入点：1a　出处：《天津医科大学》2017年硕士论文

【摘要】：研究目的:Coronin蛋白家族是在提纯细胞骨架蛋白时被发现,而Coronin 1a正是Coronin蛋白家族中研究最为广泛的一种。在细胞内,许多涉及到肌动蛋白的细胞骨架运动中Coronin 1a都作为一种辅助因子而存在。Coronin 1a还被发现与寄生虫-宿主反应,神经系统发展及信号传导等过程有关。而Coronin 1a在肿瘤发生发展中的作用一直未被阐明。前期研究发现在不同乳腺癌细胞系中Coronin1a的表达有很大差异,提示Coronin 1a可能与不同分型乳腺癌的肿瘤特性相关。本研究主要探讨Coronin 1a表达量与乳腺癌细胞增殖转移能力的关系,并分析其发挥作用的可能途径。初步阐明Coronin 1a在细胞周期、EMT过程中可能发挥的作用。研究内容:分析Coronin 1a在不同乳腺癌细胞系内的表达情况,筛选Coronin 1a低表达的细胞系;构建Coronin 1a高表达载体;建立Coronin 1a高表达的乳腺癌细胞系;通过体内和体外实验研究Coronin 1a表达对乳腺癌细胞增殖和转移能力的影响并初步探讨其潜在的分子机制。研究方法:本研究首先检测了常见乳腺癌细胞系T47D、MDA-MB-231、MCF10A、MCF-7、SK-Br-3、293中Coronin 1a的表达情况,筛选出Coronin 1a低表达的细胞系。进而通过逆转录、PCR等技术扩增目的基因Coronin 1a,并通过与真核表达载体连接,构建出Coronin 1a的真核表达载体。利用慢病毒感染向Coronin1a低表达的细胞系内介导Coronin 1a过表达质粒,即成功构建Coronin 1a的高表达乳腺癌细胞株。应用MTT,平板克隆,EDU,流式细胞术等方法探究Coronin1a与乳腺癌细胞增殖之间的关系;应用Transwell,划痕实验,EMT相关指标的Western Blot实验探究Coronin 1a与乳腺癌细胞运动转移能力之间的关系。最后通过向裸鼠皮下注射过表达Coronin 1a的细胞悬液,探究Coronin 1a对活体动物成瘤能力的影响。通过我们的研究,第一次探讨了Coronin 1a在乳腺癌增殖转移能力中所发挥的作用,并初步探讨了其可能参与的调控途径。研究结果:我们发现,Coronin 1a在MDA-MB-231细胞系和SK-Br-3细胞系中低表达,在MCF-7细胞系、MCF-10A细胞系中高表达,在T47D、293细胞系中则为阴性表达。通过构建pc DNA3.0-HA-Coronin 1a质粒并向MDA-MB-231,SK-Br-3细胞内介导转染,我们成功构建出Coronin 1a的高表达细胞株。MTT,平板克隆,EDU实验均证实Coronin 1a的过表达明显促进了乳腺癌细胞的增殖。应用流式细胞术对细胞周期进行检测发现,过表达Coronin 1a后,乳腺癌细胞中处于S期的比例明显升高。提示Coronin 1a可能通过调控细胞周期影响乳腺癌细胞的增殖活性。Transwell,划痕实验均证实Coronin 1a的过表达明显增强了乳腺癌细胞的运动转移能力。EMT相关指标的Western blot实验表明,过表达Coronin 1a是E-cadherin表达下降,N-cadherin表达升高,Vimentin与Snail表达也相应升高,提示Coronin 1a对乳腺癌细胞运动转移能力的影响可能与其促进了EMT转化过程有关。裸鼠成瘤实验证实,过表达Coronin 1a后乳腺癌细胞具有更高的成瘤能力,且Ki-67指标明显高于对照组。研究结论:Coronin 1a与乳腺癌的增殖转移能力密切相关,其可能通过影响细胞周期调控乳腺癌细胞的增殖,可能通过促进EMT过程调控乳腺癌细胞运动转移能力。
[Abstract]:Objective: To study the Coronin protein family was found in protein purification of cell skeleton, while Coronin 1a is the Coronin protein family of one of the most extensive. In cells, many involving actin cytoskeleton movements in Coronin 1A as a cofactor of.Coronin 1A has also been found to react with parasites the host, nervous system development and signal transduction and so on. Coronin 1a and its role in tumorigenesis has not been elucidated. Previous study found that the expression of Coronin1a in different breast cancer cell lines are very different, suggesting that Coronin 1A may be related to tumor characteristics in different subtypes of breast cancer. The relationship between the main to investigate the expression of 1A and Coronin in breast cancer cell proliferation and metastasis ability, and to analyze the possible ways to play role. Initially stated Coronin 1a in the cell cycle, may be in the process of EMT Role. Contents: to analyze the expression of Coronin 1a in breast cancer cell lines in the selection of Coronin 1A low expression cell line; construction of Coronin 1A high expression vector; establish breast cancer cell lines with high expression of Coronin 1A; in vivo and in vitro experimental study of Coronin 1A expression on the proliferation and metastasis of breast cancer cells and to explore its potential molecular mechanism. Methods: This study first examined the T47D common breast cancer cell line MDA-MB-231, MCF10A, MCF-7, Coronin 1A expression of SK-Br-3293 in the screening of low expression of Coronin 1A cell line. Then through reverse transcription amplification of PCR gene of Coronin 1A technology. And with the eukaryotic expression vector connection, construct the eukaryotic expression vector Coronin 1a. The infection to the low expression of Coronin1a cell line mediated by Coronin 1A using lentiviral expression plasmid, i.e. The successful construction of Coronin high expression of 1A in breast cancer cell lines. The application of MTT, clone, EDU, flow cytometry and other methods to explore the relationship between Coronin1a and breast cancer cell proliferation; application of Transwell, scratch test, the relationship between Western Blot EMT experimental indexes related to 1a and Coronin on breast cancer cell motility and metastasis. Finally, through to the over expression of Coronin 1a in nude mice subcutaneous injection of cell suspension, explore the impact of Coronin 1A on tumorigenicity in vivo animal. Through our research, first discusses the Coronin 1A transfer ability to play role in the proliferation of breast cancer, and to explore the regulatory pathways might be involved in the study. Results: we found that Coronin 1A low in MDA-MB-231 cells and SK-Br-3 cells expression in MCF-7 cells, higher expression of MCF-10A cells in T47D 293 cell line was negative expression through. Construction of PC DNA3.0-HA-Coronin plasmid and 1a to MDA-MB-231, SK-Br-3 cell mediated transfection, we successfully constructed Coronin 1A high expression cell line.MTT clone, EDU experiments have confirmed that overexpression significantly promoted the proliferation of breast cancer cells Coronin 1a. The cell cycle were detected by flow cytometry, too the expression of Coronin after 1a in breast cancer cells in S phase ratio increased significantly. Suggesting that Coronin 1A may be through the regulation of cell cycle proliferation of.Transwell breast cancer cells, scratch experiments confirmed overexpression significantly enhance the breast cancer cell metastasis Western blot experimental index movement ability of.EMT showed that Coronin 1a, expression Coronin 1a is the decreased expression of E-cadherin, increase the expression of N-cadherin, Vimentin and Snail expression increased, suggesting that Coronin 1A metastasis of breast cancer cell movement The effect may be related to promoting the EMT conversion process. The tumorigenicity experiments confirmed that overexpression of Coronin 1A breast cancer cells with tumorigenic ability higher, and the Ki-67 index was significantly higher than the control group. Conclusion: Coronin 1a and the proliferation of breast cancer metastasis, it may affect cell cycle regulation of breast the proliferation of cancer cells, could promote the EMT movement process of regulation of breast cancer cell metastasis ability.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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