ATM对乳腺癌细胞迁移和侵袭影响及其机制研究

发布时间：2019-07-05 07:02

【摘要】：目的:探讨ATM对乳腺癌细胞迁移和侵袭的影响及其调控机制。方法:(1)利用免疫组织化学方法检测乳腺癌癌旁肿瘤组织(以下简称正常乳腺组织)、侵润、侵润伴转移乳腺癌组织中p-ATM蛋白的表达;乳腺癌BT549和MDA-MB-231细胞分别经常氧(21%O2)和低氧(1%O2)处理后,细胞免疫荧光和Western Blot检测p-ATM和总ATM表达,Transwell实验检测细胞迁移和侵袭能力。(2)利用sh RNA技术和ATM特异性抑制剂Ku60019抑制乳腺癌BT549和MDA-MB-231细胞中ATM功能,Transwell实验检测细胞迁移和侵袭能力。(3)BT549细胞Ku60019处理组和溶剂对照组做磷酸化蛋白质组学分析,利用DAVID软件对靶蛋白进行GO和KEGG信号通路富集分析,筛选与迁移和侵袭相关的靶蛋白。(4)利用IP实验验证靶基因与ATM相互作用并受其调控,采用sh RNA技术敲低靶蛋白,Transwell实验检测细胞迁移和侵袭能力。(5)利用定点突变技术,Transwell实验,IP实验,细胞应力纤维染色技术验证靶蛋白特异性磷酸化位点功能。结果:(1)随着乳腺肿瘤分级的增加,p-ATM蛋白的表达逐渐增强;与常氧组相比,低氧处理后p-ATM表达量显著增加,细胞迁移和侵袭能力显著增强。(2)ATM敲低或抑制后,细胞迁移和侵袭能力显著下调。(3)磷酸化蛋白质组学分析结果显示,ATM下游靶蛋白主要参与细胞间粘附过程,调控肌动蛋白细胞骨架等信号通路。结合文献和质谱数据选择与迁移侵袭相关的靶蛋白为CTTN,其特异性磷酸位点为S113。(4)IP实验证实ATM可以磷酸化CTTN S113位点,敲低CTTN的表达,乳腺癌细胞迁移和侵袭能力显著下调。(5)利用定点突变实验发现,CTTN S113位点磷酸化可以促进癌细胞迁移和侵袭。进一步通过IP实验和细胞应力纤维染色实验发现CTTN S113位点磷酸化可以通过结合Arp2/3复合体促进细胞F-actin应力纤维形成。结论:低氧活化的ATM可以通过磷酸化CTTN S113位点调控Arp2/3复合体介导的肌动蛋白聚合促进乳腺癌细胞迁移和侵袭。
文内图片：

图片说明：低氧活化ATM促进乳腺癌细胞迁移和侵袭（A）免疫组织化学染色检测乳腺正常组织和乳腺癌组织中p-ATM表达量
[Abstract]:Objective: to investigate the effect of ATM on migration and invasion of breast cancer cells and its regulatory mechanism. Methods: (1) Immunohistochemical method was used to detect the expression of p-ATM protein in paracancerous tumor tissues (hereinafter referred to as normal breast tissues) and invasion and metastasis of breast cancer tissues. After breast cancer BT549 and MDA-MB-231 cells were treated with regular oxygen (21%O2) and hypoxia (1%O2), the expression of p-ATM and total ATM was detected by immunofluorescence and Western Blot, and the migration and invasion of breast cancer cells were detected by Transwell assay. (2) sh RNA technique and Ku60019, a specific inhibitor of ATM, were used to inhibit ATM function in BT549 and MDA-MB-231 cells of breast cancer. The ability of cell migration and invasion was detected by Transwell assay. (3) Phosphorylation proteome analysis was performed in Ku60019 treatment group and solvent control group of BT549 cells, GO and KEGG signal pathways were enriched and analyzed by DAVID software, and the target proteins related to migration and invasion were screened. (4) the interaction between target gene and ATM was verified by IP assay, and the target protein was knocked down by sh RNA technique. Transwell assay was used to detect cell migration and invasion. (5) site-directed mutagenesis, Transwell test, IP test and cell stress fiber staining were used to verify the function of specific phosphorylation sites of target proteins. Results: (1) with the increase of breast tumor grade, the expression of p-ATM protein increased gradually. Compared with normoxic group, the expression of p-ATM increased significantly and the ability of cell migration and invasion increased significantly after hypoxia treatment. (2) after ATM knockdown or inhibition, the ability of cell migration and invasion was significantly down-regulated. (3) the results of phosphorylation proteome analysis showed that the downstream target protein of ATM was mainly involved in the process of cell adhesion and regulated actin cytoskeleton and other signaling pathways. Combined with literature and mass spectrometry data, the target protein related to migration and invasion was selected as CTTN, and its specific phosphate site was S113. (4) IP assay confirmed that ATM could phosphorylation CTTN S113 site, knock down the expression of CTTN, and significantly down-regulate the migration and invasion of breast cancer cells. (5) site-directed mutation assay showed that CTTN S113 phosphorylation could promote the migration and invasion of breast cancer cells. Furthermore, IP assay and cell stress fiber staining showed that phosphorylation of CTTN S113 site could promote the formation of F-actin stress fibers by binding Arp2/3 complex. Conclusion: hypoxia activated ATM can regulate Arp2/3 complex mediated actin polymerization through phosphorylation of CTTN S113 site to promote the migration and invasion of breast cancer cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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