粘附G蛋白偶联受体GPR116调节乳腺癌转移及其机理研究

发布时间：2018-06-29 01:02

本文选题：乳腺癌转移 + GPR116　；参考：《华东师范大学》2015年博士论文

【摘要】：乳腺癌转移是其致死的最主要原因。在乳腺癌转移过程中,乳腺癌细胞的迁移运动能力对肿瘤浸润以及进入转移靶器官有着重要作用。G蛋白偶联受体(GPCRs)是最大的跨膜受体家族,其中粘附G蛋白偶联受体(aGPCRs)因胞外端含有大量粘附蛋白中的结构域而被广泛认为和细胞运动、细胞识别等相关,也据此推测其在肿瘤的转移可能具有重要功能。基于此,本文探究了粘附G蛋白偶联受体对乳腺癌转移的影响。首先,我们通过检测粘附G蛋白偶联受体在高迁移乳腺癌细胞中的基因表达水平,发现了14个高表达的候选基因；随后,利用干扰RNA抑制备选基因表达,通过细胞迁移实验的筛选,发现了粘附G蛋白偶联受体GPR116强烈的促进乳腺癌细胞的迁移。接着,我们构建了稳定干扰GPR116以及过表达GPR116的细胞株,通过体外细胞迁移实验验证了上述筛选结果。进一步地,我们通过两个独立的乳腺癌转移小鼠模型(乳腺癌肺转移模型和骨转移模型)在体内证明GPR116促进乳腺癌的转移。在机制上,首先我们发现干扰GPR116后乳腺癌细胞的stress fiber和lamelliapodia形成显著减少,细胞形态由扇状转变为细长形,细胞的极性减弱。进一步,通过pull-down实验发现小G蛋白RhoA和Racl的活性在干扰GPR116后显著下调。随后,通过回复实验证明RhoA和Rac1活性下调是造成GPR116干扰后细胞迁移能力减弱的主要原因。我们通过对G蛋白的筛选发现GPR116偶联Gαq激活RhoA和RAcl,并且回复持续激活的Gαq能够回复干扰GRP116后导致的RhoA、Racl活性下调以及细胞的迁移能力。进一步地,我们发现GPR116通过Gαq激活p63RhoGEF,后者促进RhoA和Racl的活化,调节乳腺癌细胞运动迁移中骨架F-actin的变化,最终调节乳腺癌细胞的迁移和转移。最后,通过分析临床乳腺癌病人数据发现,GPR116的表达随着乳腺癌恶性程度增加而升高,特别是在转移乳腺癌中的表达尤为显著。高表达GPR116的乳腺癌病人的复发生存率和远端转移复发生存率显著降低。因此GPR116有可能成为潜在的治疗乳腺癌转移靶点。
[Abstract]:Metastasis of breast cancer is the leading cause of death. In the process of breast cancer metastasis, the migration and movement ability of breast cancer cells plays an important role in tumor infiltration and entry into metastasis target organ. G protein coupled receptor (GPCRs) is the largest transmembrane receptor family. Adhesion G protein-coupled receptors (aGPCRs) are widely considered to be related to cell movement and cell recognition due to the presence of a large number of domains in the extracellular adhesion proteins. It is also speculated that aGPCRs may play an important role in tumor metastasis. Based on this, we investigated the effect of adhesion G-protein coupled receptor on metastasis of breast cancer. First, we detected the gene expression level of Adhesion G protein-coupled receptor in high migration breast cancer cells, and we found 14 candidate genes with high expression, and then we used interference RNA to inhibit the expression of alternative genes. Through the screening of cell migration assay, it was found that adhesion G protein coupled receptor GPR116 strongly promoted the migration of breast cancer cells. Then we constructed stable interfering GPR116 cell lines and overexpressed GPR116 cells. The results were verified by cell migration experiments in vitro. Furthermore, we demonstrated in vivo that GPR116 promotes breast cancer metastasis through two independent mouse models of breast cancer metastasis (breast cancer lung metastasis model and bone metastasis model). In mechanism, we first found that the stress fiber and lamelliapodia formation of breast cancer cells decreased significantly after GPR116 interference, cell morphology changed from fan-shaped to slender, and cell polarity decreased. Furthermore, the activity of small G protein RhoA and Racl decreased significantly after interfering with GPR116 by pull-down assay. After that, the down-regulation of RhoA and Rac1 activity was demonstrated to be the main reason for the decrease of cell migration ability after GPR116 interference. We found that GPR116 coupled G 伪 Q activated RhoA and RAcl. and that G 伪 Q which returned to continuously activated G 伪 Q could reverse the down-regulation of RhoAn Racl activity induced by GRP116 and the migration ability of GPR116 cells. Furthermore, we found that GPR116 activated p63 RhoGEF through G 伪 Q, which promoted RhoA and Racl activation, regulated the changes of F-actin in breast cancer cells, and finally regulated the migration and metastasis of breast cancer cells. Finally, by analyzing the clinical data of breast cancer patients, we found that the expression of GPR116 increased with the increase of malignant degree of breast cancer, especially in metastatic breast cancer. The recurrence survival rate and distal metastasis survival rate of breast cancer patients with high expression of GPR116 decreased significantly. Therefore, GPR116 may be a potential target for the treatment of breast cancer metastasis.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

【共引文献】