靶向脂肪酸受体GPR120调控OPCML抑制乳腺癌细胞增殖和侵袭的机制

Breast cancer is one of the most common malignant tumors in women. Its uncontrollable growth, metastasis and recurrence is the key problem which needs to be solved. In recent years, it has been found that the abnormal fatty acid metabolism in tumor microenvironment might be very important for tumor growth. The scientific problem of this project is the new strategy of targeting G protein coupled receptor 120 (GPR120) to regulate the proliferation and invasion of tumor cells and then inhibit the breast cancer. In our preliminary experiments, GPR120 was found to highly expressed in breast cancer cells, and inhibition of its expression could significantly inhibit the proliferation and invasion of breast cancer cells. This project aims to study and define that GPR120 promotes the proliferation and invasion of breast cancer cells by regulating the histone and DNA methylation of OPCML protein. Using GPR120 inhibitor and agonist, G9a inhibitor, and recombinant human OPCML protein, we expect to explore the mechanisms that GPR120 could induce the silence of OPCML gene by regulating its histone and DNA methylation, which was mediated by methytransferase G9a. The silence of OPCML gene contributes to the activation AKT/PI3K signaling, and finally leads to the proliferation and invasion of the breast cancer cells. Our study will provide novel theoretical and technical basis for breast cancer inhibition by regulating the proliferation and invasion of breast cancer cells, with GPR120 as the key target.

乳腺癌是女性常见的发病率最高的恶性肿瘤，其恶性生长、转移与复发是目前临床治疗亟待解决的难题。近年发现，肿瘤微环境中脂肪酸代谢异常可能在肿瘤细胞的生长中至关重要。本项目聚焦的科学问题是干预脂肪酸受体GPR120调控乳腺癌细胞增殖及侵袭抑制乳腺癌的新策略。我们的预初实验发现，乳腺癌细胞中脂肪酸受体GPR120高表达，干预抑制其表达可显著抑制乳腺癌细胞的增殖与侵袭。本项目拟研究和明晰GPR120通过调控细胞粘附分子OPCML的组蛋白及DNA甲基化水平而抑制其表达并促进乳腺癌细胞的增殖与侵袭的作用；采用GPR120抑制剂与激动剂、G9a抑制剂、人重组OPCML蛋白等手段，揭示GPR120通过调控甲基化蛋白G9a的表达而沉默OPCML基因，进而活化AKT/PI3K信号促进乳腺癌细胞的增殖与侵袭的作用机制；形成与建立以GPR120为核心靶标，调控干预乳腺癌细胞的增殖与侵袭抑制乳腺癌的新策略。

肿瘤微环境中游离脂肪酸靶向结合脂肪酸受体GPR120在乳腺癌的发生发展中发挥重要作用，但其具体的作用机制尚不清楚。本研究我们在乳腺癌组织样本、乳腺癌细胞株及动物模型中研究了GPR120、G9a及OPCML基因功能，系统研究了GPR120通过调控甲基化蛋白G9a的表达而沉默OPCML基因，进而调节AKT/PI3K信号通路的分子机制，并对其在乳腺癌诊断和预后方面的价值进行了探讨。（1） GPR120在乳腺癌中的表达及生物学功能研究：通过免疫组化检测GPR120在乳腺癌组织中的表达明显高于癌旁正常组织，其表达与乳腺癌组织类型相关，与ki-67表达呈正相关，而与ER表达呈负相关，但在不同分子分型中其表达无显著差异；通过细胞增殖实和迁移验证实抑制GPR120的表达后明显抑制的乳腺癌细胞的增殖和细胞迁移。（2） 甲基化蛋白G9a在乳腺癌中的研究：免疫组化检测G9a在人乳腺癌组织中呈高表达, 且在Her-2过表达型乳腺癌中呈高表达， G9a的表达与Her-2、Ki-67及E-cadherin的表达呈正相关，应用G9a抑制剂后能明显抑制乳腺癌细胞的增殖。（3）OPCML基因在乳腺癌中的研究：通过免疫组化及RT-PCR检测OPCML在乳腺癌组织及细胞中低表达，细胞功能实验证实过表达OPCML后明显抑制了乳腺癌细胞的增殖、迁移和侵袭，裸鼠体内成瘤实验再次证实OPCML具有抑制肿瘤生长的作用。通过特异性甲基化实验（MSP）验证在乳腺癌细胞和组织中OPCML启动子区普遍甲基化，OPCML启动子区甲基化为其在乳腺癌中低表达的主要原因。（4）GPR120与OPCML、G9a及PI3K/AKT信号通路的调控关系研究：抑制GPR120的表达后可明显降低G9a的表达，提高OPCML的表达，同时能减少AKT磷酸化状态。Rescue实验进一步证明GPR120对乳腺癌细胞的增殖依赖于G9a/OPCML/PI3K/AKT轴的调控。综上所述，我们的研究结果表明，GPR120上调G9a的表达，G9a通过调控OPCML启动子区甲基化沉默其表达，进而激活PI3K/AKT信号通路促进乳腺癌细胞的增殖和侵袭，对乳腺癌的早期诊断和预后评估具有重要意义。