miR-128靶向调控LAPTM4B介导乳腺癌侵袭转移及化疗耐药的研究

The metastasis and multiple-drug resistance to chemotherapy of the breast cancer is one of the main cause for failures of breast cancer treatment, and also is the bottleneck of the treatment in clinics. Studies have shown that Lysosome-associated protein transmembrane 4 beta (LAPTM4B) has important roles in multiple biological processes during cancer development and progression, including malignant transformation, apoptosis, invasion and metastasis, chemotherapy resistance through its involvement in several oncogenic signal transduction pathways, and it is a very promising tumor therapeutic target, however up to date, its regulating mechanism has not been reported at home and abroad. For the first time our previous findings have revealed that the overexpression of LAPTM4B in breast cancer was significantly associated with TNM stage、 lymph node metastasis and poor prognosis. In addition, we found that the LAPTM4B was highly expressed in MCF-7/ADR cells, and proved preliminarily that LAPTM4B gene contains the binding sites of miR-128. Therefore, we hypothesize that miR-128 negatively regulates LAPTM4B expression and thus reverses metastasis and chemotherapy resistance in breast cancer. In the project, we plan to study the effect and mechanism of miR-128 mediating the invasion and metastasis and chemotherapy resistance of breast cancer by targeting LAPTM4B through three levels of clinical patients-cell line-animal model. Based on this study, the conclusion will provide a theoretical and experimental basis for clinic to predict the prognosis and chemotherapeutic sensitivity and make the individualized therapy for patients with breast cancer.

乳腺癌的转移和化疗耐药是治疗失败的主要原因之一，亦是乳腺癌治疗的瓶颈所在。溶酶体相关4次跨膜蛋白β（LAPTM4B）与肿瘤发生、侵袭转移及化疗耐药相关，是很有前景的肿瘤治疗靶点，然而其调控机制国内外尚无报道。本课题组前期研究证实LAPTM4B在乳腺癌组织中呈高表达，且与临床分期、腋窝淋巴结转移和不良预后正相关。预实验结果显示：LAPTM4B在乳腺癌化疗耐药细胞系中呈高表达，并初步验证了miR-128和LAPTM4B的靶向关系。我们推测：LAPTM4B作为miR-128的靶基因参与乳腺癌的侵袭转移和化疗耐药。本课题拟采用miRNA过表达、绿色荧光蛋白活体标记、免疫组化等分子生物学技术，从临床患者-细胞系-动物模型三个层次明确miR-128通过靶向调控LAPTM4B介导乳腺癌侵袭转移和化疗耐药的作用机制。本课题将从新的视角阐明乳腺癌侵袭转移和化疗耐药的分子机制，为乳腺癌防治提供新思路和新靶点。

乳腺癌是全球女性最常见的恶性肿瘤和癌症死亡的主要病因之一，随着早期诊断、手术和放化疗水平的不断提高，和个体化治疗的实施，乳腺癌的死亡率已有所下降，但世界范围内乳腺癌仍是妇女癌症首要的死亡原因。肿瘤的复发转移一直以来都是肿瘤治疗难以攻克的难题之一，亦是研究的热点问题。溶酶体相关4次跨膜蛋白β（LAPTM4B）与肿瘤发生及侵袭转移密切相关，是很有前景的肿瘤治疗靶点。本项目中，我们结合临床样本的免疫组化分析明确了LAPTM4B表达与腋窝淋巴就转移的相关性；并建立LAPTM4B沉默的MCF-7细胞系以及LAPTM4B过表达的T47D细胞系，并应用MTT实验证实LAPTM4B可增强乳腺癌细胞的增殖能力，应用划痕实验证实LAPTM4B可增强乳腺癌细胞的迁移运动能力，应用Transwell小室实验侵袭实验证实LAPTM4B可增强乳腺癌细胞的侵袭能力，同时应用Western blot法检测LAPTM4B表达水平改变后对EMT相关评价指标的影响，并发现LAPTM4B影响乳腺癌细胞的侵袭转移能力可能与EMT相关。我们进一步研究调控LAPTM4B的分子机制，通过生物信息学分析，我们发现miR-128可调控LAPTM4B的表达，通过组织样本的检测我们发现miR-128的表达水平与影响乳腺癌预后的因素如淋巴结转移(P < 0.001)、临床分期(P < 0.001)、组织学分级(P < 0.001)、复发(P < 0.001)及分子亚型(P < 0.001)密切相关，多因素Cox回归分析提示miR-128表达水平与乳腺癌患者的OS和DFS均相关，亚组分析提示在三阴乳腺癌患者中，miR-128低表达明显导致患者的OS及DFS缩短(both P < 0.001)，并通过细胞体外实验和动物体内实验证实，miR-128的过表达导致三阴乳腺癌细胞系中的葡萄糖消耗降低，表现为培养基中葡萄糖摄取降低和乳酸分泌减少，抑制糖酵解的过程，同时也抑制了三阴乳腺癌的增殖能力。通过生物信息预测结果结合miR-128参与葡萄糖代谢的过程，最终我们把miR-128的靶点锁定于INSR和IRS1，且通过细胞体外实验验证miR-128通过抑制INSR和IRS1的表达来抑制TNBC细胞葡萄糖代谢和增殖。上述结果提示LAPTM4B和miR-128作为乳腺癌预防及治疗的基因靶点的可能性，为乳腺癌潜在的分子靶向治疗提供了理论基础。