Exosomes介导miR-7704传递促进乳腺癌恶性进展的机制研究

MicroRNA (miRNA) array analysis identified one oncogenic miRNA, miR-7704, that was highly expressed in exosomes from metastatic human breast cancer cell lines. Previous studies have shown that the expression of miR-7704 in exosomes from metastatic breast cancer cells is increased and miR-7704 is transferred in exosomes. MiR-7704 overexpression can promote the malignant progress of breast cancer cells. There is a negative correlation between MiR-7704 expression and E-cad expression. This project suggests that exosomes-mediated transfer of miR-7704, which targets the E-cad gene, increases tumor malignancy in breast cancer. Cell lines MCF-10A, HMLE, MDA-MB-231, BT-549 and their exosomes were selected for this study. Overexpression / silencing technique and fluorescence in situ hybridization were used to detect cell morphology,invasion ability and the target gene E-cad expression. Animal orthotopic transplantation and metastatic tumor model, clinical blood and tissue samples were used to verify the hypothesis. This study may provide a potential target for breast cancer prevention and treatment.

miR-7704是课题组利用基因芯片从乳腺癌细胞外泌体（exosomes）中筛选出与乳腺癌恶性进展相关的microRNA。前期研究发现：MDA-MB-231侵袭性乳腺癌细胞分泌的exosomes中miR-7704表达升高，且可传递miR-7704。miR-7704过表达可促进乳腺癌细胞的恶性进展，miR-7704与E-cad的表达呈负相关。本项目提出exosomes介导miR-7704传递，通过靶向作用于E-cad基因，进而调控乳腺癌恶性进展这一重要假说。本课题拟以MCF-10A、HMLE、MDA-MB-231、BT-549四种细胞以及这四种细胞来源的exosomes为研究对象，结合过表达/沉默技术，荧光原位杂交技术，观察细胞形态及侵袭能力、靶基因E-cad的表达、动物原位移植及转移瘤模型、临床血及组织标本等体外、体内研究系统验证该假说。本研究可为乳腺癌转移防治提供潜在的靶标。

乳腺癌是世界范围内最常见的女性恶性肿瘤，近来研究发现肿瘤来源的exosomes介导的miRNAs传递与乳腺癌转移的关系非常密切。MiR-7704是课题组前期通过microRNAs芯片表达谱筛选到的一条在乳腺癌组织中特异性高表达、功能未知的微小RNA。通过GEO数据库分析和组织验证显示miR-7704在乳腺癌中显著高表达。沉默miR-7704可显著抑制乳腺癌的侵袭和转移。前期研究发现：相对于低转移性乳腺癌细胞，高转移性乳腺癌细胞可以分泌更多的exosomes，并且其exosomes分泌的miR-7704含量更高，共育实验及沉默实验发现高转移性乳腺癌细胞通过其exosomes传递致瘤性miR-7704来提高受体细胞的侵袭和转移能力。软件分析结合体外生物学实验证实E-cad是miR-7704的靶基因，且miR-7704通过靶向E-cad对乳腺癌的侵袭和转移能力进行调控。体内实验再次验证高转移性乳腺癌细胞来源的exosomes可促进低转移性受体细胞的恶性进展，具体机制为exosomes通过传递致瘤性miR-7704来提高受体细胞的侵袭和转移能力。Exosomal miR-7704调控乳腺癌恶性进展的功能与机制迄今为止国内外未见报道，因此具有源头创新，为乳腺癌的诊疗提供新靶标。