miR-519d 调控自噬影响三阴性乳腺癌耐药的作用机制研究

Chemotherapy plays important role in the comprehensive therapy of triple negative breast cancer, but drug resistance is a difficult problem in the process of chemotherapy. Using miRNA array technology, we found that miR-519d significantly down-expressed in the drug-resistant triple negative breast cancer cell lines. We further found that overexpression of miR-519d could reverse the triple negative breast cancer drug resistance; we predicted Beclin1 as a potential target gene of miR-519d by employing miRNA target gene prediction softwares, and we found that overexpression of miR-519d could down-regulate the expression of Beclin1, which indicated that miR-519d might be involeved in the drug resistanceof breast cancer through regulating cell autophagy. In this study, we will further uncover the biology characteristics of miR-519d; we want to detect the effect of miR-519d on triple negative breast cancer drug resistance and cell autophagy through overexpression and downexpression strategy, we use Beclin1 as clue ,we want to explore the undergoing molecular mechanism of miR-519d induced triple negative breast cancer drug resistance turnover and regulated cell autophagy. There was few information about the relationship between miRNA and triple negative breast cance drug resistance. This study will provide a potential intervention target for reversing the drug resistant of triple negative breast cancer.

化疗在三阴乳腺癌综合治疗中占重要地位，但耐药是三阴乳腺癌化疗过程中的棘手问题。miR-519d是我们利用miRNA芯片筛选发现的、差异低表达于耐药三阴乳腺癌细胞株中的miRNA。前期发现，过表达miR-519d可逆转三阴乳腺癌细胞的耐药性；生物信息学分析自噬基因Beclin1可能为miR-519d的靶基因；过表达miR-519d不但显著下调Beclin1表达，且抑制三阴乳腺癌细胞的自噬，提示miR-519d可能通过调控自噬而参与调节乳腺癌的耐药性。研究拟探讨乳腺癌miR-519d的生物学特征；通过过表达和沉默表达，观察miR-519d对三阴乳腺癌细胞耐药、自噬的影响；以Beclin1为机制线索，应用挽救实验策略，探讨miR-519d调控自噬、诱导三阴乳腺癌细胞耐药的分子机制。miR-519d与三阴乳腺癌耐药的关系及机制未见报道，本研究将可能为乳腺癌耐药的逆转提供新的靶标。

1..本项目研究原目标是揭示miR-519d的生物学特征，应用高通量测序检测敏感株MCF-7和耐药细胞株MCF-7/ADR、MCF-7/TAX差异表达的miRNAs，选择PicTar，Target-Scan，miRanda和PITA预测明显差异表达的这些miRNAs的靶基因的交集作为分析的基因集合；通过GO功能注释、GO富集分析（Gene Ontology）和信号转导通路富集分析（Pathway Enrichment）。发现耐药细胞差异表达miR-518a-5p更显著，miR-519d并无明显差异，选取miR-518a-5p进行研究。我们发现在乳腺癌细胞系MCF-7和MDA-MB-231细胞中过表达miR-518a-5p能抑制乳腺癌细胞的侵袭和迁移功能，但对于癌细胞的增殖功能没有显著的影响。研究显示该miR可能通过负性调控基因ROKC-1起作用，其机制有待进一步研究。miR-518a-5p虽是在耐药细胞和敏感细胞中差异表达的miRNA，但它在乳腺癌敏感细胞MCF-7和MDA-MB-231中过表达并不影响细胞对于阿霉素的耐药能力。我们考虑miR-518a-5p对于细胞耐药的影响可能是通过其他机制共同作用的，其具体的分子进程有待进一步的研究。因此针对miR-518a-5p开展更加深入的研究、并进行靶标的研发已具备一定的必要性。若获得更多重要依据，将可能为逆转乳腺癌耐药提供新的线索，具有潜在巨大的经济效益，我们希望能获得进一步研究时间。