外泌体运载miRNA-224-5p介导细胞间对话抑制乳腺癌自噬的分子机制及其临床检测价值

Exosomes, are known to transfer biologically active proteins, miRNAs between cells, they have recently become the focus of intense interest as potential mediators of cell-cell communication, particularly in long-range and juxtacrine signaling events associated with progression of cancer. In our previous studies, we found that the expression of miR-224-5p was highly increased in breast cancer serum and tissue; miR-224-5p could inhibit autophagy activity; miR-224-5p level in breast cancer serum exosomes was also increased compared with controls. However, the mechanism of exosomal transfer of miR-224-5p for autophagy regulation remains to be further studied. Together with our previous results, the following aspects will be investigated by this project: 1. Collect clinical serum samples, determine the level of miR-224-5p, and analyze its clinical significance. 2. Construct cell lines with over-expression and low-expression of miR-224-5p, and prepare exosomes with different expression levels of miR-224-5p, so as to explore the role of exosomal transfer of miR-224-5p inhibiting autophagy activity at cellular level and in the animal models. 3. Clarify the target molecule downstream of miR-224-5p and the related signaling pathway involving in autophagy. This study will explore the potential clinical detection value of miR-224-5p, reveal its molecular mechanism involving in autophagy in breast cancer, and provide a theoretical basis for further research and practice.

外泌体作为细胞间生物信息传递的重要载体，能通过转运蛋白质、miRNA等改变周围或远程细胞的特性而参与肿瘤发生。前期研究发现：miR-224-5p在乳腺癌血清及组织中高表达且能抑制自噬活性，并在血清外泌体中高表达。但其是否可通过外泌体传递改变自噬活性的分子机制及其临床检测价值有待研究。据此本课题拟从以下方面展开：1.收集临床血清样本，测定样本miR-224-5p的表达水平，结合病例资料分析其检测价值。2.构建miR-224-5p过/低表达的细胞株制备其不同表达量的外泌体，在细胞水平和动物模型中探讨外泌体转运miR-224-5p抑制自噬活性的作用。3.在分子水平，研究外泌体miR-224-5p抑制自噬的下游靶分子及激活的Smad4信号通路。本研究将发掘miR-224-5p作为乳腺癌肿瘤标志物的潜在价值，揭示外泌体对其抑制自噬活性的传递作用及分子机制，为后续的研究及实践提供理论依据。

外泌体作为乳腺癌细胞间生物信息传递的重要载体，能通过转运miRNA改变周围或远程细胞的特性而参与肿瘤发生，根据我们之前的研究，miR-224-5p参与了乳腺癌（BC）的发生，但其在BC中的功能机制尚不清楚。在青年科学基金的资助下，我们探讨了miR-224-5p的表达和临床意义，研究了miR-224-5p在乳腺癌病理过程和转移中发挥的生物学作用和分子机制。研究结果表明：BC组织中miR-224-5p的表达增加，其表达水平与肿瘤恶性程度密切相关；miR-224-5p可抑制Smad4表达、促进SQSTM1表达；Smad4可下调SQSTM1，并与SQSTM1的表达呈负相关。通过双荧光素酶、ChIP等实验表明miR-224-5p可与Smad4的3'-UTR结合、Smad4与SQSTM的启动子结合发挥调节作用。细胞实验和动物实验结果均表明miR-224-5p可促进BC肿瘤发生。. 通过本项目的实施，外泌体miR-224-5p上调与乳腺癌淋巴结转移、临床分期和预后不良有关；在细胞水平和动物模型中揭示了外泌体通过转运miR-224-5p抑制乳腺癌自噬活性的作用；在分子水平，明确了外泌体miR-224-5p抑制下游靶分子及激活Smad4/SQSTM1信号通路调节乳腺癌的自噬、迁移、增殖和侵袭等生物学行为。本课题对miR-224-5p/Smad4/SQSTM1轴的研究，为乳腺癌研究提供了一个新的机制模型，并从“外泌体转运miR-224-5p”的角度解释了miR-224-5p如何促进肿瘤发生，提示miR-224-5p可作为一种潜在标志物用于乳腺癌临床预后分析，为后续的研究及实践提供理论依据。