IL-35通过下调外泌体内DSCAM-AS1促进M2型巨噬细胞极化和乳腺癌进展的机制研究

M2型巨噬细胞浸润增多是免疫逃逸和肿瘤进展的关键因素。IL-35是近年发现的免疫抑制功能强大的细胞因子。申请人前期发现IL-35环境下乳腺癌外泌体显著促进M2极化和肿瘤生长。进一步芯片分析和验证表明IL-35明显下调外泌体内LncRNA DSCAM-AS1。然而，IL-35下调外泌体内DSCAM-AS1在M2极化和肿瘤进展中分子机制尚不清楚。据此，该研究拟通过CRISPR/Cas9精准编辑DSCAM-AS1，明确IL-35是否通过下调外泌体DSCAM-AS1促进M2极化；利用RNA免疫共沉淀等解析DSCAM-AS1的ceRNA机制；构建动物模型，研究外泌体内DSCAM-AS1对体内M2极化和肿瘤进展的影响。本项目将阐明IL-35通过下调外泌体DSCAM-AS1介导M2免疫优势和肿瘤进展的分子机制，对免疫失衡导致肿瘤发展的理论进行拓展，为靶向外泌体内DSCAM-AS1治疗乳腺癌提供理论支持。

M2 macrophage polarization is essential for the maintenance of immunosuppressive tumor microenvironment (TME), which contributes to tumor progression. However, the mechanisms of M2 bias in TME is not clear yet. Interleukin-35 (IL-35) is a potent inhibitory cytokine. Our previous study showed that IL-35 was overexpressed in breast cancer cells (BCC) which promoted M2 polarization and tumor progression by regulating BCC-derived exosomes. The microarray analysis showed that long non-coding RNA (LncRNA) DSCAM-AS1 was downregulated in exosome by IL-35 which was confirmed by RT-PCR analysis in cell line sample and breast cancer tissue. Until now, the mechanisms by which the exosomal DSCAM-AS1 downregulated by IL-35 induced M2 polarization and tumor progression was unclear. In this study, we propose to demonstrate whether IL-35 induces the polarization of M2 through downregulation of exosomal DSCAM-AS1, by knockout/overexpressing DSCAM-AS1 using CRISPR/Cas9. Next, the molecular interaction mechanisms between DSCAM-AS1 and target miRNA will be confirmed by bioinformatic analysis and RNA immunoprecipitation. Furthermore, the effect of low expression of exosomal DSCAM-AS1 in tumor progression will be evaluated in in-situ tumor model and metastasis tumor model. Overall, this study will reveal the potential mechanisms involved in the polarization of M2 and tumor progression through exosomal DSCAM-AS1 downregulated by IL-35. Ultimately, the regulation process of M2 polarization demonstrated here will provide exosomal DSCAM-AS1 as a new target and new strategies in precise breast cancer treatment.