Effect of miR-200c-3p overexpression on primary human macrophages

Macrophages constitute a major part of the tumor-infiltrating immune cells and within the tumor microenvironment acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of miR-200c in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed a substantial transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and found numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. The miR-200c-mediated reduction of infiltration further correlated well with a miR-200c migration signature comprised of four miR-200c-repressed targets (PPM1F, RAB11FIB2, RDX, MSN). mRNA-seq of primary human macrophages transfected with miR-200c-3p mimic or control harvested 72 h after transfection (n = 4).

巨噬细胞（Macrophages）是肿瘤浸润免疫细胞（tumor-infiltrating immune cells）的主要组成部分，在肿瘤微环境（tumor microenvironment）中可获得交替激活的肿瘤支持表型。肿瘤细胞释放的因子对于肿瘤相关巨噬细胞（tumor-associated macrophages）的招募至关重要。本研究旨在阐明miR-200c在肿瘤细胞与巨噬细胞互作中的调控作用。为此，我们采用MCF7乳腺癌细胞与原代人巨噬细胞的共培养体系，观察到miR-200c从凋亡肿瘤细胞向巨噬细胞发生显著转移，且该过程依赖于巨噬细胞表面完整的CD36受体（CD36 receptor）。我们进一步通过mRNA测序（mRNA-sequencing）全面鉴定了巨噬细胞中miR-200c的靶基因，发现大量迁移相关mRNA可被miR-200c下调。由此，miR-200c可减弱巨噬细胞向三维肿瘤球体（3-dimensional tumor spheroids）的浸润能力。miR-200c介导的浸润减少还与由四个miR-200c抑制性靶基因（PPM1F、RAB11FIB2、RDX、MSN）组成的miR-200c迁移特征高度相关。本研究的测序样本为转染miR-200c-3p模拟物（miR-200c-3p mimic）或对照的原代人巨噬细胞，于转染后72小时收获，样本量n=4。