MiR-16对肝癌巨噬细胞表型和功能的影响及分子机制

Immune inhibition of tumor microenvironment is a key factor in tumor immune evasion. Tumor-associated macrophages play an important role in immune regulation during tumor development. Our previous study found that miR-16 was a tumor-suppressing miRNA and that miR-16 was downregulated when monocytes/macrophages were induced into M2 subset. In this project, we aim to investigate how miR-16 regulates differentiation of macrophages and verify immune evasion mediated by these macrophages in tumor microenviroment by flow cytometry, confocal laser microscopy, real-time PCR, lentiviral -transfection, as well as transgenic animal models. In addition, target genes and molecular mechanisms regulated by miR-16 in macrophage differentiation will be elucidated. We will also explore major factors that affect expression of miR-16 in tumor-associated macrophages. Results based on this study will help to clarify how miR-16 play a role in differentiation of tumor-associated macrophages and corresponding immune evasion in hepatocellular carcinoma. Thus, potentially molecular targets will be provided in immunotherapy against hepatocellular carcinoma.

肿瘤微环境的免疫抑制状态是肿瘤免疫逃逸的主要原因，而肿瘤相关巨噬细胞在其微环境的免疫调节中发挥重要作用。我们前期研究发现：MiR-16在抗肿瘤中发挥重要作用；在体外诱导的M2型巨噬细胞中，miR-16表达下调。本课题以miR-16分子为切入点，采用流式细胞术、激光共聚焦显微技术、Real-time PCR、慢病毒转染、转基因动物模型等方法，从体内、体外研究miR-16 调控肿瘤微环境中巨噬细胞的分化及在肿瘤免疫逃逸中的作用；阐明miR-16调控巨噬细胞激活的靶基因和分子机制；探讨影响巨噬细胞miR-16表达差异的主要因素。本研究有助于明确miR-16在肿瘤相关巨噬细胞分化过程中的地位及其参与肝癌免疫逃逸机制，为肝癌的免疫治疗提供有意义的潜在靶点。

肿瘤相关巨噬细胞在诱导免疫抑制性微环境中发挥重要作用。我们前期研究发现，MiR-16具有抗肿瘤活性，且M2型巨噬细胞内miR-16的表达下调。因此miR-16对巨噬细胞极化（M1/M2）的影响，及其在肝癌免疫逃逸中的作用和机制期待深入研究。.首先发现miR-16调节巨噬细胞的极化过程。巨噬细胞体外经IL-4诱导培养为M2，将慢病毒（含miR-16基因序列）感染M2巨噬细胞，CD16/32表达上调，CD206、Dectin-1表达下调；且NO、 IL-12分泌增加，IL-10产生降低。提示miR-16体外促进M2型巨噬细胞向M1型巨噬细胞转化。我们进一步证实miR-16下调M2型巨噬细胞的功能，进而影响T细胞活性，其机制可能与调节巨噬细胞PD-L1的表达有关。.利用miR-15a/16-1-/-小鼠模型，发现生理状态下幼龄（8-10周）miR-15a/16-1-/-小鼠脾脏巨噬细胞及其它免疫细胞无明显变化，但老龄（8-10月）小鼠脾脏CD19+Tim-1+细胞的频率显著增高，且IL-10分泌增多。给miR-15a/16-1-/-小鼠（8~10周）皮下移植肝癌细胞荷瘤，发现与对照小鼠相比，miR-15a/16-1-/-小鼠体内移植瘤生长速度明显减慢，肿瘤组织内M1/M2型巨噬细胞的比值增高，且CD8+ NKG2D+细胞的频率升高。.研究结果丰富了对miR-15a/16-1生物学作用的认识，证实miR-15a/16-1具有免疫调节活性，并为基于miR-15a/16-1开发临床干预措施提供重要的实验依据。