清下泻胰方通过circCDK6/hsa-miR-454-3p/CSF-1信号通路抑制肿瘤相关巨噬细胞分化的抗胰腺癌机制研究

We previously developed Qingxiaxieyi patented formula (QXXY), according to the etiology characteristics of pancreatic cancer. Clinical applications have proved that QXXY presented anti-inflammation and anti-tumor effects. By the funding of previous NSFC grants, we have confirmed that inflammatory microenvironment accelerated growth and metastasis of pancreatic cancer in vivo. QXXY successfully inhibited infiltration of cancer-promoting tumor-associated macrophages (TAMs). Microarray assay identified that QXXY repressed expression of CSF-1, the key cytokine participating in the differentiation of TAM. Reporter gene assays further indicated that the down-regulation of CSF-1 may happen at the post-transcriptional level. Bioinformatics analysis presented that circular RNA circ_000984 (circCDK6) may regulate CSF-1 expression through adsorption of several miRNAs, including hsa-miR-454-3p. By using reporter gene assays and microarray assays, we explored that QXXY was able to inhibit the transcription and expression of CDK6, the host gene of circCDK6. Since CDK6 is a crucial oncogene, we propose that QXXY may present a direct anti-cancer effect through down-regulation of CDK6. Moreover, QXXY could repress infiltration of TAM through circCDK6/hsa-miR-454-3p/CSF-1 pathway, resulting in the remodeling of inflammatory microenvironment. Therefore, QXXY may be able to present indirect anti-tumor effect through immunal regulation. Our study will explore the mechanisms involved in the anti-tumor effects of QXXY, and provide solid evidences for the further clinical applications.

申请人根据胰腺癌“湿热”的病因病机特点建立专利方：清下泻胰方，临床证实具有抗炎抗胰腺癌作用。前期研究发现，该方抑制肿瘤相关巨噬细胞（TAM）的浸润，并抑制胰腺癌细胞中癌基因CDK6的转录和表达。我们推测：该方一方面可通过抑制CDK6，直接发挥抗肿瘤作用；另一方面，由于CDK6的转录抑制，环状RNA circCDK6（circ_000984）表达随之下调，削弱了对miRNA hsa-miR-454-3p的吸附。hsa-miR-454-3p与CSF-1的3'-UTR互补结合，抑制CSF-1的表达，即circCDK6/hsa-miR-454-3p/CSF-1的ceRNA信号转导机制。CSF-1表达下降，抑制了单核细胞向TAM分化，重塑肿瘤免疫微环境。本项目拟以circCDK6/hsa-miR-454-3p/CSF-1对TAM调控为主线，通过体内外实验阐明分子机制，从而为方剂临床应用奠定理论基础。

胰腺癌患者常以“湿热”为主要证候。基于此证候，本课题组设计了包括大黄、厚朴、黄芩等组分的“清下泻胰方”。临床观察表明，此方剂与化疗联合使用治疗胰腺癌，不仅能够缓解患者的临床症状、提高生存质量，还具有显著的抗炎功效，能够下调胰腺癌患者外周血中的肿瘤相关炎症因子水平。动物实验也显示，该方与吉西他滨化疗有协同抗肿瘤作用，并且能够改善小鼠胰腺癌动物模型的体能状况。在具有炎症背景的胰腺癌动物模型中，该方显示出更显著的抗肿瘤作用，提示该方可能通过干预胰腺癌的炎症微环境，发挥抗肿瘤的作用。本项目中我们证实，清下泻胰方对胰腺癌的抗癌作用通过以下机制：一方面，清下泻胰方抑制促癌基因CDK6转录和表达，环状RNA circCDK6表达随之下调，削弱了对miRNA hsa-miR-454-3p的吸附。hsa-miR-454-3p与CSF-1的3'-UTR互补结合，抑制CSF-1的表达，即“circCDK6/hsa-miR-454-3p/CSF-1”的ceRNA信号转导机制；CSF-1表达下降，抑制了肿瘤炎症微环境中单核细胞向M2型巨噬细胞（肿瘤相关巨噬细胞，TAM）分化，进而重塑肿瘤微环境，加强抗肿瘤免疫。进一步，我们进行了拆方研究，并通过基因芯片检测了清下泻胰方对巨噬细胞极化过程中信号转导机制的影响。我们发现，清下泻胰方通过抑制单核细胞中NRP1的表达来抑制 M2巨噬细胞极化，而NRP1的下游信号通路进一步富集到JAK-STAT等调控肿瘤生物学行为的关键信号通路。由于M2巨噬细胞极化巨噬细胞参与了肿瘤的多种生物学行为的调控，我们进一步研究发现，清下泻胰方导致的M2巨噬细胞的减少，一方面抑制了胰腺癌生长和血管生成，另一方面减少了化疗抵抗的CD24 +/CD44 +/EpCAM +的胰腺癌干细胞的占比。