M2a 巨噬細胞極化過程中 REGNASE-1 調控 Ccl22 之機制探討及 REGNASE-1 缺失的轉錄組效應分析

Abstract

REGNASE-1（ZC3H12A/MCPIP1）是具核酸內切酶活性的 RNA 結合蛋白，可促進發炎相關 mRNA 降解，為維持免疫恆定之重要轉錄後調控因子，然其於 IL-4/IL-13 驅動之 M2a 巨噬細胞活化中的角色仍不清楚。本研究利用三種 Regnase-1 突變小鼠骨髓衍生巨噬細胞（全身性剔除、骨髓特異性剔除、核酸酶失活敲入），發現 Regnase-1 缺失可降低 IL-4 誘導之 Ccl22 表現，此效果不完全依賴其核酸酶活性，亦與上游 STAT6 磷酸化無明顯關聯，而與 Ccl22 3′UTR（304–608 nt）調控相關。 進一步於人類 THP-1 巨噬細胞以 siRNA 敲落 REGNASE-1 並進行 mRNA-seq，GSEA 分析顯示 REGNASE-1 缺失會抑制 type I 干擾素刺激基因程序、上調粒線體氧化磷酸化基因，並上調 deadenylation 依賴性 mRNA 降解機制。本研究另於 RAW 264.7 細胞建立 Ccl22 3′UTR-CRISPRi 螢光報告子系統，篩選出可用於後續 pooled CRISPRi 篩選之單株細胞。 綜合以上，本研究證實 REGNASE-1 透過 Ccl22 3′UTR 相關、非核酸酶依賴之機制支持 M2a 巨噬細胞 Ccl22 表現，並揭示其於干擾素訊號與 mRNA 降解調控上之更廣泛功能。

REGNASE-1 (ZC3H12A/MCPIP1) is an RNA-binding endoribonuclease that promotes decay of inflammation-associated mRNAs and maintains immune homeostasis, but its role in IL-4/IL-13-driven M2a macrophage activation remains unclear. Using bone marrow-derived macrophages from total knockout, myeloid-specific knockout, and myeloid-specific endonuclease-deficient knock-in mice, we found that Regnase-1 deficiency reduced IL-4-induced Ccl22 expression independently of STAT6 signaling, through a mechanism not fully dependent on endonuclease activity but linked to the Ccl22 3′UTR. In human THP-1-derived macrophages with siRNA-mediated REGNASE-1 knockdown, mRNA-seq and GSEA revealed suppression of type I interferon-stimulated genes and upregulation of mitochondrial OXPHOS and deadenylation-dependent mRNA decay machinery. We further established a Ccl22 3′UTR-based CRISPRi fluorescent reporter system in RAW 264.7 cells for pooled CRISPRi screening. These findings demonstrate that REGNASE-1 supports Ccl22 expression in M2a macrophages through a 3′UTR-linked mechanism that may not be fully dependent on its endonuclease activity, revealing broader roles in interferon signaling and mRNA decay regulation.