NSUN2-mediated m5C modification of SOCS3 mRNA modulates macrophage polarization in bladder cancer

Abstract Tumor-associated macrophages (TAMs) are pivotal in facilitating the progression of cancer cells. M1 and M2 are two polarization states of TAMs with opposite functions in tumor progression. While the regulatory role of N6-adenosine (m6A) methylation in macrophage polarization has been established, the function of 5-methylcytosine (m5C) remains unclear. The presence of M2 macrophages in bladder cancer and adjacent normal tissues was validated using fluorescence-activated cell sorting (FACS) and immunofluorescence (IF). The expression of reported m5C regulators (writers, readers, erasers) was screened in M2 macrophages to identify the most relevant regulators. Mechanistic insights into how m5C methylation regulates macrophage polarization were gained through RNA immunoprecipitation (RIP) and quantitative PCR. The FACS and IF results revealed that the main active state of the tumor microenvironment (TME) was the M2 subtype in bladder cancer. Next, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was identified as the most upregulated RNA m5C methylase in M2 via a qPCR assay. According to whole-transcriptome resequencing in si-NSUN2 RAW/THP-1 cells and GO analysis, SOCS3 was determined to be downstream of NSUN2. By methylating particular sites in SOCS3 mRNA, NSUN2 inhibits both the stability and nuclear export of SOCS3 mRNA, which subsequently activates the JAK2/STAT3 signaling pathway and then promotes macrophage polarization to the M2 phenotype while inhibiting M1 polarization. Additionally, this process involves the assistance and balance of the reader YBX1 and the eraser TET2. NSUN2 methylates SOCS3 mRNA to inhibit its stability and nuclear export, which consequently promotes macrophage polarization to M2.