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

Background: 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. Recent studies have revealed the regulatory roles of N6-adenosine (m6A) RNA methylation in the direction of macrophage polarization, but the role of another type of RNA methylation, 5-methylcytosine (m5C), remains unclear and needs to be explored. Methods: To validate the existence of M2, fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) assays were performed in bladder cancer and corresponding normal tissues. Several reported m5C-related regulators, including writers, readers and erasers, were subsequently detected in M2 cells to identify the most closely involved regulators. A series of assays, such as RNA immunoprecipitation (RIP) and quantitative PCR, was subsequently performed to reveal the underlying mechanism by which m5C RNA methylase participates in the regulation of macrophage polarization in bladder cancer. Results: 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 inhibited M1 polarization. Additionally, this process involves the assistance and balance of the reader YBX1 and the eraser TET2. Conclusion: NSUN2 methylates SOCS3 mRNA to inhibit its stability and nuclear export, which consequently promotes macrophage polarization to M2.