Reduced YTHDF2 inhibits PD-L1 expression by stabilizing m6A-containing SPOP mRNA in colorectal cancer

Colorectal cancer (CRC) is one of the most frequently diagnosed malignant tumors. However, clear evidence explaining the regulatory mechanisms of programmed death ligand 1 (PD-L1) in CRC has been limited. To illustrate the function of YTH N ⁶ -methyladenosine (m ⁶ A) RNA binding protein F2 (YTHDF2), we conducted a comprehensive evaluation of the expression profiling datasets from online databases and clinical samples. A subcutaneous immunodeficient mouse model was utilized to investigate the impact of YTHDF2 on CRC. Western blots, flow cytometry, PD-1/PD-L1 binding and cell killing assays were employed to detect the relationship between YTHDF2 and PD-L1. We utilized RNA sequencing, along with methylated RNA immunoprecipitation (MeRIP) and RNA binding protein immunoprecipitation (RIP) sequencing to analyze mRNA expression, m ⁶ A methylation level, and target transcripts of YTHDF2. The m ⁶ A methylation locations of mRNAs were verified using sequence-based RNA adenosine methylation site predictor (SRAMP), MeRIP-qRT-PCR, RIP-qRT-PCR, and a dual-luciferase reporter system. It was found that YTHDF2 was upregulated in CRC tissues, and patients with higher YTHDF2 expression had a worse prognosis. The in vivo model illustrated that YTHDF2 promoted CRC growth, while in vitro experiments showed that the inhibition of YTHDF2 expression did not affect cell proliferation, migration or invasion. Mechanistically, interference with YTHDF2 reduced PD-L1 expression and the binding ability between PD-1 and PD-L1. The use of RNA-seq, MeRIP-seq, RIP-seq, and bioinformatics tools confirmed speckle type BTB/POZ protein (SPOP) mRNA as a YTHDF2 target and validated its m ⁶ A methylation sites. After YTHDF2 knockdown, SPOP mRNA stability increased, causing an increase in SPOP expression and a decrease in PD-L1 expression. This study demonstrated that YTHDF2 might upregulate PD-L1 expression by destabilizing m ⁶ A-containing SPOP mRNA and promote CRC development. The biological effect of the YTHDF2-SPOP-PD-L1 axis presented a promising target for CRC treatment and provided an approach to enhance the efficacy of anti-PD-1/PD-L1 therapy.