BACKGROUND

Breast cancer (BC) is the most commonly diagnosed cancer in women. N-methyladenosine (mA) is the most prevalent internal modification in mammalian mRNAs and plays a crucial role in various biological processes. However, its function in Natural killer (NK) cells in BC remains unclear. NK cells are essential for cancer immunosurveillance. This study aims to assess mA levels in transcripts involved in the NKG2D cytotoxicity signaling pathway in NK cells of BC patients compared to controls and find out its impact on mRNA levels. Additionally, it evaluates how deliberately altering mA levels in NK cells affects mRNA and protein expression of NKG2D pathway genes and NK cell functionality.

METHODS

mA methylation in transcripts of NKG2D-pathway-related genes in BC patients and controls was determined using methylated RNA immunoprecipitation-reverse transcription-PCR (MERIP-RT-PCR). To deliberately alter mA levels in primary cultured human NK cells, the mA demethylases, FTO and ALKBH5, were knocked out using the CRISPR-CAS9 system, and FTO was inhibited using Meclofenamic acid (MA). The impact of mA alteration on corresponding mRNA and protein levels was assessed using RT-qPCR and Western blot analysis or flow cytometry, respectively. Additionally, NK cell functionality was evaluated through degranulation and Cr release cytotoxicity assays.

RESULTS

Transcripts of NKG2D, an activating receptor that detects stressed non-self tumour cells, had significantly higher mA levels in the 3' untranslated region (3'UTR) accompanied by a marked reduction in their corresponding mRNA levels in BC patients compared to controls. Conversely, transcripts of ERK2 and PRF1 exhibited significantly lower mA levels escorted with higher mRNA expression in BC patients relative to controls. The mRNA levels of PI3K, PAK1 and GZMH were also significantly elevated in BC patients. Furthermore, artificially increasing transcripts' mA levels via MA in cultured primary NK cells reduced mRNA levels of NKG2D pathway genes and death receptor ligands but did not affect protein expression or NK cell functionality.

CONCLUSION

Transcripts with higher mA levels in the 3'UTR region were less abundant, and vice versa. However, changes in mRNA levels of the target genes didn't impact their corresponding protein levels or NK cell functionality.