Regulatory Mechanism of Neurotrophin Receptor-Interacting Melanoma Antigen Coding Gene Homolog (NRAGE) Gene Methylation on Apoptosis of Breast Cancer Cell Under Tyrosine Kinases/Methyl Ethyl Ketone/Extracellular Regulated Protein Kinases Signaling Pathway

Abstract

The aim of this study was to discover the influence of Neurotrophin receptor-interacting MAGE homolog (NRAGE) gene methylation on proliferation (Pro) and apoptosis (Apo) of breast cancer cell (BCC), and its influence on TrkA/MEK/ERK signaling. BCC lines MCF-7, MDA-MB-231, and normal mammary gland cell (MGC) MCF-10 were selected. Expression of NRAGE mRNA and methylation level in cells was analyzed via reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR. Different concentrations (0, 5, 10 mol/L) of DNA methylase inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) were adopted to treat the BCC cell line. With dimethyl sulfoxide (DMSO) treatment as control, cell count, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blot were adopted to detect the Pro, Apo, relative expression (REP) of Apo-related proteins Bcl-2, Bax, and target proteins TrkA, MEK, and ERK1/2 after different treatments. The results showed that NRAGE mRNA level in MDA-MB-231 and MCF-7 was notably reduced versus MCF-10 (P < 0.05), and they could express methylated NRAGE specifically. 5-Aza-CdR can increase unmethylated NRAGE’s expression in BCC. Cell Pro level of the 5 and 10 mol/L treatments was greatly inhibited than DMSO and 0 mol/L treatments (P < 0.05). Apo rate and Apo-related proteins Bcl-2 and Bax increased obviously (P < 0.05). In addition, the phosphorylation levels of TrkA in the 5 and 10 mol/L treatments were considerably reduced (P < 0.05), while that in MEK and ERK1/2 was remarkably increased (P < 0.05). In short, NRAGE methylation can inhibit BCC’s Pro and regulate BCC’s Pro and Apo through TrkA/MEK/ERK signaling.