The role of amphiregulin in progression of melanoma

Melanoma is the most aggressive type of skin cancer, and its incidence has been rising rapidly in parallel with climate change. It is important to elucidate the mechanism of melanoma progression and to identify alternative molecular targets as it develops resistance to current treatment strategies. Amphiregulin (AREG), one of the EGFR ligands, has demonstrated oncogenic activity in various cancer types. However, the effect of AREG on the mechanisms underlying melanoma development has not yet been detailed. Therefore this study aimed to investigate the impact of AREG on cell viability, proliferation, inflammation, cell motility, and death on B16F10 cells with AREG protein suppressed by the CRISPR-Cas9 method and normal B16F10 cells. Viability analyses and acridine orange/ethidium bromide staining were performed on these cells, while cell proliferation was assessed using trypan blue. Boyden chamber for invasion and wound healing assay for cell migration were performed. CRISPR-induced nucleotide alterations in the AREG gene were validated by Sanger sequencing, AREG gene expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and protein expression by immunocytochemistry. Apoptosis by caspase-3, -8, -9, inflammation by interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa B were assessed. Cell motility-related genes matrix metalloproteinase (MMP)-2 and -9 were assessed by qRT-PCR, and their enzymatic activities were evaluated via gelatin degradation assay. The results showed that CRISPR-Cas9 suppressed gene and protein expression of AREG by inducing nucleotide changes. The suppression of AREG reduced MMP-2 and -9, which are involved in melanoma invasion and migration, and TNF-α and IL-6, which are involved in inflammation. In addition, increased expression of caspase-3, -8 and -9 genes supported cell death by apoptosis. In conclusion, suppression of AREG by CRISPR-Cas9 reduced viability, proliferation, inflammation, and cell motility and increased apoptosis in melanoma cells. Therefore, AREG can be considered an effective therapeutic target in melanoma treatment.