Abstract: 【Abstract】 Objective To investigate the effect of the methyltransferase inhibitor azacitidine （5-azaC） on the expression of homeobox A9 （HOXA9） gene in, as well as proliferation, invasion and migration of A375 cells. Methods In vitro cultured A375 cells were treated with 5-azaC at various concentrations of 1, 5, 10 and 20 μmol/L, while routinely cultured A375 cells receiving no drug intervention served as control group. Methylation-specific PCR was performed to analyze methylation status of the HOXA9 gene promoter region after the treatment with different concentrations of 5-azaC, in order to screen the optimal concentration of 5-azaC for following experiments. Cell counting kit-8 （CCK8） assay was conducted to evaluate the proliferation of A375 cells, Transwell and wound healing assays were performed to estimate the invasion and migration of A375 cells, and real-time fluorescence-based quantitative PCR （qRT-PCR） and Western blot analysis were conducted to determine the mRNA and protein expression of HOXA9 in A375 cells after 5-azaC treatment. Two-independent-sample t test was used for comparisons between two groups. Results Methylation was observed in the HOXA9 gene promoter region in A375 cells in the control group. After 5-azaC treatment, methylated and unmethylated states coexisted in the HOXA9 gene promoter region in A375 cells, and the higher the concentration of 5-azaC, the higher the degree of demethylation of the HOXA9 gene. Therefore, 20 μmol/L 5-azaC was selected to treat A375 cells for 72 hours, which served as 5-azaC treatment group in subsequent experiments. Compared with the control group, the 5-azaC treatment group showed significantly decreased cellular proliferative ability（72.46% ± 2.19% vs. 100%, t = 28.09, P < 0.001）, significantly decreased number of invasive cells （242.70 ± 29.19 vs. 466.00 ± 22.65, t = 10.47, P < 0.001）, significantly decreased migratory ability （27.56% ± 2.74% vs. 35.69% ± 2.50%, t = 3.79, P = 0.019）, significantly increased HOXA9 mRNA expression （1.73 ± 0.28 vs. 1.01 ± 0.15, t = 3.93, P = 0.017）, and significantly increased HOXA9 protein expression （0.62 ± 0.03 vs. 0.50 ± 0.01, t = 3.82, P = 0.019）. Conclusion 5-azaC can inhibit the proliferative, invasive and migratory ability of A375 melanoma cells, and one of the possible mechanisms underlying this process may be that 5-azaC reverses the methylation in the HOXA9 gene promoter region, activates HOXA9 gene expression, and participates in the regulation of biological behaviors of melanoma cells.

Key words: Melanoma, DNA methylation, Hypomethylation, HOXA9, Azacitidine, A375 cells