Genome-wide transcriptional analysis of metabolism-related genes and pathways regulated by FAH in melanoma A375 cells

Reprogramming metabolism plays an important role in tumor cells for maintaining their abnormal biologic behaviors. Therefore, special factors could regulate metabolic processes and influence the overall status of tumor cells. This phenomenon was obviously found in melanoma. Fumarylacetoacetate hydrolase (fumarylacetoacetase, FAH) is an enzyme encoded by the FAH gene located on the chromosome 15q25.1 region and contains 14 exons. FAH enzyme catalyzes the hydrolysis of 4- fumarylacetoacetase into fumarate and acetoacetate. It is the last enzyme in the subpathway from L-phenylalanine and tyrosine degradation. Mutations in the FAH gene cause type I tyrosinemia, which is a hereditary error of metabolism that is characterized by increased tyrosine levels in the blood and urine of patients. In the present study, we will explore whether FAH is an essential enzyme to promote multiple metabolic processes and elucidate the functions of FAH in melanoma. Gene microarrays and bioinformatics analysis of the differentially expressed genes (DEGs) were performed using A375 cells, and we concentrated on the biologic functions of FAH. In general, our work revealed several functional mechanisms of FAH in melanoma, which indicated FAH might be a potentially therapeutic target and an independent prognostic indicator for this disease. We used two groups of A375 cells for microarray assay and each group included three biological replicates. A375 cells transfected with siFAH for 72 hours were regarded as experimental group compared to the control group cells were transfected with negative control siRNA. The silence efficiency was verified by real-time PCR. Total RNA of two group cells were extracted using TRIzol Reagent and further purification with an RNeasy kit (Qiagen). Then total RNA quality was assessed by agarose gel electrophoresis. Subsequently, cDNA of each sample was created using One-Cycle Target Labeling and Control Reagents (Affymetrix). Then cRNA was generated using a GeneChip IVT Labeling Kit (Affymetrix). Each product was biotin-labeled, fragmented and hybridized to the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix) and washed, and stained with the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). We performed three biological replicates for each sample. Then all arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix). Data were analyzed using GeneChip Operating Software 1.4.