Mechanisms regulating melanoma by the ubiquitin-conjugating enzyme E2S: a bioinformatics analysis / 中华皮肤科杂志

ABSTRACT

Objective:To identify genes interacting with the ubiquitin-conjugating enzyme E2S (UBE2S) in melanoma, and to explore the molecular mechanisms regulating the biological behavior of melanoma cells by UBE2S.Methods:Cultured A375 melanoma cells were divided into 2 groups to be transfected with LV-UBE2S-RNAi (14011-1) -containing lentivirus (gene knockdown group) and a negative control lentivirus CON053 (negative control group), respectively, and the UBE2S gene-knockdown cell line and negative control cell line were established. RNA was extracted from the two cell lines, purified, fragmented, and hybridized with GeneChip probes, followed by washing and dyeing, and then microarray data were obtained. Ingenuity pathway analysis (IPA) software was used to further analyze the gene expression profiling data to identify potential genes interacting with UBE2S. Western blot analysis was performed to verify the downstream molecules regulated by UBE2S. A two-tailed t test was used to screen for differentially expressed genes. Results:A total of 512 differentially expressed genes with a|fold change| > 2 and a P value < 0.05 were screened out between the gene knockdown group and negative control group, including 247 upregulated genes and 265 downregulated genes. IPA of differentially expressed genes revealed that interferon signaling and liver X receptor (LXR) /retinoid X receptor (RXR) activation pathways were significantly activated, while eukaryotic initiation factor-2 (EIF2) signaling and nuclear factor-κB (NF-κB) signaling pathways were inhibited. Among the upstream regulators, IFNA2 was predicted to be strongly activated, and NF-κB (complex) to be strongly inhibited. Based on the above bioinformatics analysis results, it was suggested that the UBE2S gene may exert some effects in melanoma cells by regulating the expression of IFITM1, STAT1, ISG15 and TNFRSF11B genes. Western blot analysis showed that IFITM1 protein was not expressed in A375 cells before and after the UBE2S gene knockdown. After the UBE2S gene knockdown, ISG15 protein expression was upregulated by 19.94 times, STAT1 protein expression was upregulated by 1.47 times, and TNFRSF11B protein expression was downregulated by 79.1%. Conclusion:UBE2S may interact with STAT1, ISG15 and TNFRSF11B to regulate the biological behavior of melanoma cells.