Differential Gene Expression and Methylation Analysis of Melanoma in TCGA Database to Further Study the Expression Pattern of KYNU in Melanoma

Abstract

Background: The aim of this study was to analyze and compare melanoma gene expression profiles in TCGA database through the application of different genes to explore the pathogenesis of melanoma. Furthermore, we confirmed the extent of the role of KYNU in melanoma and whether it can be a potential target for the diagnosis and treatment of melanoma. Methods: The gene expression profiles of melanoma samples were downloaded from TCGA database, and matrix files were synthesized to screen differential genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis and GCDA broad institute were used to analyze common gene locus mutations and expression changes in melanoma, as well as methylation. In addition, the expression patterns of KYNU in melanoma were quantified by immunohistochemistry, Western blotting, qRT-PCR, software such as GEO DataSets and the Human Protein Atlas, and meta-analysis of skin diseases. KYNU was overexpressed in keratinocytes (HaCaT and HEKα) and melanoma cells (A375 and H1205-lu). CFDA-SE, Annexin V–PI double staining, and PI single staining were used to investigate the mechanism of KYNU in melanoma and its effects on melanoma proliferation, apoptosis, invasion, and migration. Results: The main signaling pathways involved in melanoma were EGF/EGFR–RAS–BRAF–MEK–ERK–CyclinD1/CDK4, Ras–PI3K–PTEN–PKB/AKT, and p14/p16 (CDKN2A)–MDM2–p53–p21–cyclinD1/CDK4/6–Rb/E2F. Moreover, MITF, KIT, CDH1. NRAS, AKT1, EGFR, TP53, KIT, and CDK4 were elevated in melanoma, whereas PTEN, cAMP, and BCL2 were reduced in melanoma. The copy number of tumor-promoting genes increased, while the copy number of tumor suppressor genes decreased. Changes in the copy number of the above tumor genes enriched in chromosomes were found through SNP gene mutations. The genes whose expression was negatively regulated by DNA methylation in melanoma included KRT18, CDK2, JAK3, BCL2, MITF, MET, CXCL10, EGF, SOX10, SOCS3, and KIT. The mutation rate of KYNU was high according to TCGA database. The KYNU level was decreased in melanoma. Overexpression of KYNU can promote changes in apoptotic BCL-2, metabolic KYN, 3-HAA, invasion and migration MMP9, E-cadherin, and other related proteins in melanoma. Fluorescence staining and flow analysis showed that a slower proliferation rate led to a stronger fluorescence intensity. In melanoma tumor cells with a low expression of KYNU, overexpression of KYNU could promote tumor cell apoptosis. IL-10 induced immunoregulatory changes in melanoma. The expression of MMP9 and AMPK decreased in A375, but the change in BCL-2 was not obvious. The expression of BCL-2 decreased significantly in H1205-lu. A375 showed cell-cycle arrest, indicating that IL-10 could slow down the cell cycle of melanoma. Conclusions: These results provide insights into the pathologic mechanisms of melanoma target genes and KYNU as a biomarker and potential therapeutic factor for melanoma.

The biological interactions between kynurenine and AhR in melanocytes: in vitro studies

“…A similar molecular mechanism of KYN activity was previously revealed in melanoma A375 and RPMI-7951 cells (Walczak et al 2020a ). Moreover, bioinformatics analysis confirmed the interactions between the kynurenine pathway, cyclin D1, and CDK4 in melanoma (Wang et al 2022 ). Interestingly, the gene expression data suggested that the biological activity of KYN was the result of its influence on gene expression and protein products.…”

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“…This practicality and fast turnaround enable prompt clinical testing. The versatility of IHC for evaluating para n-embedded and frozen tissue slices allows important historical materials to be studied in addition to new samples 38,39 . This study is limited by the small number of patients who were analysed.…”

5-Methylcytosine immunohistochemistry for predicting cutaneous melanoma prognosis

“…This practicality and fast turnaround enable prompt clinical testing. The versatility of IHC for evaluating paraffin-embedded and frozen tissue slices allows important historical materials to be studied in addition to new samples 38 , 39 . This study is limited by the small number of patients who were analysed.…”