Comprehensive upstream and downstream regulatory analyses identify miR-675-3p as a potential prognostic biomarker in melanoma

Abstract

This study assessed miR-675-3p-related regulatory mechanisms in melanoma and the clinical relevance of such regulatory activities. We downloaded miRNA mature strand expression RNA-Seq, phenotypic, and DNA methylation data pertaining to the TCGA Melanoma cohort. Differentially expressed miRNAs (DEMs) between metastatic and primary melanoma patient tissues were then identified, and miR-675-3p expression in melanoma patient peripheral blood was confirmed using the GSE20994 GEO dataset, while its expression in melanoma cell lines was evaluated via qRT-RCR. The clinical and prognostic implications of miR-675-3p in melanoma were assessed, and miR-675-3p target genes were identified using bioinformatics tools. Functional roles of this miRNA were explored via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. We identified 3 and 22 miRNAs that were up- and downregulated, respectively, in metastatic melanoma samples relative to primary melanoma samples. Upregulation of miR-675-3p was associated with poorer overall patient survival, tumor histologic grade, and Clark's level. Consistently, miR-675-3p was also overexpressed in the peripheral blood of melanoma patients relative to healthy controls, and in melanoma cell lines relative to control cells. Gene regulatory networks indicated that 32 transcription factors control miR-675-3p expression, and that it, in turn, regulates 10 target genes. KEGG analyses indicated that these genes were associated with cell cycle, transcriptional misregulation in cancer, TGF-beta signaling, and HIF-1 signaling pathways. Gain-of-function assays revealed that miR-675-3p could promote cell proliferation via accelerating cell cycle progression. Western blotting results indicated that miR-675-3p could active TGF-beta and HIF-1 signaling. Through upstream and downstream analyses of miR-675-3p-related regulatory activity, we confirmed that this miRNA participates in key melanoma-related processes and offers value as a prognostic biomarker in melanoma patients.