Integrative analysis of bulk and single-cell RNA-seq reveals the molecular characterization of the immune microenvironment and oxidative stress signature in melanoma

Abstract

BackgroundThe immune microenvironment and oxidative stress of melanoma show significant heterogeneity, which affects tumor growth, invasion and treatment response. Single-cell and bulk RNA-seq data were used to explore the heterogeneity of the immune microenvironment and oxidative stress of melanoma. MethodsThe R package Seurat facilitated the analysis of the single-cell dataset, while Harmony, another R package, was employed for batch effect correction. Cell types were classified using Uniform Manifold Approximation and Projection (UMAP). The Secreted Signaling algorithm from CellChatDB.human was applied to elucidate cell-to-cell communication patterns within the single-cell data. Consensus clustering analysis for the skin cutaneous melanoma (SKCM) samples was executed with the R package ConsensusClusterPlus. To quantify immune infiltrating cells, we utilized CIBERSORT, ESTIMATE, and TIMERxCell algorithms provided by the R package Immuno-Oncology Biological Research (IOBR). Single nucleotide variant (SNV) analysis was conducted using Maftools, an R package specifically designed for this purpose. Subsequently, the expression levels of PXDN and PAPSS2 genes were assessed in melanoma tissues compared to adjacent normal tissues. Furthermore, in vitro experiments were conducted to evaluate the proliferation and reactive oxygen species expression in melanoma cells following transfection with siRNA targeting PXDN and PAPSS2. ResultsMalignant tumor cell populations were reclassified based on a comprehensive single-cell dataset analysis, which yielded six distinct tumor subsets. The specific marker genes identified for these subgroups were then used to interrogate the Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) cohort, derived from bulk RNA sequencing data, resulting in the delineation of two immune molecular subtypes. Notably, patients within the cluster2 (C2) subtype exhibited a significantly more favorable prognosis compared to those in the cluster1 (C1) subtype. An alignment of immune characteristics was observed between the C2 subtype and unique immune functional tumor cell subsets. Genes differentially expressed across these subtypes were subsequently leveraged to construct a predictive risk model. In vitro investigations further revealed elevated expression levels of PXDN and PAPSS2 in melanoma tissue samples. Functional assays indicated that modulation of PXDN and PAPSS2 expression could influence the production of reactive oxygen species (ROS) and the proliferative capacity of melanoma cells. ConclusionThe constructed six-gene signature can be used as an immune response and an oxidative stress marker to guide the clinical diagnosis and treatment of melanoma.