China ; Consensus ; Humans ; Melanoma/genetics*

Humans ; Melanoma ; diagnosis ; genetics ; Precision Medicine

Humans ; Melanoma/genetics* ; Molecular Biology ; Mucous Membrane

OBJECTIVES: Malignant melanoma is a highly malignant and heterogeneous skin cancer. Although immunotherapy has improved survival rates, the inhibitory effect of tumor microenvironment has weakened its efficacy. To improve survival and treatment strategies, we need to develop immune-related prognostic models. Based on the analysis of the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Sequence Read Archive (SRA) database, this study aims to establish an immune-related prognosis prediction model, and to evaluate the tumor immune microenvironment by risk score to guide immunotherapy. METHODS: Skin cutaneous melanoma (SKCM) transcriptome sequencing data and corresponding clinical information were obtained from the TCGA database, differentially expressed genes were analyzed, and prognostic models were developed using univariate Cox regression, the LASSO method, and stepwise regression. Differentially expressed genes in prognostic models confirmed by real-time reverse transcription PCR (real-time RT-PCR) and Western blotting. Survival analysis was performed by using the Kaplan-Meier method, and the effect of the model was evaluated by time-dependent receiver operating characteristic curve as well as multivariate Cox regression, and the prognostic model was validated by 2 GEO melanoma datasets. Furthermore, correlations between risk score and immune cell infiltration, Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) score, immune checkpoint mRNA expression levels, tumor immune cycle, or tumor immune micro-environmental pathways were analyzed. Finally, we performed association analysis for risk score and the efficacy of immunotherapy. RESULTS: We identified 4 genes that were differentially expressed in TCGA-SKCM datasets, which were mainly associated with the tumor immune microenvironment. A prognostic model was also established based on 4 genes. Among 4 genes, the mRNA and protein levels of killer cell lectin like receptor D1 (KLRD1), leukemia inhibitory factor (LIF), and cellular retinoic acid binding protein 2 (CRABP2) genes in melanoma tissues differed significantly from those in normal skin (all P<0.01). The prognostic model was a good predictor of prognosis for patients with SKCM. The patients with high-risk scores had significantly shorter overall survival than those with low-risk scores, and consistent results were achieved in the training cohort and multiple validation cohorts (P<0.001). The risk score was strongly associated with immune cell infiltration, ESTIMATE score, immune checkpoint mRNA expression levels, tumor immune cycle, and tumor immune microenvironmental pathways (P<0.001). The correlation analysis showed that patients with the high-risk scores were in an inhibitory immune microenvironment based on the prognostic model (P<0.01). CONCLUSIONS The immune-related SKCM prognostic model constructed in this study can effectively predict the prognosis of SKCM patients. Considering its close correlation to the tumor immune microenvironment, the model has some reference value for clinical immunotherapy of SKCM.
Humans ; Melanoma/genetics* ; Skin Neoplasms/genetics* ; Tumor Microenvironment ; Prognosis

ObjectiveThe objective of the study was to summarize the role of DNA methylation in the development and metastasis of uveal melanoma (UM).

Data SourcesThe relevant studies in MEDLINE were searched.

Study SelectionIn this review, we performed a comprehensive literature search in MEDLINE using "uveal melanoma" AND ("DNA methylation" OR "epigenetics") for original research/review articles published before February 2018 on the relationship between DNA methylation and UM. References of the retrieved studies were also examined to search for potentially relevant papers.

ResultsPrevious studies on the relationship between DNA methylation and UM covered many genes including tumor suppressor genes (TSGs), cyclin-dependent kinase genes, and other genes. Among them, the TSG genes such as RASSF1A and p16INK4a, which encodes a cyclin-dependent kinase inhibitor, are relatively well-studied genes. Specifically, a high percentage of promoter methylation of RASSF1A was observed in UM cell lines and/or patients with UM. Promoter methylation of RASSF1A was also associated with the development of metastasis. Similarly, a high percentage of promoter hypermethylation of p16INK4a was found in UM cell lines. DNA promoter methylation can control the expression of p16INK4a, which affect cell growth, migration, and invasion in UM. Many other genes might also be involved in the pathogenesis of UM such as the Ras and EF-hand domain containing (RASEF) gene, RAB31, hTERT, embryonal fyn-associated substrate, and deleted in split-hand/split-foot 1.

ConclusionsOur review reveals the complex mechanisms underlying the tumorigenesis of UM and highlights the great needs of future studies to discover more genes/5'-C-phosphate-G-3' sites contributing to the development/metastasis of UM and explore the mechanisms through which epigenetic changes exert their function in UM.

Cell Transformation, Neoplastic ; genetics ; DNA Methylation ; genetics ; Epigenesis, Genetic ; genetics ; Humans ; Melanoma ; genetics ; Promoter Regions, Genetic ; genetics ; Uveal Neoplasms ; genetics

Endothelin-3 (ET-3) is aberrantly expressed in both metastatic melanoma tissues and cultured melanoma cells. Our previous work showed that ET-3 could promote survival of metastatic melanoma cells via its altered expression. In this study, we investigated the mechanisms responsible for these gene-induced phenotypes in melanoma cells. An ET-3 gene sequence-specific shRNA vector pLVTHM-ET3-RNAi was constructed and transfected into human malignant melanoma cells A375 and MMRU, and the resultant molecular events and cellular changes were examined. As compared with the empty-vector group, cell proliferation was slowed down, and the growth inhibition rates were 38.9% in A375 cells and 38.4% in MMRU cells after transfection. In addition, cell invasion capability was also inhibited, with a reduction of 62.2% in A375 cells and 54.3% in MMRU cells. The percentage of apoptotic cells was found to increase. Meanwhile, in both cell lines, secreted protein acidic and rich in cysteine (SPARC) levels were down-regulated together with inhibition of its upstream signaling molecule, NF-κB. Thus, the current results suggested that down-regulated expression of ET3 attenuates the malignant behaviors of human melanoma cells partially by decreasing the expression of SPARC and NF-κB.
Cell Line, Tumor ; Endothelin-3 ; genetics ; Gene Silencing ; Humans ; Melanoma ; genetics ; pathology ; Osteonectin ; genetics

Humans ; RNA-Binding Protein EWS/genetics* ; Melanoma/genetics* ; Oncogene Proteins, Fusion/genetics*

To explore the activity of the pmel core promoter of Bashang long-tail chickens, we constructed dual-luciferase expression vectors and transiently transfected into DF1 cells with Lipofectamine 2000. We measured the luciferase activity with the dual-luciferase detection kit. The 1 268 bp fragment in 5-flanking region of the pmel gene in Bashang long-tail chickens was cloned. The region from -1 200 bp to +68 bp included 2 CpG islands and multiple transcription factor binding sites. We constructed 9 expression vectors with different promoter regions and a mutant vector of the core promoter region of the pmel gene of Bashang long-tail chickens. The core promoter region from -840 bp to +68 bp was identified in the pmel gene. The region from -590 to -525 bp negatively regulated the pmel gene during the transcription process. The -840--590 bp and -525--266 bp regions were positive regulatory regions. The polymorphic sites (-456, -435, -410, -374 and -341) had a significant effect on the promoter activity of the pmel gene.
Animals ; Chickens ; genetics ; Cloning, Molecular ; CpG Islands ; Luciferases ; Promoter Regions, Genetic ; gp100 Melanoma Antigen ; genetics

In previous studies we reported that the expression of HLA-DR on melanoma cell lines was differentially modulated by IFN- gamma and that the transcription rate was responsible for this differential modulation. We have also reported the nucleotide sequence variations in the promoter region of HLA-DR genes, and proposed that differences in the promoter activity by the sequence variations of the HLA-DR promoters might contribute to such a differential transcriptional regulation at the promoter level. In this study, in order to assess whether the sequence variations of the HLA-DR promoters affect the factor binding and exert influence on the promoter activity, nuclear factor binding to our previous six HLA-DRA and fourteen HLA-DRB promoter clones was evaluated with the nuclear protein extracted from a B-lymphoblastoid cell line (BLCL), BH, together with the chloramphenicol acetyltransferase (CAT) reporter assay. In the HLA-DRA promoters, clone 35 containing one bp nucleotide sequence variation at the octamer binding site (OCT) (GATTTGC to GATCTGC) showed relatively weak factor binding. In the HLA-DRB promoters, clusters I, III, and IV of our previous HLA-DRB promoter homologues, containing one bp nucleotide sequence variation (GATTCG) in their Y boxes exhibited weak factor binding and CAT activity compared to other clusters (GATTGG) that showed strong factor binding and CAT activity. This data suggests chat the binding patterns of transcription factors influenced by the nucleotide sequence variations of the HLA-DR promoter could affect the promoter activity and the DNA sequence elements in the HLA-DR promoter could mediate transcriptional regulation.
Base Sequence/genetics ; HLA-DR Antigens/genetics* ; Human ; Melanoma/pathology* ; Melanoma/genetics* ; Molecular Sequence Data ; Nuclear Proteins/metabolism* ; Promoter Regions (Genetics)/physiology* ; Promoter Regions (Genetics)/genetics* ; Tumor Cells, Cultured ; Variation (Genetics)*

OBJECTIVETo detect tumor cells in the peripheral blood of patients with hepatocellular carcinoma (HCC) by using the mRNA of the MAGE-1 and MAGE-3 genes as specific tumor markers.

METHODSPeripheral blood was obtained from 25 HCC patients and 20 healthy volunteers. The mRNA of the MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR. The MAGE-1 and MAGE-3 transcripts in the tumor tissues of these HCC patients were also detected by RT-PCR.

RESULTSOf the 25 HCC patients, MAGE-1 and MAGE-3 mRNA were positive in 44% (11/25) and 36% (9/25) of PBMCs respectively, and in 68% (17/25) and 56% (14/25) of HCC tissues respectively. In the PBMCs of the 25 HCC patients, 16 (64%) samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMCs from the patients whose tumors did not express the MAGE genes, nor in the PBMCs from the 20 healthy donors. The positive rate of MAGE mRNA in the PBMCs was closely correlated with the TNM stages and the diameter of tumors, but there was no correlation between the positive rate of MAGE mRNA in PBMCs and tumor differentiation degree or serum alpha-FP level. Of 9 HCC patients whose serum alpha-FP was normal or slightly elevated (< 50 ng/ml), 6 were MAGE-1 and/or MAGE-3 mRNA positive in their PBMCs.

CONCLUSIONMAGE-1 and MAGE-3 mRNA could be specifically detected with high percentage in the PBMCs of HCC patients by our method. They can be used as specific tumor markers for the detection of the circulating HCC cells, and the detection results may be helpful to evaluate the prognosis of HCC patients.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; Humans ; Leukocytes, Mononuclear ; Liver Neoplasms ; genetics ; Melanoma-Specific Antigens ; Neoplasm Proteins ; RNA, Messenger ; genetics