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Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases and benign melanocytic naevi

ABSTRACT: Perturbations in microRNA (miRNA) expression profiles has been reported for cutaneous malignant melanoma (CMM). With regards to a rapidly growing number of newly discovered miRNA sequences, the availability of up-to-date miRNA expression profiles for primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM) and benign melanocytic naevi (BMN) is limited. Patients with PCMM (n=9), CMMM (n=4) and BMN (n=8) were included in the study. Specimens were obtained during surgery from the center of the tumors (lesional). An exploratory microarray analysis was performed by miRNA expression profiling based on miRBase V.16. Additionally, the expression levels of selected miRNA candidates were confirmed by TaqMan real-time quantitative polymerase chain reaction (RT-PCR). New miRNA candidates previously not described to be associated with CMM were found to be potentially dysregulated in CMM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE34460 | GEO | 2012/11/02

SECONDARY ACCESSION(S): PRJNA151195

REPOSITORIES: GEO

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Project description:Perturbations in microRNA (miRNA) expression profiles has been reported for cutaneous malignant melanoma (CMM). With regards to a rapidly growing number of newly discovered miRNA sequences, the availability of up-to-date miRNA expression profiles for primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM) and benign melanocytic naevi (BMN) is limited. Patients with PCMM (n=9), CMMM (n=4) and BMN (n=8) were included in the study. Specimens were obtained during surgery from the center of the tumors (lesional). An exploratory microarray analysis was performed by miRNA expression profiling based on miRBase V.16. Additionally, the expression levels of selected miRNA candidates were confirmed by TaqMan real-time quantitative polymerase chain reaction (RT-PCR). New miRNA candidates previously not described to be associated with CMM were found to be potentially dysregulated in CMM. Nine patients with PCMM (PCMM1-PCMM9), four patients with CMMM (CMMM1-CMMM4) and eight patients with BMN (BMN1-BMN8) were enrolled in the present study. All cutaneous specimens were harvested in the operating room, immediately stored in RNAlater (Quiagen, Hilden, Germany) and kept at - 80 M-BM-0C until RNA isolation. Total RNA including miRNAs was isolated using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys protocol. RNA concentration and purity was determined by NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). RNA integrity control (RIN) was determined by means of capillary electrophoresis with the 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA). To be further considered for Microarray analysis a RIN M-bM-^IM-% 7.0 indicating medium to good RNA quality was defined as inclusion criteria. For assessment of labeling and hybridization efficiencies total RNA samples were spiked with in-vitro synthesized oligonucleotides by using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). A total of 100 ng total RNA per sample were introduced into a labeling reaction. The spiked total RNA was treated with alkaline calf intestine phosphatase (CIP). The dephosphorylated RNA was labeled with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturerM-BM-4s instructions using T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (pCp). The Cyanine-3-labeled miRNA samples were prepared for One-Color based hybridization with Complete miRNA Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturerM-BM-4s instructions. Labeled miRNA samples were hybridized at 55M-BM-0C for 20 hrs on separate Human miRNA Microarrays Release 16.0 (8x60K format, (Agilent Technologies, Santa Clara, USA). Afterwards, microarrays were washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies, Santa Clara, USA) followed by drying with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturerM-BM-4s instructions.

Project description:Background: Cutaneous malignant melanoma (CMM) is one of the most highly fatal cancers; its pathogenesis requires study. We recognize that abnormalities in lipid metabolism may be associated with the risk of CMM development; nevertheless, the role of lipid metabolism in the proliferation, invasion, and metastasis of CMM remains unclear. Methods: Lipid metabolism-related genes were identified in MsigDB. A lipid metabolism-related predictive model was constructed in TCGA-SKCM using univariate Cox regression analysis, consensus cluster analysis, WGCNA analysis, LASSO regression analysis, and outcomes-related genes were identified. Immunohistochemistry, qPCR, and western blotting were used to identify essential genes, construct stable lentivirus strains, and study the biological role of PLEKHA5 in CMM cells. Transcriptomic and proteomic sequencing, protein-protein molecular docking, CO-IP, and GST pull-down were performed on the cells with knockout essential genes to explore this gene’s upstream and downstream regulatory mechanisms. After analyzing the results of lipid metabolomics sequencing, the effects of the essential lipids related to CMM cells after the gene knockdown were studied. Results: A predictive model was established, Risk Score = -0.009 * UBE2L6 + 0.033 * PLEKHA5 + 0.024 * LHB + 0.036 * CARM1 + 0.016 * PRXL2B + 0.131 * PLA2G4D. PLEKHA5 was identified as an essential gene, and PLEKHA5 was positively correlated with poor outcomes in CMM patients. PLEKHA5 is more highly expressed than melanocytes in CMM, which promotes the proliferation, invasion, and metastasis of CMM. FCRLA is the downstream gene of PLEKHA5, upregulated in CMM, and the primary enrichment pathway is the TNFα-NFκB signaling pathway. TNFα is also an essential cytokine that promotes CMM proliferation and metastasis. Hex1Cer(d18:1/26:2) and Cer(d18:2/26:2) in the downstream were significantly decreased, while ChE(0:0), ChE(18:0) and TG(18:0/16:0/19:0) in the downstream were significantly increased. The cholesterol ester inhibitor avasimibe inhibits CMM proliferation, metastasis, and invasion. Conclusion: The predictive model of CMM related to lipid metabolism established in this study has good predictive ability. PLEKHA5 is an essential biomarker related to lipid metabolism in CMM. TNFα-PLEKHA5-FCRLA is a signal axis that promotes proliferation, metastasis, and invasion of CMM. Its downstream product, the cholesterol ester inhibitor avasimibe, may treat CMM.

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Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases and benign melanocytic naevi

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