human epidermal melanocyte pellets  (PromoCell)

Journal: Theranostics

Article Title: MicroRNA-23a-3p Inhibits Mucosal Melanoma Growth and Progression through Targeting Adenylate Cyclase 1 and Attenuating cAMP and MAPK Pathways

doi: 10.7150/thno.30516

Figure Lengend Snippet: miR-23a-3p is downregulated in mucosal melanoma (MM) tissues and correlates with poor prognosis. miR-23a-3p expression was validated by qRT-PCR in 117 FFPE tissue samples and was normalized to the mean level in 12 normal mucosal nevus tissues. U6 was used as an endogenous control. A fold change in relative miR-23a-3p expression >2 or <1/2 was defined as significant. Waterfall plot (A) and pie chart (B) displaying relative miR-23a-3p levels in MM tissues. (C) Relative expression of miR-23a-3p in three MM cell lines and normal human epithelial melanocytes (HEMs) cells was determined by qRT-PCR. (D) miR-23a-3p levels in MM tissues from patients at different TMM stages. (E) The expression level of miR-23a-3p in metastatic MM and non-metastatic MM. (F) Kaplan-Meier curve for OS in the cohort of 117 MM patients. Patients were divided into two groups according to the ratio of the miR-23a-3p level in each tumor sample to the mean level in control nevi, and the median ratio value was chosen as the cutoff point. (G) Kaplan-Meier curve for DFS in 100 patients, excluding patients first diagnosed at stage IV.

Article Snippet: Pelleted normal human epidermal melanocytes (HEMs) cell pellets were purchased from Sciencell, and miRNA expression was detected according to the TaqMan microRNA assay protocol (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy

doi:

Figure Lengend Snippet: (A) Heatmap of differentially expressed miRNAs in melanoma patient tissue samples comprising primary melanomas, lymph node metastases and distant cutaneous metastases (ou) (n=24), generated using expression data from TaqMan® low density miRNA arrays. Colors represent normalized Ct values (blue for high Ct values indicates low expression and red for low Ct values indicates high expression of miRNAs). Dendrograms are based on hierarchical clustering with Euclidean distance. (B) miR-638 expression in PM and MM and (C) PM of varying thickness (mm). (D) miR-126* expression in PM and MM and (E) PM of varying thickness (mm). Paired t- test and Pearson's coefficient (Pearson's r) were used to determine significance of correlation between miR-638 expression and tumor thickness. (F, G) Expression levels of miR-638 were analysed in primary melanocytes (n=3) in comparison with (F) PM and MM tissue samples (n=27). (G) miR-638 expression in 9 different melanoma cell lines (HT144, 1F6, Mewo, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, A375 and BRO). All miRNA expression analyses were performed using TaqMan® qRT-PCR technology. RNU48 expression was used as universal reference control for miRNAs.

Article Snippet: Freshly isolated normal human epidermal melanocyte pellets were purchased from PromoCell, Heidelberg, Germany (C-14043).

Techniques: Generated, Expressing, Comparison, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy

doi:

Figure Lengend Snippet: (A) Melanoma cells were transiently transfected with miR-638, antagomir-638 or control-antagomiR and analyzed using whole-genome cDNA microarrays. The Venn diagram at the top displays the overlap between down-regulated and up-regulated genes upon miR-638 and antagomiR-638 overexpression, respectively (n = 3,199). The middle Venn diagram displays the intersection of miRWalk database predicted miR-638 targets and strongly differentially regulated mRNAs (total log 10 fold change (FC) > 2; n = 86). Finally, a subset of targets predicted by at least three different algorithms as high-confidence targets of miR-638 regulation was identified and used in subsequent analyses (n = 30). (B) Heatmap of differentially regulated putative target genes for miR-638. Colors represent fold changes of relative gene expressions of miR-638 or antagomiR-638-transfected cells in comparison with mock transfected control cells (blue color indicates high expression and red color indicates low expression). (C) TP53INP2 mRNA expression analysis for primary melanocytes (P-mel 1 and 2), human fibroblasts (Hu.Fib), and melanoma cell lines, respectively, using TaqMan® gene expression assays. Normalized Ct values were quantitated and compared with the mean TP53INP2 expression values, respectively, across all cell lines. Results are expressed as percentage of relative expression. (D) SK-Mel-147 cells were transfected with a scrambled-control (Co) or miR-638. Fourty eight hours after transfection TP53INP2 mRNA expression was analysed using TaqMan® gene expression assays. GAPDH was used as reference control (mean ± S.E.M). (E) SK-Mel-147 melanoma cells were transfected with scrambled-control (Co) or miR-638. After 48 h of transfection, protein expression for TP53INP2 was analysed using immunoblotting. β-actin was used as loading control. (F) Reporter assay in SK-Mel-147 cells co-transfected with a scrambled miRNA control or miR-638 and with either wild type or single (1X) or double (2X) mutated TP53INP2 3′-UTR cloned in a dual-luciferase constructs (mean ± S.E.M; n= 4). (G) XTT cell proliferation assay using SK-Mel-147 cells transiently overexpressing control plasmid or TP53INP2 cDNA co-transfected with a control miRNA or miR-638 (mean ± S.E.M; n=3). The UV-absorptions were measured at 24 h and 48 h at 492 nm (H) Matrigel invasion assays were performed for SK-Mel-147 cells transiently overexpressing a control plasmid or TP53INP2 cDNA co-transfected with a control miRNA or miR-638. Microscopic pictures were taken at 48 h (mean ± S.E.M, n=3). All biological assays were performed in triplicates and repeated twice individual transfections and assay measurements.

Article Snippet: Freshly isolated normal human epidermal melanocyte pellets were purchased from PromoCell, Heidelberg, Germany (C-14043).

Techniques: Transfection, Control, Over Expression, Comparison, Expressing, Gene Expression, Western Blot, Reporter Assay, Clone Assay, Luciferase, Construct, Proliferation Assay, Plasmid Preparation