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

Journal: Cells

Article Title: Amphiregulin Regulates Melanocytic Senescence

doi: 10.3390/cells10020326

Figure Lengend Snippet: Analysis of amphiregulin (AREG) expression in senescent normal human epidermal melanocytes (NHEM)/BRAFm cells. ( A – D ) NHEMs were transduced with lentiviruses expressing oncogenic B-Raf V600E (BRAFm) or copepod green fluorescent protein (copGFP, NHEM/Mock) cDNA. Arrays with n = 3 sample pairs were analyzed , and the volcano blot was generated via bioinformatical analyses ( https://amp.pharm.mssm.edu/biojupies/analyze ) ( A ), AREG detection by qRT-PCR ( n = 11) ( B ), western blot analysis ( n = 10) ( C ), and ELISA ( n = 7) ( D ). The expression level of AREG was determined by Western blot analysis 7 days after infection using β-actin as a loading control. The ELISA was performed with cell culture supernatant. ( E – G ) AREG expression in vivo was detected in normal melanocytes ( n = 6) compared to nevi ( n = 6), using qRT-PCR ( E ); example of immunofluorescence staining of nevi tissue ( n = 10) and normal skin ( n = 5) (AREG: red; nucleus: blue) ( F ). AREG expression analyzed by in silico analysis of microdissected nevi ( n = 23) and primary melanoma tissues ( n = 57) generated by RNASeq analysis (Accession: GSE112509) ( G ). (*: p < 0.05; **: p < 0.01; ***: p < 0.005).

Article Snippet: As described previously [ ], normal human epidermal melanocytes (NHEM, Lonza, Basel, Switzerland) were grown in MGM-4 BulletKit medium (Lonza).

Techniques: Expressing, Transduction, Generated, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Control, Cell Culture, In Vivo, Immunofluorescence, Staining, In Silico

Journal: Cells

Article Title: Amphiregulin Regulates Melanocytic Senescence

doi: 10.3390/cells10020326

Figure Lengend Snippet: Schematic overview: senescent function of AREG in melanocytes.

Article Snippet: As described previously [ ], normal human epidermal melanocytes (NHEM, Lonza, Basel, Switzerland) were grown in MGM-4 BulletKit medium (Lonza).

Techniques:

Journal: Cancer Management and Research

Article Title: Long Noncoding RNA CCAT1 Functions as a Competing Endogenous RNA to Upregulate ITGA9 by Sponging MiR-296-3p in Melanoma

doi: 10.2147/CMAR.S252635

Figure Lengend Snippet: The expression level of CCAT1 in melanoma tissues and cells. ( A and B ) RT-qPCR assay was performed to examine the expression level of CCAT1 in 40 pairs of melanoma tissues and adjacent normal tissues, as well as in melanoma cell lines (A375, SK-MEL-28, and A875) and normal human epidermal melanocytes (NHEM cells). * P < 0.05.

Article Snippet: Melanoma cell lines (A375, SK-MEL-28, and A875) and normal human epidermal melanocytes (NHEM) were obtained from China center for type culture collection (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR