Fig 1.
miR-1469 expression in melanoma tissue and melanoma cell lines.
(A) Normalized expression level of miR-1469 in FFPE tissue in ulcerated tumors relative to non-ulcerated tumors, determined by Nanostring (1.34 fold change, p = 0.0482). (B). miR-1469 expression in FFPE tissue from ulcerated primary cutaneous melanomas relative to non-ulcerated tumors, as assessed by qPCR (11.81 mean fold change, p = 0.043). (C) qPCR for miR-1469 expression following transfection in total RNA isolated from CHL1, MEL39, and A375 cell lines after 24 hours of transfection (* = p <0.05).
Fig 2.
miR-1469 expression reduces migration of melanoma cells.
(A) Transwell migration by melanoma cell lines CHL1, MEL39, and A375 when transfected with miR-1469, expressed as percent migration relative to untreated cells (no treatment). * = p<0.0332. (B) Representative images of Transwell migration assay, imaged at 20x magnification.
Fig 3.
miR-1469 expression inhibits melanoma cell invasiveness.
(A) Relative percent invasion of CHL1, MEL39, and A375 melanoma cell lines when normalized to untreated cells using Matrigel transwell invasion assays. (* = p<0.0332, ** = p<0.0021, *** = p<0.0002, **** = p<0.0001) (B) Representative images at 20x magnification of Matrigel invasion assay for each cell line and treatment condition.
Fig 4.
miR-1469 transfection of melanoma cell lines does not alter viability or proliferation.
(A) Trypan blue staining for assessment of viability following transfection of melanoma cell lines with miR-1469 relative to scramble-transfected and untransfected cells at 48 hours. (B) MTS Proliferation assays performed at 24, 48, and 72 hours following transfection of melanoma cell lines with miR-1469 miR relative to scramble-transfected or untransfected cells.
Fig 5.
miR-1469 expression in melanoma does not significantly alter susceptibility of tumor cells to apoptosis via regulation of MCL1.
(A) 24 hours after melanoma cell lines had been transfected with miR-1469 or a scramble miR or left untransfected, they were treated with 1 uM staurosporine for 0, 1, 3, 16, or 24 hours and assessed for apoptosis as determined by flow cytometry for Annexin V positive cells. (B) Representative flow cytometric scatter plots depict percent Annexin V positive cells (x axis) in CHL1 cells following 1 hour exposure to 1uM staurosporine. The percentage of Annexin V positive cells is indicated in red. (C) Protein expression of MCL1 in CHL1, MEL39, and A375 cell lines 72 hours following transfection, determined by immunoblot. GAPDH was used as a loading control. Densitometry indicated above as a ratio relative to untreated (no tx) cells for each cell line, normalized to GAPDH.