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Fig. 2 | BMC Cancer

Fig. 2

From: MicroRNA-195 acts as an anti-proliferative miRNA in human melanoma cells by targeting Prohibitin 1

Fig. 2

MicroRNA-195 modulates PHB1 expression in melanoma cells and in a gene reporter assay. UACC-62 melanoma cells were transfected with either miR-control/mir-195. miR-195 mimics transfection produced a significant reduction of PHB1 (P ≤ 0.0001) (a) mRNA and (b) protein levels compared to miR-control. For RT-qPCR experiments, ACT-B mRNA was used as an endogenous control and the data were analyzed using the 2 (−∆∆Ct) method; for immunoblotting ACT-B was also used as loading control. Protein quantification (fold-change based on the control) is indicated above the blots. In (c) and (d) miR-195 levels 48 and 72 h after transfection, respectively. RNU48 was used as an endogenous control and the data were analyzed using the 2 (−∆∆Ct) method. (e) Schematic representation of the PHB1–3’UTR region. pmiR-GLO-PHB1–3’UTR wild type (PHB1-WT) was submitted to a mutagenesis assay to delete the miR-195 binding-site sequence (PHB1-del195). HeLa cells were transiently co-transfected with either pmiR-GLO-PHB1–3’UTR-WT/pmiR-GLO-PHB1–3’UTR-del195 in the presence of miRNA-control/miR-195 mimics. After 48 h, Firefly and Renilla luciferase activity was measured and normalized. Results shown that miR-195 decreased luciferase activity by about 40% (P ≤ 0.0001). Statistical analysis was carried out using the unpaired t test and data are reported as means ± SD. Representative examples of at least three independent experiments are reported. *P ≤ 0.05; ****P ≤ 0.0001

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