Figure 6

MiR-377 targets E2F3. (A) The putative binding sites of miR-377 in the E2F3 3' UTR are shown (taken from TargetScan at http://www.targetscan.org/). (B) mel-14PA melanoma cells stably expressing HTR control (dark grey) or expressing miR-377 (light grey) were transfected with psiCHECK-E2F3-WT-3’ UTR-luciferase or psiCHECK- E2F3-mut-3'UTR-luciferase; mutation in site 1 (position 10–16 of the 3′UTR), or mutation in sites 1 + 2 (positions 10–16 and 951–957 of the 3′UTR), or mutation in sites 1 + 2 + 3 (positions 10–16 and 951–957 and 1251–1257 of the 3′UTR) of the E2F3 3′UTR. The results are presented as the ratio of expression of renilla/luciferase that was normalized relative to control-transfected cells for each of the 3′UTR constructs. *signifies p < 0.05. (C) E2F3 mRNA levels in different melanoma cell lines relative to NHEM as assessed by qRT-PCR. (D) E2F3 protein levels in different melanoma cell lines as assessed by WB. (E) E2F3 mRNA (left panel) and protein (right panel) levels in mel-14PA over-expressing HTR control vector or vector expressing miR-377 as assessed by qRT-PCR and WB, respectively. (F) mel-14PA melanoma cells stably expressing HTR control or expressing miR-377 were transfected with E2F-BS-luciferase and renilla vector as an internal control. The resulting cell extracts were analyzed by luciferase reporter assay and values normalized to renilla activity. Data is represented as mean ± SEM from three independent experiments. *signifies p < 0.05.