miR-489-3p Inhibits SIX1 Expression by Targeting Its 3′ UTR
(A) Immunoblot analysis of 293T cells transfected with negative control (NC, a universal negative control of RNA) or miR-23-3p, miR-140-5p, miR-208-3p, miR-365-3p, miR-342-3p, miR-489-3p, and miR-548a-3p mimics. Bar graphs display transfected miRNAs expression determined by qRT-PCR. (B and C) A375 and SK-MEL-2 cells were transfected with NC or miR-489-3p mimics (B) or scramble or miR-489-3p inhibitor (C). Scramble was a negative control for miRNA inhibitors. Bar graphs under the immunoblot reveal corresponding expression levels of transfected miRNAs testing by qRT-PCR. (D) RT-PCR analysis of SIX1 mRNA levels in the above melanoma cell lines transfected with the indicated constructs. (E) miRNA luciferase reporter assays of A375 and SK-MEL-2 cells transfected with wild-type or mutated SIX1 reporter and miR-489-3p mimics. Red font indicates the putative miR-489-3p binding sites within human SIX1 3′ UTR. Red and italicized font indicates mutated miR-489-3p binding sequences in the SIX1 3′ UTR (*p < 0.05, **p < 0.01). Each experiment was repeated at least twice, and one representative result was given. The data shown are the average values with error bars representing the SE (A–D).