Figure 1

Plk1 is regulated by miR-509 at mRNA and protein levels in smooth muscle cells. (A) Human airway smooth muscle (HASM) cells were transfected with either 20 nM miR-control (miR-Ctrl) or miR-100, or they were untransfected, for 3 days. Blots of the HASM cells were probed with antibodies against Plk1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are mean ± SD (n = 4). NS, not significant. (B) All three online miR search tools predict 3′UTR of human Plk1 as a target of miR-509. (C) Sequence alignment between miR-509 and 3′UTR of human Plk1. (D) Blots of untransfected HASM cells and cells transfected with either miR-Ctrl or miR-509 for 3 days were probed with antibodies against Plk1 and GAPDH. Data are mean ± SD (n = 4, **p < 0.01). (E) HASM cells were transfected with either 20 nM miR-Ctrl or miR-509, or they were untransfected for 3 days. Plk1 mRNA in the cells was assessed by RT-qPCR (reverse transcription quantitative real-time polymerase chain reaction). Data are mean ± SD (n = 4, **p < 0.01). (F) HASM cells were untransfected, or transfected with either 20 nM control RNA or miR-509 inhibitor for 2 days. miR-509 inhibitor is small, chemically modified single-strand RNA molecules designed to specifically bind to and inhibit endogenous miR-509. Blots of the cells were probed with antibodies against Plk1 and GAPDH. Data are mean ± SD (n = 4, **p < 0.01). (G) mRNA of Plk1 in untransfected cells and cells transfected with either 20 nM control RNA or miR-509 inhibitor for 2 days was assessed by RT-qPCR. Data are mean ± SD (n = 7, *p < 0.05). UT, untransfected cells. (H) Relative luciferase activity in cells cotransfected with plasmids encoding either wild-type (W) or mutant (M) Plk1 3′UTR plus miR-Ctrl or miR-509. Data are mean ± SD (n = 5, **p < 0.01). One-way ANOVA was used for statistical analysis.