Fig. 5.

miR-221/222 reduce L-type Ca²⁺ channel current and Kcnj5 (GIRK4) protein amount by targeting Cacna1c or Kcnj5 3′-UTR. a To evaluate if the miRNAs bind to the 3′-UTR dual luciferase constructs containing the 3′-UTR from the L-type Ca²⁺ channel subunits, the potassium channel subunits, the seed sequence or an empty vector was transfected in HEK293 cells either with or without scramble or miR-221 mimic. miR-221 mimic reduced the luciferase activity of the Cacna1c-II, Cacna1c-III, and the Kcnj5 construct. b While miR-222 mimic reduced the luciferase activity for Cacnb2, Cacna1c-I, Cacna1c-III, Kcnj5, and Kcnd2 significantly (N = 4–9 experiments/group). c HL-1 cells were transfected either with scrambled or mimics for miR-221. After 48 h, the mRNA for Cacnb2 and Cacna1c was reduced (N = 5–6 wells/group, relative change compared to scramble). d To confirm the effect on L-type Ca²⁺ channel, we performed patch clamp analysis in HL-1 cells transfected with scramble, miR-221 or miR-222 mimics. miR-221 and -222 mimic reduced the I_Ca,L current (n = 19–46 cells/group, N = 3–5 experiments). Representative current tracings for control and mimic are given. e Western blot analysis for GIRK1, GIRK4, and Kcnd2 was performed in HL-1 cells treated either with scrambled (control) or miR-221 mimics (N = 3 per group). f HL-1 cells were transfected either with scramble, miR-221 or miR-222 mimics and GIRK4-dependent ion flux was measured by fluorescence changes of a thallium-sensitive dye. In HL-1 cells transfected with miR-221/222 mimics, the area under the curve and thereby the ion flux over time were significantly reduced compared to control cells. (N = 4 experiments, n = 3 wells)