Progesterone Changes the Pregnancy-Induced Adaptation of Cardiomyocyte Kv2.1 Channels via MicroRNA-29b

Figure 5

Validation of Kv2.1 miR-29b targeting. (a) Schematic illustration of the hypothesis that progesterone facilitated Kv2.1 channel expression through miR-29b. (b) Structure diagram of the luciferase reporter of kcnb1 and its mutant. Seed match regions of kcnb1 and miR-29b are indicated as vertical lines. The mutation site of kcnb1 mRNA is indicated in blue. (c) qRT–PCR analysis of miR-29b in the ventricles of pregnant mice with/without P4 injections. (d) qRT–PCR analysis of miR-29b in the H9C2 cells treated with 5 μM P4 for 48 h. The H9C2 cells cultured without treatment served as negative controls (Ctl). (e) Luciferase assay for kcnb1. miR-29b mimics were cotransfected with the wild-type 3-UTR of kcnb1 (kcnb1-WT) or its mutant (kcnb1-M) into 293 cells. Luciferase activities were measured after 24 h of culture ( individual experiments). qPCR (f), WB (g), and immunocytochemistry analysis (h) of Kv2.1 expression were conducted in the H9C2 cells from the different transfection groups that were cultured under 5 μM P4 conditions. The H9C2 cells transfected with scrambled RNA and cultured without P4 exposure were used as controls (NC). and ^## ( individual experiments; at least 6–10 fields from each group were imaged and scored in a blinded fashion). Comparisons of H9C2 data were conducted by a one-way ANOVA followed by the Bonferroni post hoc or Student’s test. P4: progesterone; qRT–PCR: quantitative real-time polymerase chain reaction; WB: western blot.

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