Figure 4. miR-22 inhibits MEF2-ALP activity by targeting STK40. (A) Bioinformatics prediction of the binding sites between miR-22 and STK40. (B) Dual-luciferase reporter gene assay showing the binding between miR-22 and STK40; * p < 0.05 vs. WT NC group. (C) mRNA expression of STK40, MEF2 and ALP and miR-22 expression in HFSCs in response to transfection with the miR-22 mimic, NC mimic, miR-22-inhibitor or NC-inhibitor determined by RT-qPCR. * p < 0.05 vs. NC-mimic; # p < 0.05 vs. NC-inhibitor. (D) STK40, MEF2 and ALP protein expression in HFSCs normalized to GAPDH in response to transfection with miR-22 mimic, NC mimic, miR-22-inhibitor or NC-inhibitor determined by Western blot assay. (E) The expression of miR-22 during HFSC differentiation determined by RT-qPCR; * p < 0.05 vs. day 0. Measurement data were expressed as mean ± standard deviation. Unpaired t test was adopted to analyze the differences between two experimental groups, while one-way ANOVA was utilized to compare data among multiple groups, followed by Tukey’s post hoc test. Cell experiments were conducted 3 times independently.