FIGURE 3.

miR‐454 negatively modulates NEDD4‐2 expression in H9c2 cells. A, Bioinformatics analysis of binding site between miR‐454 and NEDD4‐2. B, Correlation analysis of the expression of miR‐454 and NEDD4‐2 in 72 patients with HF determined with immunohistochemistry. C, The mRNA expression of NEDD4‐2 in non‐treated H9c2 cells or H9c2 cells treated with H₂O₂ for 12 h as measured by RT‐qPCR; *P < .05. D, The protein expression of NEDD4‐2 in non‐treated H9c2 cells or H9c2 cells treated with H₂O₂ as measured by Western blot assay; *P < .05. E, Dual‐luciferase reporter gene assay examining whether miR‐454 binds to NEDD4‐2 mRNA; *P < .05. F, Cellular lysates from H9c2 cells were used for RIP with Ago2 antibody. miR‐454 and NEDD4‐2 mRNA were detected using RT‐qPCR. In panels G‐I, H9c2 cells transfected with mimic NC, miR‐454 mimic, inhibitor NC or miR‐454 inhibitor. G, The expression of miR‐454 in H9c2 cells determined by RT‐qPCR 48 h after transfection; */^# P < .05. H, The mRNA expression of NEDD4‐2 in H9c2 cells determined by RT‐qPCR 48 h after transfection; */^# P < .05. I, The protein expression of NEDD4‐2 in H9c2 cells determined by Western blot assay 48 h after transfection; */^# P < .05. Measurement data are displayed as mean ± standard deviation. Independent sample t‐tests were used for comparing data between the two groups, and one‐way ANOVA for comparison of data among multiple groups. Pearson's correlation analysis was performed for observation of the correlation between miR‐454 and NEDD4‐2 mRNA expression. Each cell experiment was repeated three times