MiR-181c-5p Promotes Inflammatory Response during Hypoxia/Reoxygenation Injury by Downregulating Protein Tyrosine Phosphatase Nonreceptor Type 4 in H9C2 Cardiomyocytes

Figure 6

Reduction of PTPN4 mediated the proinflammatory effect of miR-181c-5p in H9C2 cardiomyocytes. (a) Transfection of cells with PTPN4 siRNA (PTPN4^KD) resulted in significant reduction of PTPN4 protein level in H9C2 cardiomyocytes. (b) Representative Western blots of phosphorylated IκBα (Ser^32/36), IκBα, phosphorylated p65 (Ser⁵²⁶), p65, and β-tubulin in the scramble siRNA or PTPN4 siRNA transfected H9C2 cardiomyocytes with or without H/R stimulation. (c) mRNA expression of NFκB-dependent genes, including IL-1β, IL-6, and TNFα in the scramble siRNA or PTPN4 siRNA-transfected H9C2 cardiomyocytes with H/R stimulation. mRNA levels are expressed as fold changes against those mRNA expressions in scramble siRNA-transfected H9C2 cardiomyocytes with no stimulation. (d) Representative Western blots of phosphorylated IκBα (Ser^32/36), IκBα, phosphorylated p65 (Ser⁵²⁶), p65, and GAPDH in the H9C2 cardiomyocytes cotransfected with miR-181c-5p antagomir and PTPN4 siRNA and subjected to H/R stimulation. Protein presence of phosphorylated IκBα (Ser^32/36), IκBα, and phosphorylated p65 (Ser⁵²⁶) was normalized to IκBα, β-tubulin/GAPDH, and p65, respectively. Data are shown as ; vs. CTL or vs. NC agomir (NC) or no treatment group, ^# vs. PTPN4^WT or vs. H/R+miR-181c-5p antagomir, ^$ vs. H/R (two-tailed unpaired Student’s -test in (a, c), two-way ANOVA followed by Bonferroni test in (b), and one-way ANOVA followed by Bonferroni test in (d)), .