Upregulation of miR-29b-3p protects cardiomyocytes from hypoxia-induced apoptosis by targeting TRAF5

Fig. 1. Hypoxia induces cell injury and downregulation of miR-29b-3p. AC16 cells were incubated in a hypoxic incubator containing 1% O2, 5% CO2 and 94% N2 to induce hypoxia. a – The cell viability of hypoxic and normoxic cells was measured using the CCK-8 assay. b – Apoptosis of hypoxic and normoxic cells was determined with a flow cytometry assay. c – The expression of miR-29b-3p was measured using quantitative real time PCR. Each experiment was repeated three times. ***p < 0.001 vs. normoxia

Fig. 2. Hypoxia-induced AC16 cell injury is attenuated by miR-29b-3p overexpression. AC16 cells were transfected with miR-29b-3p or miR-NC before culture under hypoxic conditions. Non-transfected AC16 cells were cultured under normoxic conditions as a blank group. a – The expression of miR-29b-3p was detected using quantitative real time PCR. b – CCK-8 assay was performed to measure cell viability. c and d – Flow cytometry with Annexin V/PI staining and Hoechst 33258 staining were used to assess cell apoptosis. Each experiment was repeated three times. ***p < 0.001 vs. miR-NC or blank

Fig. 3. MiR-29b-3p targets the 3’UTR of TRAF5. a – The predicted miR-29b-3p-binding sites located in the 3’UTR of TRAF5. b – The effect of miR-29b-3p on luciferase activity was detected using the dual-luciferase reporter assay. The pmirGLO reporter vector containing the WT or MUT TRAF5 was co-transfected with miR-29b-3p or miR-NC into AC16 cells for 48 h. c and d – The mRNA and protein expressions of TRAF5 in AC16 cells transfected with miR-29b-3p or miR-NC and then subjected to hypoxia for 24 h were determined using quantitative real time PCR and western blot analysis. Each experiment was repeated three times. ***p < 0.001

Fig. 4. Knockdown of TRAF5 potentiated the protective effects of miR-29b-3p against hypoxia-induced cell injury. AC16 cells were transfected with siNC, siTRAF5 or miR-29b-3p, or co-transfected with miR-29b-3p and siTRAF5 before culture under hypoxic conditions. a – Expression of TRAF5 mRNA was detected using quantitative real time PCR. b – CCK-8 assay was performed to measure cell viability. c – Flow cytometry with Annexin V/PI staining was used to assess cell apoptosis. Each experiment was repeated three times. **p < 0.01, ***p < 0.001