Down-regulation of miR-30b-5p protects cardiomyocytes against hypoxia-induced injury by targeting Aven

Fig. 1. Inhibition of miR-30b-5p expression in hypoxic cardiomyocytes. a AC16 cells were exposed to hypoxia for 6, 12 and 24 h, respectively, and then harvested for analysis by quantitative real-time PCR. b The expression of miR-30b-5p was determined in AC16 cells transfected with miR-30b-5p inhibitor or NC, followed by 12 h hypoxia treatment. Cells cultured under normoxia were used as the control. *: hypoxia vs. normoxia; #: hypoxia + inhibitor vs. hypoxia + NC; **p < 0.01, ***p < 0.001, ##p < 0.01

Fig. 2. Inhibition of miR-30b-5p improves cell survival of hypoxic cardiomyocytes. AC16 cells were transfected with miR-30b-5p inhibitor or NC, followed by exposure to hypoxia for 12 h. Cell survival of cardiomyocytes was determined by MTT (a) and LDH (b) assays. LDH (%) means the percentage of LDH in cell lysates. *: hypoxia vs. normoxia; #: hypoxia + inhibitor vs. hypoxia + NC; **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001

Fig. 3. Inhibition of miR-30b-5p suppresses hypoxia-induced apoptosis in cardiomyocytes. a Representative captures of flow-cytometric data demonstrating the percent early apoptosis (Annexin V+/PI-) and late apoptosis (Annexin V+/PI+) in AC16 cells grown under normoxia or hypoxia with or without miR-30b-5p inhibitor. b Quantitation of A. c Protein expression of Bax and Bcl-2 was detected by western blot analysis in AC16 cells grown under normoxia or hypoxia with or without miR-30b-5p inhibitor. GAPDH was used as an internal control. *: hypoxia vs. normoxia; #: hypoxia + inhibitor vs. hypoxia + NC; ***p < 0.001, ##p < 0.01

Fig. 4. miR-30b-5p targets the 3′-UTR of Aven. a Sequence alignment of miR-30b-5p and 3′-UTR of Aven. b Dual-luciferase reporter assay. AC16 cells were co-transfected with miR-30b-5p inhibitor and a luciferase reporter containing the Aven 3′-UTR or mutant Aven 3′-UTR and incubated for 48 h. Relative luciferase activities were detected by the dual luciferase assay system. ***p < 0.001 vs. NC. The mRNA (c) and protein (d) expression levels of Aven were detected by qRT-PCR and Western blot analysis, respectively. *: hypoxia vs. normoxia; #: hypoxia + inhibitor vs. hypoxia + NC; ***p < 0.001, ##p < 0.01, ###p < 0.001

Fig. 5. Knockdown of Aven partially reversed the protective effects of miR-30b-5p silencing. AC16 cells were co-transfected with miR-30b-5p inhibitor and siAven and then exposed to hypoxia for 12 h. a Protein expression levels of Aven were detected by Western blot analysis. Cell survival of cardiomyocytes was determined by MTT (b) and LDH (c) assays. d Cell apoptosis was evaluated by flow cytometry assay. *: inhibitor + si-NC vs. si-NC; #: inhibitor + siAven vs. inhibitor + si-NC; **p < 0.01, ***p < 0.001, ###p < 0.001