The protective effects of the miR-129-5p/keap-1/Nrf2 axis on Ang II-induced cardiomyocyte hypertrophy

Figure 1. The overexpression of miR-129-5p inhibits Ang II-induced cardiomyocyte hypertrophy. The expression of miR-129-5p (A) was detected through qRT-PCR. The expression of keap-1 mRNA was detected through qRT-PCR (B) and western blot (C), respectively. Immunofluorescence staining showed the morphological changes of cardiomyocytes in different groups (D). Scale is 10 µm. The expression of myocardial hypertrophy marker genes Nppa and Myh7 was detected through qRT-PCR (E). The (3H)-leucine incorporation assay was conducted to value cardiomyocyte hypertrophy (F). **, P<0.01. The bars show the mean ± standard deviation (SD) for each of the three independent experiments. miR, micro-RNA; NC, negative control; Ang II, angiotensin II; mRNA, message ribonucleic acid.

Figure 2. The overexpression of miR-129-5p suppresses Ang II-induced oxidative stress. The activities of oxidative stress–related enzymes SOD (A), CAT (B), and MDA (C), NO (D) and the production of ROS (E) were detected using the corresponding detection kits in accordance with the manufacturer’s instructions. **, P<0.01. The bars show the mean ± standard deviation (SD) for each of the three independent experiments. miR, micro-RNA; NC, negative control; Ang II, angiotensin II; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; NO, nitric oxide; ROS, reactive oxygen species.

Figure 3. Keap-1 is a target of miR-129-5p. A bioinformatics analysis showed predictive complementary sequences between keap-1 and miR-129-5p (A). A luciferase reporter gene assay was conducted to further demonstrate the targeting relationship between keap-1 and miR-129-5p (B). qRT-PCR (C) and western blot (D) were employed to detect the expression of keap-1. GAPDH was used as an internal reference. **, P<0.01. The bars show the mean ± standard deviation (SD) for each of the three independent experiments. WT, wild type; MUT, mutant; miR, micro-RNA; keap-1, kelch-like ECH-associated protein 1; NC, negative control; Ang II, angiotensin II; si, small interfering RNA.

Figure 4. MiR-129-5p inhibitor promotes cardiomyocyte hypertrophy and oxidative stress by elevating keap-1. The expression of keap-1 was detected through western blot (A). The morphological change of cardiomyocyte was observed through immunofluorescence staining (B). Scale is 10 µm. The expression of Nppa and Myh7 was detected through qRT-PCR (C). The (3H)-leucine incorporation assay was conducted to value cardiomyocyte hypertrophy (D). The activities of oxidative stress-related enzymes SOD (E), CAT (F), and MDA (G), NO (H) and the production of ROS (I) was detected using corresponding detection kits following the manufacturer’s instructions. *, P<0.05; **, P<0.01. The bars show the mean ± standard deviation (SD) for each of the three independent experiments. si, small interfering RNA; miR, micro-RNA; NC, negative control; Ang II, angiotensin II; keap-1, kelch-like ECH-associated protein 1; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; NO, nitric oxide; ROS, reactive oxygen species.

Figure 5. Keap-1 mediated the inactivation of Nrf2 pathway by the miR-129-5p inhibitor. Cells were treated with 200 µM NK-252 for 2 hours to activate the Nrf2 pathway (A,B). The expression of nuclear Nrf2 in the indicated group was detected through western blot. Histone H3 was used as the internal reference (C). The expression of Nppa and Myh7 was detected through qRT-PCR (D). The (3H)-leucine incorporation assay was conducted to value cardiomyocyte hypertrophy (E,F,G,H). The activities of oxidative stress–related enzymes SOD (E), CAT (F), and level of MDA (G), and NO (H) were detected using corresponding detection kits following the manufacturer's instructions. *, P<0.05; **, P<0.01. The bars show the mean ± standard deviation (SD) for each of the three independent experiments. si, small interfering RNA; miR, micro-RNA; keap-1, kelch-like ECH-associated protein 1; NC means negative control; Ang II, angiotensin II; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; NO, nitric oxide.

Figure 6. The Nrf2 pathway is involved in the protective effects of microRNA-129-5p in Ang II-induced cardiomyocyte hypertrophy by targeting keap-1. miR, micro-RNA; mRNA, message ribonucleic acid; keap-1, kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid2-related factor 2; ARE, antioxidant response element.