MiR-133b-3p attenuates angiotensin II-induced cardiac hypertrophy through the inhibition of apoptosis by targeting CDIP1

Figures

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  Figure 1 Ang II induced cardiac hypertrophy and apoptosis (A) Representative B-mode images and cardiac function indicators (EF%, LVFS%, LVPWd, and LVPWs) measured by echocardiography in sham and Ang II-infused mice. (B,C) H&E and Masson staining of the left ventricle in sham and Ang II-infused mice. Scale bar: 500 μm. (D) Representative images of the left ventricle of sham and Ang II-infused mice were captured via transmission electron microscopy. Scale bar: 5 μm. (E) Heart-to-body weight (HW/BW) ratios of sham and Ang II-infused mice. (F) Representative images of WGA staining (red) of the left ventricle in sham and Ang II-infused mice. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (G) Western blot analysis of ANP and BNP expressions in ventricle samples from sham and Ang II-infused mice. (H) Representative immunofluorescence staining of cTnT (green) in cardiomyocytes treated with Ang II. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (I) Western blot analysis of ANP and BNP expressions in cardiomyocytes treated with Ang II. (J) Immunohistochemical staining of Bax, Bcl-2 and cleaved caspase-3 in sham and Ang II-infused mice. Scale bar: 500 μm. (K) Western blot…

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  Figure 2 The expression of miR-133b-3p is downregulated in cardiac hypertrophy (A,B) qRT-PCR analysis of miR-133b-3p expression in Ang II-infused mice (A) and cardiomyocytes (B) treated with Ang II. The data are presented as the mean ± SD. ***P < 0.001, ****P < 0.0001.

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  Figure 3 MiR-133b-3p inhibits Ang II-induced cardiomyocyte hypertrophy and apoptosis (A) qRT-PCR analysis of miR-133b-3p expression in cardiomyocytes transfected with the miR-133b-3p mimic, miR-133b-3p inhibitor or negative control (miR-133b-3p NC). (B) Western blot analysis of ANP and BNP expressions in cardiomyocytes transfected with the miR-133b-3p mimic, miR-133b-3p inhibitor or miR-133b-3p NC in the presence of Ang II. (C) Immunofluorescence staining of cTnT (green) in cardiomyocytes transfected with the miR-133b-3p mimic, miR-133b-3p inhibitor or miR-133b-3p NC in the presence of Ang II. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (D) Western blot analysis of Bax, Bcl-2 and cleaved caspase-3 expressions in cardiomyocytes transfected with the miR-133b-3p mimic, miR-133b-3p inhibitor or miR-133b-3p NC in the presence of Ang II. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 4 CDIP1 is a target negatively regulated by miR-133b-3p (A) Bioinformatics analysis of the binding site between the 3′UTR of CDIP1 and miR-133b-3p. (B,C) Dual-luciferase reporter assay using the WT-CDIP1 3′UTR reporter and MUT reporter in the presence of the miR-133b-3p mimic. (D,E) Western blot analysis of CDIP1 expression in Ang II-infused mice and cardiomyocytes treated with Ang II. (F) Western blot analysis of CDIP1 expression in cardiomyocytes transfected with the miR-133b-3p mimic or miR-133b-3p inhibitor in the presence of Ang II. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 5 Effects of CDIP1 on cardiomyocyte hypertrophy and apoptosis (A) Western blot and qRT-PCR analysis of CDIP1 expression in cardiomyocytes transfected with CDIP1 siRNAs (si-CDIP1) or negative controls (si-NC). (B) Western blot analysis of ANP and BNP expressions in cardiomyocytes transfected with si-CDIP1-3, si-CDIP1-3 + miR-133b-3p mimic or si-CDIP1-3 + miR-133b-3p inhibitor in the presence of Ang II. (C) Immunofluorescence staining of cTnT (green) in cardiomyocytes transfected with si-CDIP1-3, si-CDIP1-3 + miR-133b-3p mimic or si-CDIP1-3 + miR-133b-3p inhibitor in the presence of Ang II. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (D) Western blot analysis of Bax, Bcl-2 and cleaved caspase-3 expressions in cardiomyocytes transfected with si-CDIP1-3, si-CDIP1-3 + miR-133b-3p mimic or si-CDIP1-3 + miR-133b-3p inhibitor in the presence of Ang II. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6 MiR-133b-3p attenuates angiotensin II-induced cardiac hypertrophy through the inhibition of apoptosis by targeting CDIP1 Downregulation of miR-133b-3p promotes Ang II-induced cardiomyocyte hypertrophy and apoptosis. Mechanistically, miR-133b-3p directly binds to the CDIP1 3′UTR, thereby promoting CDIP1 expression and subsequently enhancing Ang II-induced cardiomyocyte apoptosis.