miR-154-5p Functions as an Important Regulator of Angiotensin II-Mediated Heart Remodeling

Figure 1. miR-154-5p is upregulated during AngII-induced cardiac remodeling. (a) Representative images of mouse hearts and the ratio of heart weight to body weight (HW/BW) in sham- and AngII-treated (2.5 mg/kg per day, 14 days) mice (n = 6). In this time point, the heart was in the period of cardiac hypertrophy. (b) Histological analysis of the surface area of mouse cardiomyocytes using WGA staining (n = 5). (c) Images of Masson's trichrome-stained sections of the mouse heart (n = 5). (d) Images of TUNEL staining and the average data obtained from heart sections (n = 5). (e) Cleaved caspase-3 and caspase-3 expression, as assayed by Western blotting (n = 6). (f) Conservation of the miR-154-5p sequence between mice, rats, and humans is shown. (g) The cardiac expression of miR-154-5p in AngII-treated and sham-treated mice. (h) Distribution of miR-154-5p in various tissues from C57BL/6J mice (n = 6). miR-154-5p expression is upregulated in a dose-dependent (i) and time-dependent (j) manner in AngII-treated mice (n = 5). (k) The surface area of cardiomyocytes (control group and treated 100 nM AngII for 24 h) was identified using α-actinin staining (green), and nuclei were stained with…

Figure 2. miR-154-5p induces cardiac hypertrophy and apoptosis by promoting oxidative stress and inflammatory reactions in cardiomyocytes. (a) Images of the cell area labeled with Actin-Tracker Green (n = 50). (b) Results from the real-time PCR analysis of the expression of the ANF, BNP, and β-MHC mRNAs in cardiomyocytes transfected with normal control scrambled RNA (NC) and miR-154-5p mimics (n = 3). (c) Images of TUNEL staining and quantification of the percentage of apoptotic cells (n = 4). (d) Western blots and the quantification of cleaved caspase-3 and caspase-3 levels (n = 3). (e) Images of the cell area labeled with Actin-Tracker Green staining and quantitative analysis of the control, AngII, normal control inhibitor RNA with AngII (NCi+AngII), and miR-154 inhibitor with AngII (miR-154i+AngII) groups (n = 50). (f) Images and quantitative analysis of TUNEL staining (n = 4). (g) DHE staining was used to analyze ROS levels (n = 4). (h) Images of double immunofluorescence staining showing the NF-κB (green) localization. (i) Western blot and quantitative analysis of caspase-3 and cleaved caspase-3 levels (n = 3). (j) Average levels of ANF, BNP, and β-MHC mRNA expression (n = 3). ∗P <…

Figure 3. miR-154-5p directly targets Arsb. (a) A binding site for miR-154-5p in the 3′-UTR of Arsb was analyzed using miRNA target prediction databases, including miRBase, miRanda, TargetScan, and PicTar. The mutated sequence of the binging site is marked in red. (b) Luciferase activity was analyzed in HEK-293A cells 24 h after transfection with the indicated plasmids: miR-154-5p+vectors, miR-154-5p+Arsb 3′UTR, and miR-154-5p+mutated Arsb 3′UTR (n = 4). (c, d) The level of the Arsb protein was examined using Western blot analysis in the NC, miR-154-5p mimic, NCi, and miR154-5p inhibitor groups (n = 3). (e) Protein level of Arsb in sham and AngII mice explored by Western blot (n = 3). (f) Protein level of Arsb in AngII-treated primary cardiomyocytes transfected with NCi and miR-154-5p inhibitor (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4. Upregulation of Arsb reverses the effects of miR-154-5p on cardiomyocyte hypertrophy and apoptosis. (a) Expression of markers of cardiac hypertrophy in cardiomyocytes transfected with scramble, si-Arsb, as measured using real-time PCR (n = 3). (b) Western blot images showing the levels of Arsb, cleaved caspase-3, and caspase-3 in cardiomyocytes subjected to different treatments (n = 3). (c) Expression of markers of cardiac hypertrophy in cardiomyocytes transfected with NC, miR-154-5p, miR-154-5p+vector, and miR-154-5p+Arsb, as measured using real-time PCR (n = 3). (d) Western blot images showing the levels of Arsb, cleaved caspase-3, and caspase-3 in cardiomyocytes subjected to different treatments (n = 3). (e, f) Western blot images showing the levels of the p-JNK, JNK, p-p38, p38, p-STAT3, and STAT3 proteins in the Con, AngII, AngII+vector, and AngII+Arsb groups (n = 3) and in the NC, miR-154-p, miR-154-5p+vector, and miR-154-5p+Arsb groups (n = 3).

Figure 5. miR-154-5p is involved in AngII-induced cardiac remodeling mediated by the AT1 receptor in vivo and in vitro. (a) Representative images of mouse hearts and the ratio of heart weight to body weight (HW/BW) in control (Con), telmisartan- (Tel-), AngII-, and AngII+telmisartan- (AngII+Tel-) treated mice (n = 6). (b) Histological analysis of the surface area of cardiomyocytes in the Con, Tel-, AngII-, and AngII+Tel-treated groups using WGA staining (n = 8). (c) Images of Masson's trichrome-stained sections of the hearts from Con, Tel-, AngII-, and AngII+Tel-treated mice (n = 5). (d) TUNEL staining and quantitative analysis of sections of the hearts from Con, Tel-, AngII-, and AngII+Tel-treated mice (n = 6). (e) Expression of markers of cardiac remodeling in the myocardium of different groups of treated mice, as measured using real-time PCR (n = 5). (f) Western blot and quantitative analysis of cleaved caspase-3 and caspase-3 levels in the hearts of Con, Tel-, AngII-, and AngII+Tel-treated mice (n = 5). (g) Cardiac expression of miR-154-5p, as detected using real-time PCR (n = 3). ∗P < 0.05 compared with the Con group; #P < 0.05 compared with the AngII group. (h) Expression of markers…