MicroRNA Let-7a, -7e and -133a Attenuate Hypoxia-Induced Atrial Fibrosis via Targeting Collagen Expression and the JNK Pathway in HL1 Cardiomyocytes

Figures

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  Figure 1. Differential expressions of proteins, mRNAs and microRNAs in hypoxic cardiomyocyte cells. (A) HL-1 cells were incubated under hypoxic conditions (1% O2) for the indicated times. The indicated proteins were detected in cellular homogenates by Western blot analysis. (B) qRT-PCR analysis of fibrotic mRNAs was performed. Error bars represent means ± SD from three independent experiments. * p < 0.01 vs. normoxia; vs. hypoxia + scramble control mimic (NC), n ≥ 3. (C) Heat map showing the results of hierarchical clustering present the distinct microRNA expression profiles between hypoxia and controls. (D) Venn diagram showing the overlap between hypoxia-responsive microarray miRNAs or collagen-specific miRNAs in HL-1 cardiomyocytes under hypoxia stress. The three circles represent predicted miRNA target genes found in the list of reported miRNA websites. (E) qRT-PCR analysis of mmu-miR-133a, mmu-let-7a and mmu-let-7e miRNA expression in HL-1 cell. Mean ± SD, n ≥ 3, * p < 0.05.

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  Figure 2. The miR133a and miR-let-7 family attenuated ECM markers through targeting collagen COL1A and COL3A. (a,c) The UCSC Genome Browser was used to align potential miRNA binding sites and mmu-miR-133a, mmu-let-7a and mmu-let-7e binding sites within the COL1A 3′ UTR and COL3A 3′ UTR. The COL1A 3′ UTR and COL3A 3′ UTR luciferase reporter constructs containing mutated target sites are shown with changes in the central nucleotides that should abolish binding of mmu-miR-133a or mmu-let-7 to the predicted target sites. miRNA binding sites were predicted using TargetScan and miRWalk. (b,d) Cells were transfected with a firefly luciferase and β-gal reporter construct, which contained 3′ UTR of COL1A or COL3A mRNA, along with either miRNA mimic, miRNA inhibitor or negative control (NC). For the mutant firefly luciferase reporter, putative miR-133a, miR-let-7a or miR-let-7e binding sites in 3′ UTR regions were also detected and normalized to beta-galactosidase reporter. Data are presented as mean ± standard error from at least three separate experiments. * p < 0.05 compared with the NCs. (COL1A = collagen type 1A1, COL3A = collagen type 3A1, UCSC = University of California, Santa Cruz).

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  Figure 3. Regulation of collagen type 1A1 and 3A1 by miR-133a and let-7 family under hypoxic stress. (a) The HL-1 cells transfected with miRNA mimic were exposed to a hypoxic microenvironment and the levels of miR-133a, miR-let-7a and miR-let-7e were determined for transfection effectiveness from each group. (b) Western blotting analysis and quantification (c) of COL1A and COL3A protein expression in HL-1 cells transfected with the indicated mimic miRNA. (d) qRT-PCR analysis of COL1A1, COL1A2 and COL3A mRNA expressions in HL-1 cells transfected with the indicated mimic miRNA. Mean ± SD, n ≥ 3, * p < 0.05. (e) Effect of extracellular matrix shown by immunofluorescent green color expression in miR-133a and miR-let-7 family mimics treated in hypoxic HL-1 cells using an immunofluorescence assay. HL-1 cells were transfected with miR-133a mimic, miR-let-7a or miR-let-7e scramble control mimic (NC) and the expressions of COL1A and COL3A under hypoxic conditions were detected by immunostaining. Scale bar: 50 μm. Secondary antibody control slides for each group showed no COL1A or COL3A staining. The green intensity was quantified using ImageJ software and represents means ± standard error of the…

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  Figure 4. Post-translation repression of miR-let-7a, miR-let-7e and miR-133a by the JNK/SAPK pathway. (a) JNK pathway protein levels were evaluated by Western blotting and quantification (b) and HL-1 cells were treated with hypoxia. (c) Expressions of miR-133a, miR-let-7a and miR-let-7e were evaluated in HL-1 cells treated with SP600125 under hypoxic conditions. (d) Expressions of COL1A and COL3A proteins were analyzed by Western blotting and quantification. Mean ± SD, n ≥ 3, * p < 0.05 vs. normoxia; # p < 0.05 vs. hypoxia.

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  Figure 5. A proposed model of miRNA-mediated regulation of cardiac fibrosis under hypoxia by modulating JNK pathway.