microRNA-221 Inhibits Latent TGF-β1 Activation through Targeting Thrombospondin-1 to Attenuate Kidney Failure-Induced Cardiac Fibrosis

Figures

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  Figure 1. miR-221 Inhibits Latent TGF-β1 Mediated Cardiac Fibroblast Activation Adult rat cardiac fibroblasts (cFB) were serum starved for 24 h and then treated with 80 ng/mL L-TGF-β1 for 5-day with transfections of miR-221 mimic (miR-221) or mimic control (MC). (A) Immunofluorescence images of cFB stained with α-SMA (green), phalloidin (red) and DAPI (blue). Scale bar = 100 μm. (B) Collagen secretion in cFB culture supernatant measured by Sircol assay. (C) Western blot measurement for α-SMA, phosphorylated (P-), and total (T-) SMAD3, with β-actin as loading control. Values were expressed as mean ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 versus L-TGF-β1+MC; ∗∗p < 0.01, ∗∗∗p < 0.001 versus Ctrl by by one-way ANOVA and Bonferroni post hoc analysis. Experiments performed three times in triplicates.

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  Figure 2. miR-221 Downregulates Thbs1 via Direct 3′ UTR Binding Adult rat cFB were serum starved for 24 h and then treated with 80 ng/mL L-TGF-β1 for 5-day with transfections of miR-221 or MC. Expressions of (A) Thbs1 by qRT-PCR and (B) TSP1 by western blot. (C) Diagram of the alignment of human THBS1 and rat Thbs1 and the indications of miR-221 binding sites (BS). Human conventional miR-221 BSs shown as hollow arrowheads, rat unconventional miR-221 BSs shown as solid arrowheads. (D) Schematic of rat Thbs1 3′ UTR binding sites sequence and mutations (from shaded to red). (E) 3′ UTR luciferase reporter assay using a 221 sensor (psiCHECK2 vector carrying perfect complimentary sequence to miR-221) as a positive control. Values were expressed as mean ± SD. ##p < 0.01, ###p < 0.001 versus L-TGF-β1+MC; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus Ctrl by by one-way ANOVA and Bonferroni post hoc analysis (A and B) and unpaired t test (E). Experiments performed three times in triplicate.

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  Figure 3. miR-221 Inhibits Latent TGF-β1 Activation Adult rat cFB reaching 80% confluence were incubated in serum free medium for 2–7 days, with or without transfections of miR-221 or MC. (A) Western blot of TSP1 in 40× concentrated cFB supernatants from 1-, 2-, and 7-day culture. Samples loaded by equal volume. (B) Total TGF-β1 bioactivity in acid activated supernatant and cFB lysate assessed by TMLC. Results were expressed in relative luminescence units (RLUs). (C) Culture protocol to assess endogenous L-TGF-β1 activation. (D) Endogenous L-TGF-β1 activation assessed by cFB and TMLC co-culture. (E) Total endogenous TGF-β1 production in cFB. (F) Tgfb1 mRNA expression assessed by qRT-PCR. (G) miR-221 expression by stem-loop qPCR in TGF-β1 treated versus Ctrl. Values were expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 versus MC by by one-way ANOVA and Bonferroni post hoc analysis. Experiments performed three times in triplicates.

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  Figure 4. miR-221 Inhibits TSP1 Upregulation and Reduces TGF-β1 Signaling in the Heart Heart tissues were harvested from Sham or Nx animals at day 3 and 7 post-Nx. (A) Thbs1 mRNA expression by qRT-PCR. Protein expressions of (B) TSP1 and (C) phosphorylated (P-) and total (T-) SMAD3. HSP90 and β-actin served as loading control, respectively. (D) Scheme of miR-221 treatment. Arrows indicating i.v. injections of 1 mg/kg mimics or PBS. Short-term: rats with Nx received 1 mg/kg miR-221 mimics or PBS injection i.v. at day-6 post-Nx and hearts were harvested 24 h later. (E) Stem-loop qPCR measurement of miR-221. (F) qRT-PCR measurement of Thbs1 mRNA. Western blot measurement of (G) TSP1 protein expression and (H) phosphorylated (P-) and total (T-) SMAD3 in the heart. Values were expressed as mean ± SD. #p < 0.05, ##p < 0.01 versus Nx Ctrl; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus Sham by by one-way ANOVA and Bonferroni post hoc analysis. n = 3–6 each group.

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  Figure 5. miR-221 Reduces Cardiac Fibrosis and Myofibroblast Activation in the Heart Rats received 1 mg/kg miR-221 mimics (Nx miR-221) or PBS i.v. injection (Nx Ctrl) for 3 times at day 3, 7, and 14 post-Nx. Hearts were harvested by the end of 8 weeks. Middle sections of 1/3 the heart were used for histological analysis. (A) Picro-sirius red staining of the heart was performed as follows (i) cross-section, scale bar = 2 mm; (ii) zoom-in images, scale bar = 200 μm; (iii) quantified percentage of fibrosis with ImageJ. (B) Heart weight (HW) normalized by tibial length (TL). (C) (i) Immunofluorescence staining with α-SMA (green) and DAPI (blue), Scale bar = 100 μm; (ii) Quantification of the α-SMA+myoFBs expressed as percentage of cells. Values were expressed as mean ± SD. #p < 0.05 versus Nx Ctrl; ∗p < 0.05, ∗∗p < 0.01 versus Sham by by one-way ANOVA and Bonferroni post hoc analysis. n = 6 per group.

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  Figure 6. miR-221 Improves Survival and Cardiac Function In Vivo Rats received 1 mg/kg Nx miR-221 or PBS i.v. injections (Nx Ctrl) 3 times on day 3, 7, and 14 post-Nx and were kept for 8 weeks. (A) A Kaplan-Meier curve of survival rate. n = 6 for Sham, n = 17 for Nx Ctrl, n = 10 for Nx miR-221. (B) Body weight measurement at 8 weeks. (C) Left ventricle (LV) function measured by Millar pressure sensor catheterized through right carotid artery into LV. BP, blood pressure; d, diastole, s, systole; LVDP, LV developed pressure; HR, heart rate; ± dP/dt, maximum and minimum rate changes of LV pressure; Tau, LV relaxation time constant; EDP, end diastolic pressure. Values were expressed as mean ± SD. ∗∗p < 0.01 versus Nx Ctrl by one-way ANOVA and Bonferroni post hoc analysis. n = 6 each group.

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  Figure 7. miR-221 Targets Thbs1 but Does Not Alter Kidney Fibrosis in the End of 8 Weeks Post-Nx Rats with Nx received 1 mg/kg miR-221 mimics or PBS i.v. injection at day 6 post-Nx and kidneys were harvested 24 h later and dissected into infarct and remote region for RNA and protein analysis. (A) Kidney miR-221 levels were measured by stem-loop qPCR. Expressions of (B) Thbs1 mRNA by qRT-PCR and (C) TSP1 protein by western blot. Rats received 1 mg/kg Nx miR-221 or PBS i.v injections (Nx Ctrl). for 3 times on day 3, 7, and 14 post-Nx and were kept for 8 weeks. Plasma and urine were collected for kidney function analysis, kidneys for histology. (D) Picro-sirius red staining of kidney sections for assessment of fibrosis. Scale bar = 200 μm. (E) Measurements of plasma creatinine, urea nitrogen, and urine protein. Values were expressed as mean ± SD. #p < 0.05, ##p < 0.01 versus Nx Ctrl; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus Sham by one-way ANOVA and Bonferroni post hoc analysis. n = 3–6 each group.