miR-19a-3p enhances TGF-β1-induced cardiac fibroblast activation via targeting BAMBI

Figures

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  Figure 1. miR-19a-3p levels were increased in the myocardium of cardiac fibrosis and the TGF-β1-induced NRCFs. A–M: Eight-week-old male mice were subjected to transverse aortic constriction (TAC) or sham surgery for four weeks. A: Representative gross morphology of the hearts. Scale bar, 1 mm. n = 6. B: Heart weight-to-body weight ratio (HW/BW) of the mice. n = 6. C: Representative graphs of Masson's trichrome staining. Scale bar, 100 μm. n = 6. D–G: Quantitative reverse transcription-PCR (qRT-PCR) was performed to analyze the expression of fibrotic markers, including Col1a2 (D), Col3a1 (E), Acta2 (F), and Tgfb1 (G) using Hprt as the internal reference. n = 6. H–K: Protein levels of TGF-β1 (H and I) and BAMBI (J and K) in sham- or TAC-treated mouse hearts were detected by Western blotting. The grayscale values of protein bands were statistically analyzed and normalized to the internal control, β-tubulin. n = 6. L: Immunofluorescence staining of vimentin and BAMBI in sham- or TAC-treated mouse hearts. Scale bar, 40 μm. M: The levels of miR-19a-3p in sham- or TAC-treated mouse hearts were examined by qRT-PCR. n = 6. N: The levels of miR-19a-3p in heart tissues from five patient hearts with…

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  Figure 2. miR-19a-3p was involved in the TGF-β1-induced NRCF differentiation. The miR-19a-3p mimic or inhibitor was transfected into NRCFs for 48 h. The transfected cells were cultured in DMEM containing 1% FBS before being incubated for a further 24 h with or without 10 ng/mL TGF-β1. A–D: The levels of α-SMA following transfection with miR-19a-3p mimic (A and B) or miR-19a-3p inhibitor (C and D) were detected by Western blotting. The grayscale values of protein bands were statistically analyzed and normalized to the internal control, GAPDH. n = 3. E: Immunofluorescent expression of α-SMA in NRCFs. Scale bar, 50 μm. F: The extent of NRCF activation, as indicated by the proportion of cells with positive α-SMA. n = 3. All data are presented as the mean ± standard deviation. Statistical analyses were performed by one-way ANOVA followed by Tukey's tests for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviations: TGF-β1, transforming growth factor β1; NRCF, newborn rat ventricular cardiac fibroblast; α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DAPI, diamidino-phenyl-indole.

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  Figure 3. Down-regulation of miR-19a-3p restricted TGF-β1-induced NRCF proliferation, migration, and collagen deposition. NRCFs were transfected with miR-19a-3p mimic or inhibitor for 48 h, and then cells were stimulated with or without 10 ng/mL TGF-β1 for an additional 24 h. A: The proliferation of NRCFs was investigated by the EdU incorporation assay. Scale bar, 50 μm. B: The histogram displayed the proliferation rates as indicated by the proportion of EdU-positive cells. n = 3. C: Measurement of NRCF migration by the wound closure assay. Scale bar, 100 μm. D: The degree of NRCF motility was displayed by a histogram of the migration rate. n = 3. E–H: Quantitative reverse transcription-PCR was performed to examine the expression levels of fibrotic markers including Col1a2 (E and G) and Col3a1 (F and H). Hprt served as the internal reference. n = 3. I–L: Western blotting was used to assess the level of collagen Ⅰ following the transfection with miR-19a-3p mimic (I and J) or miR-19a-3p inhibitor (K and L). Grayscale values of the protein bands were statistically analyzed. n = 3. Data are presented as mean ± standard deviation. Statistical analyses were performed by one-way ANOVA followed by…

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  Figure 4. miR-19a-3p enhanced the TGF-β1 signaling by targeting BAMBI. A: TargetScanHuman 8.0 predicted miR-19a-3p binding sites in the BAMBI 3ʹ UTR. B: The pGL3-luciferase reporter vectors carrying either wild-type (WT) or mutant (Mut) BAMBI 3ʹ UTR were transfected into HEK 293T cells. Further treatment for the cells included either miR-19a-3p mimic or a negative control (NC). The activity of luciferase was detected 24 h later. n = 4. C–E: NRCFs were transfected with miR-19a-3p mimic or inhibitor. The levels of miR-19a-3p were detected by quantitative reverse transcription-PCR, using U6 as the internal reference (C). n = 3. Western blotting was performed to examine the BAMBI protein levels (D). E: Quantitative analysis of the protein expression levels in panel D using GAPDH as an internal reference. n = 3. F–K: NRCFs were transfected with either miR-19a-3p mimic or its inhibitor. After 48 h of transfection, the cells were treated with TGF-β1 for an additional 24 h. The protein levels of p-SMAD2/3, SMAD2/3, and BAMBI were detected by Western blotting and were quantitatively analyzed with β-tubulin as the internal reference. n = 3. Data are presented as mean ± standard deviation.…

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  Figure 5. Elevated BAMBI ameliorated miR-19a-3p-induced fibroblast collagen expression through TGF-β1 signaling. In addition to miR-19a-3p mimic or negative control (NC), pcDNA3.1-BAMBI-3×Flag or pcDNA3.1-GFP were transfected into HT1080 cells. After 48 h of transfection, the cells were treated with TGF-β1 for an additional 24 h. A: Western blotting was used to identify the protein levels of p-SMAD2/3, SMAD2/3, and Collagen Ⅰ. B: SMAD2/3 was used as the internal reference to quantify the level of p-SMAD2/3. C: Grayscale values for collagen Ⅰprotein were statistically analyzed using β-tubulin as the internal reference. D and E: Quantitative reverse transcription-PCR was performed to detect the expression levels of COL1A2 and COL3A1. HPRT was used as the internal reference gene. n = 3. Data are presented as mean ± standard deviation. Statistical analyses were performed by one-way ANOVA followed by Tukey's tests for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviations: TGF-β1, transforming growth factor β1; BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor.

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  Figure 6. miR-19a-3p enhances TGF-β1-induced cardiac fibroblast activation via targeting BAMBI. BAMBI acts as a pseudo receptor of TGF-β1 to block the phosphorylation of SMAD2/3. TGF-β1 increases miR-19a-3p expression, which suppresses BAMBI and promotes the phosphorylation of SMAD2/3 and cardiac fibroblast activation under pressure overload. Abbreviations: TGF-β1, transforming growth factor β1; BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor.