miR‐409‐3p Regulated by GATA2 Promotes Cardiac Fibrosis through Targeting Gpd1

Figure 1. miR-409-3p expression is upregulated in cardiac fibrosis. (a) Dysregulated miRNAs in heart tissues of post-MI mice versus sham mice whose roles in cardiac fibrosis are unknown (n = 3). (b) miR-409-3p expression of post-MI mice versus sham mice determined by qRT-PCR (n = 5). (c, d) Protein levels of fibrotic biomarkers and miR-409-3p expression in two cell models of cardiac fibrosis induced by Ang II or TGF-β (n = 5). ∗P < 0.05 and ∗∗∗P < 0.001 versus respective controls.

Figure 2. Deficiency of miR-409-3p reverses MI-induced cardiac fibrosis and cardiac dysfunction. C57BL/6 mice were injected with miR-409-3p antagomir or nc antagomir via tail vein for three consecutive days and subjected to sham or MI surgery for 3 weeks. (a) The expression of miR-409-3p in heart tissues measured by qRT-PCR. (b) Masson's trichrome staining and quantification of collagen deposition in heart sections. (c) Cardiac function recorded by echocardiography. (d) Proliferation of cardiac fibroblasts determined by immunofluorescent staining of Ki67 and Vimentin. (e) Fibroblast-to-myofibroblast differentiation determined by immunofluorescent staining of α-SMA and Vimentin. (f) Expression of fibrotic markers (α-SMA, Col1a1, and Col3a1) measured by qRT-PCR. (g) Protein levels of CTGF, α-SMA, Col1a1, and Col3a1 in heart tissues (n = 5). Scale bar: 100 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus respective controls.

Figure 3. miR-409-3p mimic promotes cardiac fibrosis in vitro. (a) Expression of miR-409-3p in cultured NRCFs transfected with miR-409-3p mimic (n = 5). (b, c) mRNA and protein levels of fibrosis-related genes in NRCFs transfected with miR-409-3p mimic and subjected to Ang II stimulation (n = 5 for qRT-PCR, n = 6 for western blot). (d) Proliferation and differentiation degree of NRCFs determined by immunofluorescent staining of EdU and α-SMA (n = 5). (e, f) Expression of fibrotic genes in NRCFs transfected with miR-409-3p mimic in the presence or absence of TGF-β treatment (n = 5 for qRT-PCR, n = 6 for western blot). (g) Proliferation and differentiation degree of NRCFs evidenced with EdU/α-SMA staining (n = 5). Scale bar: 200 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus respective controls.

Figure 4. Inhibition of miR-409-3p suppresses cardiac fibrosis in NRCF. (a) miR-409-3p level in NRCFs transfected with miR-409-3p inhibitor measured by qRT-PCR (n = 5). (b, c) qRT-PCR and western blot were, respectively, conducted to detect levels of fibrosis-related genes in NRCFs transfected with miR-409-3p inhibitor and subjected to Ang II stimulation (n = 5 for qRT-PCR, n = 6 for western blot). (d) Proliferation and differentiation degree of NRCFs determined by immunofluorescent staining of EdU and α-SMA (n = 5). (e, f) Expression of fibrotic genes in NRCFs transfected with miR-409-3p inhibition in the presence or absence of TGF-β treatment (n = 5 for qRT-PCR, n = 6 for western blot). (g) NRCF proliferation and differentiation into myofibroblasts evidenced by EdU/α-SMA staining (n = 5). Scale bar: 200 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus respective controls.

Figure 5. miR-409-3p affects cardiac fibrosis by targeting Gpd1. (a) Three prediction databases were used to search for the potential target gene of miR-409-3p. (b) Dysregulated genes in heart tissues of post-MI mice versus sham mice (n = 3). (c) Direct target of miR-409-3p identified by luciferase reporter assays (n = 3). (d) Protein levels of Gpd1 in NRCFs treated with miR-409-3p mimic or inhibitor (n = 6). (e) Expression of Gpd1 in heart tissues from post-MI mice versus sham mice detected by western blot (n = 5). (f) Protein levels of Gpd1 in two cell models of cardiac fibrosis induced by Ang II or TGF-β (n = 6). (g) Silence efficiency of Gpd1 siRNA detected by qRT-PCR and western blot (n = 5). (h–j) The effect of Gpd1 knockdown in NRCFs transfected with miR-409-3p inhibitor on fibrotic gene expression, cell proliferation, and differentiation into myofibroblasts (n = 5 for qRT-PCR and immunofluorescent staining, n = 6 for western blot). Scale bar: 200 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus respective controls.

Figure 6. GATA2 is a fibrosis-associated positive regulatory transcription factor of miR-409-3p. (a) Transcription factors of miR-409-3p were searched in the TransmiR database. (b, c) miR-409-3p expression in NRCFs transfected with GATA2 overexpression plasmid or siRNA (n = 5). (d) Expression of GATA2 in heart tissues from post-MI mice compared to sham mice (n = 5). (e) Protein levels of GATA2 in NRCFs treated with Ang II or TGF-β (n = 6). (f–h) The effect of Gpd1 knockdown in NRCFs overexpressing GATA2 on fibrotic gene expression, cell proliferation, and differentiation into myofibroblasts (n = 5 for qRT-PCR and immunofluorescent staining, n = 6 for western blot). Scale bar: 200 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus respective controls.