MicroRNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβRII

Figures

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  Figure 1. MiR‐30c was decreased in atrial fibrosis. (A, B) Masson's trichrome staining and quantitative analysis in left atrium of Sham (n = 6) and AAC (n = 7) groups. The blue staining indicates interstitial fibrosis. Interstitial fibrosis was increased in AAC rats. **P < .01 compared with the Sham group (Scale bar, 100 μm). (C) The level of miR‐30c was down‐regulated in the left atrium of AAC rat model by qRT‐PCR. *P < .05 compared with the Sham group. (D, E) Representative western blot and quantification analysis revealed that the protein levels of TGFβRII were up‐regulated in left atrium of the AAC rat models. *P < .05 compared with the Sham group. (F) The expression levels of miR‐30c and TGFβRII in CFs with TGF‐β1 (5 ng/mL) were down‐regulated and up‐regulated by qRT‐PCR, respectively. *P < .05 compared with Control group. All experiments were repeated 3 times and data were presented as the mean ± SEM (n = 3, each)

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  Figure 2. MiR‐30c exhibited an anti‐fibrotic effect on CFs. (A) CFs were transfected with a miR‐30c mimic or miR‐30c negative control (NC) for 72 hours. NC was used as a mimic negative control. The level of miR‐30c was increased in CFs transfected with miR‐30c mimic by qRT‐PCR. # P < .05 compared with NC group. (B) After transfecting with a miR‐30c mimic or NC, the CFs were stimulated with TGF‐β1 (5 ng/mL) for 24, 48 or 72 hours. Overexpression of miR‐30c inhibited CF proliferation performed with a CCK8‐Kit assay. # P < .05, ## P < .01 compared with NC group; *P < .05 compared with NC + TGF‐β1 group. (C–E) qRT‐PCR, western blot and quantification analysis of levels of α‐SMA and vimentin in CFs transfected with the miR‐30c mimic or negative control in the presence or absence of TGF‐β1 (5 ng/mL). # P < .05 compared with NC group; *P < .05, **P < .01 compared with NC + TGF‐β1 group. (F) Representative photomicrographs of the migrating cells after transfection and staining by crystal violet (Scale bar, 100 μm). (G) The effect of the miR‐30c mimic on the migration of the cells was quantified. # P < .05 compared with NC group; *P < .05 compared with NC + TGF‐β1 group. (H–J) The mRNA and protein…

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  Figure 3. The miR‐30c inhibitor reversed the anti‐fibrotic effects on CFs. (A) CFs were transfected with a miR‐30c inhibitor or miR‐30c inhibitor negative control (NC) for 72 hours. The level of miR‐30c was decreased in CFs transfected with miR‐30c inhibitor by qRT‐PCR. NC was used as an inhibitor negative control. # P < .05 compared with NC group. (B) After transfecting with a miR‐30c inhibitor or NC, the CFs were stimulated with TGF‐β1 (5 ng/mL) for 24, 48 or 72 hours. A miR‐30c inhibitor promoted CF proliferation evaluating by a CCK8‐Kit assay. # P < .05, ## P < .01, compared with NC group; *P < .05, **P < .01, compared with NC + TGF‐β1 group. (C–E) The levels of α‐SMA and vimentin were increased in CFs transfected with the miR‐30c inhibitor in the presence or absence of TGF‐β1 (5 ng/mL) via qRT‐PCR, western blot and quantification analysis. # P < .05 compared with NC group; *P < .05, **P < .01, compared with NC + TGF‐β1 group. (F) Representative photomicrographs of the migrating cell after transfection and staining by crystal violet (Scale bar, 100 μm). (G) The effect of the miR‐30c inhibitor on the migration of the cells was quantified. ## P < .01, compared with NC group; **P < .01…

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  Figure 4. MiR‐30c targets TGFβRII. (A) Diagram of miR‐30c binding site in TGFβRII 3′UTR. TGFβRII 3′UTR mutant was constructed to evaluate miR‐30c binding. (B) After 293T cell cotransfection, luciferase activity was measured using a Dual Luciferase Report Assay Kit. The luciferase activity was significantly decreased in TGFβRII‐WT + miR‐30c group compared with that in TGFβRII‐WT + miR‐30c NC and TGFβRII‐mu + miR‐30c. ### P < .001, compared with pGL3‐TGFβRII‐WT + NC group. && P < .01, compared with pGL3‐TGFβRII‐mu + miR‐30c group. (C–E) qRT‐PCR and western blot analyses of CFs transfected with the miR‐30c mimic or negative control for 72 hours in the absence or presence of TGF‐β1. The miR‐30c mimic decreased the mRNA and protein levels of TGFβRII. # P < .05, ## P < .05 compared with NC group; **P < .01, ***P < .01, compared with NC + TGF‐β1 group. (F–H) The level of TGFβRII was increased after transfection with a miR‐30c inhibitor as measured by qRT‐PCR and western blot. # P < .05 compared with NC group; *P < .05 compared with NC+TGF‐β1 group. All experiments were repeated 3 times, and all data were presented as the mean ± SEM (n = 3, each)

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  Figure 5. In vivo overexpression of miR‐30c via AAV9 attenuates atrial fibrosis. (A) After administering AAV9‐miR‐30c into the inferior vena cava for 8 weeks, the level of miR‐30c in the left atrium was increased by qRT‐PCR. **P < .01, compared with AAC group. (B–D) The levels of TGFβRII were determined by qRT‐PCR and western blot. The mRNA and protein levels of TGFβRII were decreased in the AAC+miR‐30c group. *P < .05, compared with AAC group. (E, F) Masson's trichrome staining and quantitative analysis in left atrium demonstrated that overexpression miR‐30c prevented atrial fibrosis (Scale bar, 100 μm). **P < .01, compared with AAC group. (G, H) The protein levels of Col I, Col3α1 and α‐SMA were measured by western blot. In vivo, overexpression miR‐30c inhibited the expression of Col I, Col3α1 and α‐SMA. *P < .05, **P < .01, compared with AAC group. (Sham, n = 6; AAC, n = 6; AAC + miR‐30c, n = 7; AAC + Control, n = 7)

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  Figure 6. MiR‐30c regulates atrial fibrogenesis. TGF‐β1 decreased miR‐30c expression. Overexpression of miR‐30c in cardiac fibroblasts inhibited TGFβRII expression, thus preventing cell proliferation, differentiation and migration as well as collagen synthesis and atrial fibrogenesis