CircMACF1 alleviates myocardial fibrosis after acute myocardial infarction by suppressing cardiac fibroblast activation via the miR-16-5p/SMAD7 axis

Figures

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  Figure 1.. CircMACF1 was repressed in AMI tissues and activated CFs. The blood samples were collected from 35 AMI patients and 35 healthy people. (A) The expression of circMACF1 in AMI patients was assessed by using qPCR. CFs were treated with TGF-β1 (10 ng/mL) or Ang II (100 nM) for 48 hours. (B) CircMACF1 expression in activated CFs was examined by qPCR. The circMACF1 from CFs was treated with RNase R (3 U/μg) and incubated for 60 minutes at 40°C. (C) Resistance of circMACF1 to RNase R digestion was assessed via real-time PCR. (D) qPCR was used to measure the nuclear and cytoplasmic distribution of circMACF1. All the data are presented as the mean ± standard deviation. *P < .05. AMI = acute myocardial infarction, CFs = cardiac fibroblasts, circMACF1 = circular microtubule actin crosslinking factor 1, qPCR = real time fluorescent quantitative PCR, TGF = transforming growth factor.

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  Figure 2.. CircMACF1 overexpression suppressed the activation, proliferation and migration of TGF-β1-treated CFs. CFs were transfected with circMACF1 overexpression plasmid (100 nM) and its negative control (NC; 100 nM) for 48 hours. (A) qPCR was used to measure the level of circMACF1. CFs were transfected with NC and circMACF1 overexpression plasmids (100 nM) before the treatment with TGF-β1 (10 ng/mL). (B) Fibrosis-related protein expression levels were measured via qPCR. (C) The protein levels of fibrosis-related proteins were examined by western blotting assay. (D) Immunofluorescence was performed to investigate α-SMA expression. (E) CCK-8 was carried out to investigate cell viability. (F) Cell migration was investigated by Transwell assay. All the data are presented as the mean ± standard deviation. *P < .05. CFs = cardiac fibroblasts, circMACF1 = circular microtubule actin crosslinking factor 1, qPCR = real time fluorescent quantitative PCR, TGF = transforming growth factor.

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  Figure 3.. CircMACF1 acted as a sponge of miR-16-5p. The blood samples were collected from 35 AMI patients and 35 healthy people. (A) MiR-16-5p expression in AMI patients was measured by qPCR. (B) The correlation analysis of miR-16-5p and circMACF1 in AMI patients. CFs were transfected with miR-NC, miR-16-5p mimics (50 nM), inhibitor NC and miR-16-5p inhibitor (100 nM) for 48 hours. (C) MiR-16-5p expression in transfected CFs was detected by qPCR. (D) Bioinformatics software was used to predict the binding sites between miR-16-5p and circMACF1. (E) RIP confirmed the interaction of circMACF1 and miR-16-5p. (F) A dual luciferase reporter gene assay was used to measure the luciferase activity. CFs were transfected with circMACF1 ovexexpression plasmid (100 nM) or silencing plasmid (50 nM) for 48 hours. (G, H) qPCR was used to measure miR-16-5p expression without or with TGF-β1 treatment (10 ng/mL) in circMACF1 transfected cells. All the data are presented as the mean ± standard deviation. * P < .05. AMI = acute myocardial infarction, CFs = cardiac fibroblasts, circMACF1 = circular microtubule actin crosslinking factor 1, qPCR = real time fluorescent quantitative PCR, RIP = RNA…

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  Figure 4.. SMAD7 could be a target gene of miR-16-5p. The blood samples were collected from 35 AMI patients and 35 healthy people. (A) The expression of SMAD7 in AMI patients was investigated by qPCR. (B) MiR-16-5p was negatively correlated with SMAD7 in AIM. (C) CircMACF1 was positively correlated with SMAD7 in AMI. (D) Bioinformatics software predicted the binding sites between miR-16-5p and SMAD7. CFs were transfected with miR-16-5p mimics (50 nM) or miR-16-5p inhibitor (100 nM) for 48 hours. (E) Luciferase activity was examined via dual luciferase reporter gene assay. (F, G) The regulation of SMAD7 mRNA or protein levels by miR-16-5p were examined through qPCR or western blotting. CFs were transfected with miR-16-5p inhibitor (100 nM) and/or si-circMACF1 (50 nM) before the treatment with TGF-β1 (10 ng/mL) for 48 hours. (H, I) The mRNA level or protein expression of SMAD7 was investigated via qPCR or western blotting. All the data are presented as the mean ± standard deviation. *P < .05. AMI = acute myocardial infarction, CFs = cardiac fibroblasts, circMACF1 = circular microtubule actin crosslinking factor 1, qPCR = real time fluorescent quantitative PCR, SMAD7 = mothers against…

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  Figure 5.. CircMACF1 inhibited the activation, migration and proliferation of TGF-β1-treated CFs by regulating the miR-16-5p/Smad7 axis. CFs were transfected with SMAD silencing plasmid (50 nM) for 48 hours. (A) SMAD7 levels were measured by qPCR. (B) The protein level of SMAD7 was measured by western blotting. CFs were transfected with miR-16-5p inhibitor (100 nM) and/or si-circMACF1 or si-SMAD7 (50 nM) before the treatment with TGF-β1 (10 ng/mL) for 48 hours. (C, D) Fibrosis-related protein levels were measured via qPCR and western blotting. (E) Immunofluorescence was performed to measure α-SMA expression. (F) Cell proliferation was measured through CCK-8 assay. (G) Cell migration was measured by transwell assay. All the data are presented as the mean ± standard deviation. * P < .05. CCK-8 = cell counting kit-8, CFs = cardiac fibroblasts, circMACF1 = circular microtubule actin crosslinking factor 1, qPCR = real time fluorescent quantitative PCR, SMAD7 = mothers against decapentaplegic homolog 7, TGF = transforming growth factor.