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. 2021 Mar 3;54(4):e10692.
doi: 10.1590/1414-431X202010692. eCollection 2021.

hsa-miR-4443 inhibits myocardial fibroblast proliferation by targeting THBS1 to regulate TGF-β1/α-SMA/collagen signaling in atrial fibrillation

Jingwen Xiao  1 , Yan Zhang  1 , Yuan Tang  2 , Hengfen Dai  3 , Yu OuYang  1 , Chuanchuan Li  1 , Meiqin Yu  2
Affiliations
Free PMC article

hsa-miR-4443 inhibits myocardial fibroblast proliferation by targeting THBS1 to regulate TGF-β1/α-SMA/collagen signaling in atrial fibrillation

Jingwen Xiao et al. Braz J Med Biol Res. .
Free PMC article

Abstract

Fibrosis caused by the increase in extracellular matrix in cardiac fibroblasts plays an important role in the occurrence and development of atrial fibrillation (AF). The aim of this study was to investigate the role of hsa-miR-4443 in AF, human cardiac fibroblast (HCFB) proliferation, and extracellular matrix remodeling. TaqMan Stem-loop miRNA assay was used to measure hsa-miR-4443 expression in patients with persistent AF (n=123) and healthy controls (n=100). Patients with AF were confirmed to have atrial fibrosis by late gadolinium enhancement. At the cellular level, after hsa-miR-4443 mimic and inhibitor were transfected with HCFBs, proliferation, apoptosis, migration, and invasion were analyzed. Lastly, hsa-miR-4443-targeted gene and transforming growth factor (TGF)-β1/α-SMA/collagen pathway were evaluated by dual-luciferase reporter assay and western blot, respectively. In patients with AF, hsa-miR-4443 decreased significantly and collagen metabolism level increased significantly. Logistic regression analysis showed that low hsa-miR-4443 level was a risk factor of AF (P<0.001). The receiver operating characteristic curve revealed that hsa-miR-4443 was useful for predicting AF (area under the curve: 0.828, sensitivity: 0.71, specificity: 0.78, P<0.001). In HCFBs, hsa-miR-4443 targeted thrombospondin-1 (THBS1) and downregulated TGF-β1/α-SMA/collagen pathway. The inhibition of hsa-miR-4443 expression promoted HCFB proliferation, migration, invasion, myofibroblast differentiation, and collagen production. The significant reduction of hsa-miR-4443 can be used as a biomarker for AF. hsa-miR-4443 protected AF by targeting THBS1 and regulated TGF-β1/α-SMA/collagen pathway to inhibit HCFB proliferation and collagen synthesis.

Figures

Figure 1
Figure 1. hsa-miR-4443 decreased in patients with atrial fibrillation (AF). A, Serum hsa-miR-4443 expression level was determined by qRT-PCR. B, ROC analysis was performed to evaluate the diagnostic efficacy of serum hsa-miR-4443 for persistent AF. Horizontal lines indicate median and interquartile range. ***P<0.001 vs control (Mann-Whitney U test, two-sided).
Figure 2
Figure 2. hsa-miR-4443 affected the biological function of human cardiac fibroblast (HCFBs). A, HCFBs were transfected with hsa-miR-4443 mimic or inhibitor. hsa-miR-4443 expression affected cell proliferation (B), apoptosis (C), migration (D), and invasion (E) (scale bars, 100 μm). All data are reported as means±SD of at least three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control (one-way ANOVA analysis with two-sided LSD post hoc test). NC: negative control.
Figure 3
Figure 3. hsa-miR-4443 targeted THBS1 to regulate transforming growth factor (TGF)-β1/α-SMA/collagen signaling in human cardiac fibroblast (HCFBs). A, Binding sequences between hsa-miR-4443 and THBS1 3′-UTR transcripts, and dual-luciferase activity assay revealed hsa-miR-4443 targeted THBS1. B, hsa-miR-4443 promoted THBS1 mRNA degradation. C, The expression of THBS1, TGF-β, Smad2/3, and LTBP1 protein was examined in HCFBs. D, p-Smad2 and p-Smad3 expression levels were detected. E, α-SMA, collagen I, and collagen III protein expression levels were compared with the control group. All data are reported as means±SD of at least three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control (Student's t-test or one-way ANOVA analysis with two-sided LSD post hoc test). NC: negative control.
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