The long noncoding RNA THBS1-AS1 promotes cardiac fibroblast activation in cardiac fibrosis by regulating TGFBR1
Junteng Zhou, Geer Tian, Yue Quan, Qihang Kong, Fangyang Huang, Junli Li, Wenchao Wu, Yong Tang, Zhichao Zhou, Xiaojing Liu
Junteng Zhou, Geer Tian, Yue Quan, Qihang Kong, Fangyang Huang, Junli Li, Wenchao Wu, Yong Tang, Zhichao Zhou, Xiaojing Liu
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Research Article Cardiology

The long noncoding RNA THBS1-AS1 promotes cardiac fibroblast activation in cardiac fibrosis by regulating TGFBR1

Abstract

Cardiac fibrosis is associated with an adverse prognosis in cardiovascular disease that results in a decreased cardiac compliance and, ultimately, heart failure. Recent studies have identified the role of long noncoding RNA (lncRNA) in cardiac fibrosis. However, the functions of many lncRNAs in cardiac fibrosis remain to be characterized. Through a whole-transcriptome sequencing and bioinformatics analysis on a mouse model of pressure overload–induced cardiac fibrosis, we screened a key lncRNA termed thrombospondin 1 antisense 1 (THBS1-AS1), which was positively associated with cardiac fibrosis. In vitro functional studies demonstrated that the silencing of THBS1-AS1 ameliorated TGF-β1 effects on cardiac fibroblast (CF) activation, and the overexpression of THBS1-AS1 displayed the opposite effect. A mechanistic study revealed that THBS1-AS1 could sponge miR-221/222 to regulate the expression of TGFBR1. Moreover, under TGF-β1 stimulation, the forced expression of miR-221/222 or the knockdown TGFBR1 significantly reversed the THBS1-AS1 overexpression induced by further CF activation. In vivo, specific knockdown of THBS1-AS1 in activated CFs significantly alleviated transverse aorta constriction–induced (TAC-induced) cardiac fibrosis in mice. Finally, we demonstrated that the human THBS1-AS1 can also affect the activation of CFs by regulating TGFBR1. In conclusion, this study reveals that lncRNA THBS1-AS1 is a potentially novel regulator of cardiac fibrosis and may serve as a target for the treatment of cardiac fibrosis.

Authors

Junteng Zhou, Geer Tian, Yue Quan, Qihang Kong, Fangyang Huang, Junli Li, Wenchao Wu, Yong Tang, Zhichao Zhou, Xiaojing Liu

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Figure 5

The identification of miR-221/222 as sponged targets of lncRNA THBS1-AS1 and TGFBR1.

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The identification of miR-221/222 as sponged targets of lncRNA THBS1-AS1...
(A) The gene expression of TGFBR1 in cardiac fibroblasts after siRNA-mediated THBS1-AS1 knockdown by qPCR (n = 3). An unpaired, 2-tailed t test was used. (B) The gene expression of TGFBR1 in cardiac fibroblasts after adenovirus-mediated THBS1-AS1 overexpression by qPCR (n = 6). An unpaired, 2-tailed t test was used. (C) The predicted binding sites of THBS1-AS1 and miR-221/222. (D) The expression of miR-221 and miR-222 in cardiac fibroblasts after TGF-β1 treatment (n = 3). An unpaired, 2-tailed t test was used. (E) The expression of miR-221 and miR-222 in cardiac fibroblasts after THBS1-AS1 knockdown (n = 3). An unpaired, 2-tailed t test was used. (F) The schematic diagram of plasmid. (G) The verification of miR-221/222 as sponged targets of lncRNA THBS1-AS1 by the dual-luciferase reporter assay (n = 6). An unpaired, 2-tailed t test in the left panel and 1-way ANOVA followed by a Bonferroni post hoc test in the right panel were used. (H) The predicted binding sites of TGFBR1 and miR-221/222. (I) The verification of TGFBR1 as a target gene of miR-221/222 by the dual–luciferase reporter assay (n = 6). One-way ANOVA, followed by a Bonferroni post hoc test, was used. (J) The gene expression of TGFBR1 in cardiac fibroblast transfected with miR-221/222 mimics alone or transfected with miR-221/222 mimics and AAV-mediated THBS1-AS1 overexpression (n = 3). One-way ANOVA, followed by a Bonferroni post hoc test, was used. (K) The gene expression of TGFBR1 in cardiac fibroblast transfected with miR-221/222 antagomir alone or transfected with miR-221/222 antagomir and siRNA-mediated THBS1-AS1 knockdown (n = 6). One-way ANOVA, followed by a Bonferroni post hoc test, was used. The results are presented as means ± SEM; *P < 0.05.