LncRNA-Safe contributes to cardiac fibrosis through Safe-Sfrp2-HuR complex in mouse myocardial infarction

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  Figure 1. Safe is a fibroblast-enriched lncRNA and associated with cardiac fibrosis. (A) Heatmap showing 389 lncRNAs differentially expressed in tissues from the infarction zone (IZ) and non-infarcted remote zone (RZ) at day 14 post MI (fold ≥ 2, p<0.05). (B) qRT-PCR validation of four upregulated lncRNAs and four downregulated lncRNAs in cardiac samples from IZ and RZ of infarcted hearts (n=3). (C) qRT-PCR analysis of Safe expression in healthy (sham) or infarcted myocardium at indicated days post MI (n=3). (D) qRT-PCR detection of Safe expression in adult cardiomyocytes (aCM), adult cardiac fibroblasts (aCF) and adult endothelial cells (aEC). Myh7 (myosin heavy chain 7), FSp1 (fibroblast specific protein-1) and Pecam1 (Platelet endothelial cell adhesion molecule-1) were used as markers of aCM, aCF and aEC, respectively (n=3). (E) qRT-PCR detection of Safe, Col1a1 and α-SMA expression in TGF-β-treated cardiac fibroblasts (n=3). (F) Representative western blot analysis and relative densitometric quantification of COL1A1 and α-SMA protein levels in cardiac fibroblasts with TGF-β treatment (n=3). (G) Agarose gel electrophoresis of the 5' and 3' RACE amplification products. (H) Schematic…

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  Figure 2. Suppression of Safe prevents TGF-β-induced cardiac fibrosis in vitro. (A) mRNA expression of Safe, Col1a1, α-SMA and Bmp1 in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Safe knockdown (n=3). (B) Western blot analysis and relative densitometric quantification of COL1A1 and α-SMA protein levels in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Safe knockdown (n=3). (C) Representative images of immunofluorescence staining for α-SMA (n=5) and quantification of the relative cell areas of cardiac fibroblasts after treatments as indicated (n=40). The nuclei were counterstained with DAPI (blue). Scale bar indicates 50 μm. (D) Representative images of collagen gel contraction for 24 hours and quantification of collagen area inside the dashed circles (n=3). Scale bar indicates 0.5 cm. (E) BMP1 protein enzyme activity in the supernatant of cultured fibroblasts after indicated treatments (n=3). The excitation wavelength is 320 nm, and the emission wavelength is 405 nm. (F) ELISA assay of COL1A1 protein in the supernatant of cultured fibroblasts after indicated treatments (n=3). (G) CCK-8 assay of cardiac fibroblasts with TGF-β-untreated or TGF-β-treated showing…

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  Figure 3. Inhibition of Safe ameliorates MI-induced cardiac fibrosis. (A) Representative M-mode echocardiographic images obtained from MI mice injected with scramble control shRNA (Ctr) or shSafe lentiviral particles at day 28 after surgery (n=10 in each group). (B) Left ventricular ejection fraction (EF) of MI mice as assessed via echocardiography in indicated time points (n=10). (C) Left ventricular fractional shortening (FS) of MI hearts in indicated time points (n=10). (D) Schematic diagram of the slice, starting from left ventricular exposure, each level is 500 μm apart. Representative images of Masson's trichrome-stained MI hearts, and quantification of fibrosis (% area) showing a significant decrease of fibrosis areas in MI hearts injected with shSafe lentiviral particles (n=8). Scale bar indicates 2 mm. (E) qRT-PCR analysis of Col1a1 and α-SMA in the infarction zone of MI hearts (n=3). (F) Western blot analysis and relative densitometric quantification of COL1A1 and α-SMA protein levels in MI hearts after Safe inhibition (n=3). Data are presented as mean ± SEM; Student's t-test or two-way repeated-measures ANOVA; *p < 0.05.

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  Figure 4. Safe and Sfrp2 mutually regulates each other's RNA stability in fibroblasts. (A) Schematic diagram of RNA-RNA interaction between Safe and Sfrp2 at their reverse complementary regions (462 nucleotides in length). Thick blocks and thin blocks represent the coding sequence (CDS) and untranslated regions (UTRs), respectively. (B) qRT-PCR detection of Sfrp2 expression in left ventricles of sham and MI mice at indicated days after surgery (n=3). (C) qRT-PCR detection of Sfrp2 expression in the infarct zone (IZ), border zone (BZ), and remote zone (RZ) of infarcted hearts after 14 days following MI surgery (n=3). (D) qRT-PCR detection of Sfrp2 expression in aCM, aCF and aEC (n=3). (E) qRT-PCR analysis showing increased expression of Sfrp2 in cardiac fibroblasts after TGF-β treatment (n=3). (F) mRNA expression of Sfrp2, Col1a1, α-SMA and Bmp1 in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Sfrp2 knockdown (n=3). (G) Representative western blot analysis and relative densitometric quantification of SFRP2, COL1A1 and α-SMA protein levels in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Sfrp2 knockdown (n=3). (H) qRT-PCR analysis showing decreased expression of…

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  Figure 5. Sfrp2 overexpression disturbed the regulatory effects of shSafe in cardiac fibroblasts. (A) qRT-PCR analysis showing mRNA expression of Safe, Sfrp2, Col1a1, α-SMA and Bmp1 by Sfrp2 overexpression in Safe-silenced cardiac fibroblasts (n=3). (B) Representative western blot analysis and relative densitometric quantification showing up-regulated expression of SFRP2, COL1A1, and α-SMA proteins by Sfrp2 overexpression in Safe-silenced cardiac fibroblasts (n=3). (C) Representative images of immunofluorescence staining for α-SMA (red, n=5) and quantification of the relative cell area of cardiac fibroblasts after treatments as indicated (n=40). Scale bar indicates 50 μm. (D) Representative images of collagen gel contraction for 24 hours and quantification of collagen area inside the dashed circles (n=3). Scale bar indicates 0.5 cm. (E) BMP1 protein enzyme activity in the supernatant of cultured fibroblasts after indicated treatments (n=3). The excitation wavelength is 320 nm, and the emission wavelength is 405 nm. (F) ELISA assay of COL1A1 protein in the supernatant of cultured fibroblasts after indicated treatments (n=3). (G) CCK-8 assay of cardiac fibroblasts showing restored cell…

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  Figure 6. Sfrp2 overexpression disturbed the protective effect of shSafe on cardiac function post MI. (A) Representative M-mode echocardiographic images obtained from MI mice after Safe knockdown, in combination with Sfrp2 overexpression (n=10 in each group). (B) Sfrp2 overexpression led to a reduced improvement of EF in shSafe-injected MI mice at day 28 post-surgery (n=10). (C) shSafe-mediated increase in FS in MI hearts was inhibited by Sfrp2 overexpression (n=10). (D) Representative images of Masson's trichrome-stained MI hearts, and quantification of relative fibrosis areas (n=8). Scale bar indicates 2 mm. (E) qRT-PCR analysis showing increased expression of Safe, Sfrp2, Col1a1 and α-SMA in left ventricles of shSafe-injected MI hearts after Sfrp2 overexpression (n=3). (F) Western blot analysis and relative densitometric quantification of SFRP2, COL1A1 and α-SMA protein levels in shSafe-injected MI hearts after Sfrp2 overexpression (n=3). All data are presented as mean ± SEM; Student's t-test, one-way ANOVA or two-way repeated-measures ANOVA, *p < 0.05.

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  Figure 7. Binding of HuR to the Safe-Sfrp2 duplex accelerates RNA stabilization of both Safe and Sfrp2. (A) Schematic presentation of predicated HuR binding sites and corresponding RNA probes for EMSA assay in the complementary region of Safe and Sfrp2 RNA. (B) EMSA assay indicating specific binding of nuclear proteins of cardiac fibroblasts to the 26-nucleotide RNA duplexes corresponding to nucleotide at the 1408 nt protein binding site of Safe. (C) EMSA supershift assay revealing the in vitro interaction between HuR protein and the 26-nucleotide RNA duplexes in nuclear extracts. RNA immunoprecipitation (RIP) assay using HuR antibody and IgG (isotype control) showing enrichment of Safe (D) and Sfrp2 (E) RNAs to HuR protein. (F) qRT-PCR analysis showing decreased expression of HuR, Safe and Sfrp2 in cardiac fibroblasts after HuR knockdown (n=3). (G) Representative western blot analysis and relative densitometric quantification of HuR and SRRP2 protein levels in cardiac fibroblasts with or without HuR inhibition (n=3). (H) Dual luciferase assay showing shHuR inhibited Firefly luciferase activities of pGL3-control vectors carrying the 3'-end of Safe (462-nucleotide in length) (n=3). (I) Dual…

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  Figure 8. Mechanism of lncRNA-Safe-mediated cardiac fibrosis. Pathological stimuli such as TGF-β and myocardial infarction facilitate the expression of lncRNA-Safe in cardiac fibroblasts. In cooperation with HuR protein, Safe can complementarily binds to the 3'-UTR of Sfrp2 mRNA, and promote Sfrp2 RNA stabilization and protein expression, which in turn accelerates the transformation and proliferation of myofibroblasts, eventually leading to excessive secretion of extracellular matrix proteins.