Long Non-Coding RNA 554 Promotes Cardiac Fibrosis via TGF-β1 Pathway in Mice Following Myocardial Infarction

Figures

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  FIGURE 1. LncRNA 554 is upregulated after myocardial infarction and is enriched in cardiac fibroblasts. Two weeks after MI, the hearts of mice were stained by hematoxylin-eosin staining and Masson’s trichrome staining (A). Scale bars, 100 μm n = 8 mice in each group; (B) qRT-PCR were conducted to detect the expression of lncRNA 554 in the border zone of mice at different time (0, 3, 7, 14, 28 days) after MI. **p < 0.01 vs 0 day, n = 8; (C) qRT-PCR were conducted to detect lncRNA 554 levels in CMs and CFs. *p < 0.05 vs CMs, n = 5. Representative fluorescent in situ hybridization images; (D) and corresponding statistical chart; (E) showing the expression abundance and cellular localization of lncRNA 554 in CFs. Scale bars, 50 μm n = 10.

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  FIGURE 2. Knockdown of lncRNA 554 attenuates the function of cardiac fibroblasts and reduces the expression of TGF-β1 and its downstream (A) qRT-PCR was used to detect the expression of lncRNA 554 in CFs after siRNA transfection. Control, untreated group; NC, scrambled control siRNA transfection group; Si1-4, siRNA transfection group. **p < 0.01 vs NC, n = 5; (B) The CFs was transfected with si1 with different time (24 h, 48 h), and then qRT-PCR was conducted to detect the level of lncRNA 554. **p < 0.01 vs NC, n = 5; (C) After silencing of lncRNA 554, the level of ECM mRNAs (α-SMA, Col 1, Col 3, Fn 1, CTGF) were analyzed by qRT-PCR. **p < 0.01 vs NC, n = 5; (D) and (E) The efficiency of healing in lncRNA 554 silencing group and NC group was analyzed by wounding healing assay at different time (0, 12 and 24 h). **p < 0.01 vs NC, n = 5. Scale bars, 250 μm; (F) After silencing of lncRNA 554, qRT-PCR was conducted to detect the level of TGF-β1 pathway (TGF-β1, Smad 2, Smad 3). **p < 0.01 vs NC, n = 5.

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  FIGURE 3. LncRNA 554 regulates cardiac fibrosis via TGF-β1 signal pathway in vitro (A) Representative images of GFP fluorescence intensity of different MOI (10, 50 and 100) show the infection efficiency of lentivirus. n = 5. Scale bars, 250 μm; (B) The level of lncRNA 554 overexpression by lentivirus was analyzed by qRT-PCR. *p < 0.05 vs NC, n = 5; (C) The level of ECM mRNA in CFs treated with different concentration TGF-β1 inhibitor (TEW-7197) (0 , 1, 5 and 10 μM). *p < 0.05 vs 0 μM, n = 5; (D) The CFs were treated with lentivirus-NC, lentivirus-lncRNA554 and (or) TEW-7197. qRT-PCR was conducted to detect ECM levels (Col 1, Col 3, Fn 1). **p < 0.01 vs NC, ##p < 0.01 vs Len-Lnc 554, n = 5. NC or Len-NC, empty lentivirus group; Len-Lnc554, lncRNA 554 overexpression group.

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  FIGURE 4. Knockdown of lncRNA 554 mitigates cardiac fibrosis of infarcted hearts. To knockdown lncRNA 554, the hearts were injected with lentivirus containing control or lncRNA 554 shRNA and subjected to qRT-PCR and Masson’s trichrome staining in 14 days after MI (A) The expression of lncRNA 554 was detected by qRT-PCR in post-MI hearts. **p < 0.01 vs SHAM, ##p < 0.01 vs MI + Len-scramble, n = 8. The expression of Col 1; (B), Col 3; (C) and; Fn 1 (D) mRNA in peri-infarct area. **p < 0.01 vs SHAM, ##p < 0.01 vs MI + Len-scramble, n = 8; (E) and (F) Representative images of Masson’s trichrome staining and a quantification of Masson’s trichrome staining show the degree of fibrosis. ****p < 0.0001 vs SHAM, ##p < 0.01 vs MI + Len-scramble, n = 8.

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  FIGURE 5. Knockdown of lncRNA 554 improves cardiac function of infarcted hearts. The hearts were injected with lentivirus containing control or lncRNA 554 shRNA after MI. Two weeks after MI, ventricular parameters were measured and analyzed by echocardiography, including LVAWd; (A), LVAWs; (B), LVIDd; (C), LVIDs; (D), left ventricular EF; (E), and FS; (F). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs SHAM group; #p < 0.05, ##p < 0.01 vs MI + Len-Scramble group, n = 8. EF: ejection fraction; FS: fractional shortening; LVAWd: left ventricular anterior wall end diastolic thickness; LVAWs: left ventricular anterior wall end systolic thickness; LVIDd: left ventricular internal diastolic diameter; LVIDs: left ventricular internal systolic diameter.