Long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense RNA 1 regulates myocardial fibrosis in diabetes through the Hippo-Yes-associated protein signaling pathway

Abstract

Aims/introduction: Fibrosis is the principle reason for heart failure in diabetes. Regarding the involvement of long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) in diabetic myocardial fibrosis, we explored its specific mechanism.

Materials and methods: Human cardiac fibroblasts (HCF) were treated with high glucose (HG) and manipulated with plasmid cloning deoxyribonucleic acid 3.1-ZEB1-AS1/microribonucleic acid (miR)-181c-5p mimic/short hairpin RNA specific to sirtuin 1 (sh-SIRT1). ZEB1-AS1, miR-181c-5p expression patterns, cell viability, collagen I and III, α-smooth muscle actin (α-SMA), fibronectin levels and cell migration were assessed by reverse transcription quantitative polymerase chain reaction, cell counting kit-8, western blot and scratch tests. Nuclear/cytosol fractionation assay verified ZEB1-AS1 subcellular localization. The binding sites between ZEB1-AS1 and miR-181c-5p, and between miR-181c-5p and SIRT1 were predicted and verified by Starbase and dual-luciferase assays. The binding of SIRT1 to Yes-associated protein (YAP) and YAP acetylation levels were detected by co-immunoprecipitation. Diabetic mouse models were established. SIRT1, collagen I, collagen III, α-SMA and fibronectin levels, mouse myocardium morphology and collagen deposition were determined by western blot, and hematoxylin-eosin and Masson trichrome staining.

Results: Zinc finger E-box binding homeobox 1 antisense 1 was repressed in HG-induced HCFs. ZEB1-AS1 overexpression inhibited HG-induced HCF excessive proliferation, migration and fibrosis, and diminished collagen I, collagen III, α-SMA and fibronectin protein levels in cells. miR-181c-5p had targeted binding sites with ZEB1-AS1 and SIRT1. SIRT1 silencing/miR-181c-5p overexpression abrogated ZEB1-AS1-inhibited HG-induced HCF proliferation, migration and fibrosis. ZEB1-AS1 suppressed HG-induced HCF fibrosis through SIRT1-mediated YAP deacetylation. ZEB1-AS1 and SIRT1 were repressed in diabetic mice, and miR-181c-5p was promoted. ZEB1-AS1 overexpression improved myocardial fibrosis in diabetic mice, and reduced collagen I, collagen III, α-SMA and fibronectin protein levels in myocardial tissues.

Conclusion: Long non-coding ribonucleic acid ZEB1-AS1 alleviated myocardial fibrosis through the miR-181c-5p-SIRT1-YAP axis in diabetic mice.

Keywords: Myocardial fibrosis; Yes-associated protein; Zinc finger E-box binding homeobox 1 antisense 1.

Figure 1

Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) inhibited high glucose (HG)‐induced fibrosis in human cardiac fibroblast (HCF) cells. HCF cells were treated by HG to simulate diabetes in vitro. With plasmid cloning deoxyribonucleic acid 3.1‐NC as the overexpression negative control (oe‐NC), plasmid cloning deoxyribonucleic acid 3.1‐ZEB1‐AS1 (oe‐ZEB1‐AS1) was introduced into HCF cells. (a) Long non‐coding ribonucleic acid (LncRNA) ZEB1‐AS1 expression was assessed using reverse transcription quantitative polymerase chain reaction. (b) Cell viability was detected using cell counting kit‐8. (c) Cell migration was detected using scratch test. (d) Collagen I, collagen III, α‐smooth muscle actin (α‐SMA) and fibronectin levels were determined using western blot. The cell experiment was independently repeated three times, and the data are expressed as mean ± standard deviation. One‐way anova was used for multigroup data comparisons, followed by Tukey's test. **P < 0.01. OD, optical density.

Figure 2

Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) modulated sirtuin 1 (SIRT1) level by targeting microribonucleic acid (miR)‐181c‐5p. (a) Localization prediction of long non‐coding ribonucleic acid (LncRNA) ZEB1‐AS1. (b) LncRNA ZEB1‐AS1 level in the nucleus and cytoplasm was determined by reverse transcription quantitative polymerase chain reaction. (c) The binding sites between ZEB1‐AS1 and miR‐181c‐5p were predicted by the bioinformatics website. (d) The targeted binding relationship between ZEB1‐AS1 and miR‐181c‐5p was validated using dual‐luciferase assay. (e) miR‐181c‐5p expression was determined by reverse transcription quantitative polymerase chain reaction. (f) The binding sites between miR‐181c‐5p and SIRT1 were predicted on the bioinformatics website. (g) The targeted relationship between miR‐181c‐5p and SIRT1 was confirmed using dual‐luciferase assay. (h) SIRT1 expression was assessed using western blot. The cell experiment was repeated three times independently, and the data are expressed as mean ± standard deviation. The independent t‐test was applied for pairwise data comparisons, and one‐way anova was used for multigroup data comparisons, followed by Tukey's test. **P < 0.01. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MUT, mutant type; NC, negative control; oe, overexpression; WT, wild type.

Figure 3

Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) inhibited high glucose (HG)‐induced fibrosis of human cardiac fibroblasts (HCFs) by targeting microribonucleic acid (miR)‐181c‐5p. miR‐181c‐5p mimics (miR‐mimics) were introduced into HG‐induced HCFs with overexpressed ZEB1‐AS1, with mimic‐NC (miR‐NC) as the control. (a) Cell viability was detected using cell counting kit‐8. (b) Cell migration was detected by scratch test. (c) Collagen I, Collagen III, α‐smooth muscle actin (α‐SMA) and Fibronectin levels were determined by western blot. The cell experiment was repeated three times independently, and data are expressed as mean ± standard deviation. One‐way anova was used for multigroup data comparisons, followed by Tukey's test. *P < 0.05, **P < 0.01. NC, negative control; oe, overexpression.

Figure 4

Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) inhibited high glucose (HG)‐induced fibrosis of human cardiac fibroblasts (HCFs) by upregulating sirtuin 1 (SIRT1). The expression of SIRT1 was knocked down on the basis of ZEB1‐AS1 overexpression in HCFs. (a) SIRT1 expression was assessed by western blot. (b) Cell viability was detected by cell counting kit‐8. (c) The migration of cells was detected by scratch test. (d) Collagen I, collagen III, α‐smooth muscle actin (α‐SMA) and fibronectin levels were determined using western blot. The cell experiment was independently repeated three times, and data are expressed as mean ± standard deviation. The one‐way anova was used for multigroup data comparisons, followed by Tukey's test. *P < 0.05, **P < 0.01. NC, negative control; oe, overexpression; sh, short hairpin.

Figure 5

Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) deacetylated Yes‐associated protein (YAP) by upregulating sirtuin 1 (SIRT1). The binding of (a) SIRT1 and YAP, and (b) YAP acetylation level were detected using co‐immunoprecipitation (IP). The cell experiment was independently three times. IgG, immunoglobulin G; NC, negative control; oe, overexpression; sh, short hairpin.

Figure 6

Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) repressed high glucose (HG)‐induced fibrosis of human cardiac fibroblasts (HCFs) through sirtuin 1 (SIRT1)‐mediated Yes‐associated protein (YAP) deacetylation. HCFs were treated with 400 nmol/L trichostatin A (TSA) on the basis of ZEB1‐AS1 overexpression for 24 h. (a) The expression of SIRT1 was determined using western blot. (b) The YAP acetylation level was detected using co‐immunoprecipitation. (c) Cell viability was detected using cell counting kit‐8. (d) The migration of cells was detected by scratch test. (e) Collagen I, collagen III, α‐smooth muscle actin (α‐SMA) and fibronectin levels were determined using western blot. The cell experiment was repeated independently thrice, and data are expressed as mean ± standard deviation. The one‐way anova was employed for multigroup data comparisons, followed by Tukey's test. *P < 0.05, **P < 0.01. DMSO, dimethyl sulfoxide; oe, overexpression.

Figure 7

Overexpression of zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1) improved myocardial fibrosis in diabetic mice. Diabetic mice were induced by intraperitoneal administration with 50 mg/kg/day streptozotocin. (a, b) ZEB1‐AS1 and microribonucleic acid (miR)‐181c‐5p expression patterns were determined using reverse transcription quantitative polymerase chain reaction. (c) SIRT1 expression was assessed by western blot. (d, e) Myocardial morphology and collagen deposition were observed by hematoxylin–eosin and Masson trichrome staining. (f) Collagen I, collagen III, α‐smooth muscle actin (α‐SMA) and fibronectin protein levels in myocardial tissues were determined by western blot, n = 6. Data are expressed as mean ± standard deviation. One‐way anova was employed for multigroup data comparisons, followed by Tukey's test. *P < 0.05, **P < 0.01. DM, diabetic mouse model; lncRNA, long non‐coding ribonucleic acid; NC, negative control; oe, overexpression.