LncRNA GUSBP5-AS promotes EPC migration and angiogenesis and deep vein thrombosis resolution by regulating FGF2 and MMP2/9 through the miR-223-3p/FOXO1/Akt pathway

Figures

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  Figure 1. Effects of lncRNA GUSBP5-AS on the treatment of DVT. (A) Representative images of the thrombosis in GFP-GUSBP5-AS-EPC group and LV5-NC group were observed. (B) Weight of the venous thrombi at day 7 after the transplantation. Data are expressed as mean ± SEM (n = 8 mice per group), **P < .01 compared to the corresponding negative controls. (C) Representative images of recruitment of GFP-positive EPCs in DVT (×200). (D) Percentage of green fluorescent area in DVT. **P < .01. (E) The expression levels of CD34 and MMP2 in thrombosis were detected by immunofluorescence.

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  Figure 2. Effects of lncRNA GUSBP5-AS on angiogenesis of EPCs in vivo and in vitro, EPC migration and invasion. (A) In vivo angiogenesis was evaluated at Day 7 after subcutaneous injection of Matrigel-mixed EPCs into nude mice. (B) HE staining: lncRNA GUSBP5-AS knockdown and overexpression, respectively, decreased and increased tube formation by EPCs in vivo (original magnification, ×200). (C) In vitro tube formation assay. Scale bar=100μm. (original magnification, ×100). LncRNA GUSBP5-AS knockdown and overexpression, respectively, decreased and increased tube number. ***P< .001. (D) Wound healing assay showing the effects of lncRNA GUSBP5-AS on EPC migration. Scale bar=100μm. (original magnification, ×100). LncRNA GUSBP5-AS knockdown and overexpression, respectively, decreased and increased cell migration in EPCs. **P< .01. (E) Effects of lncRNA GUSBP5-AS on cell invasion ability were analysed. Scale bar=100μm. (original magnification, ×200). Data are represented as mean ± SEM. ***P< .001. (F) Effect of lncRNA GUSBP5-AS on actin cytoskeleton structure in cultured EPCs. Cells were fixed, permeabilized, stained with rhodamine–phalloidin and DAPI, and visualized by confocal microscopy;…

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  Figure 3. GUSBP5-AS have a central role in proliferation. (A) Cell proliferation ability of GUSBP5-AS in lncRNA GUSBP5-AS knockdown and overexpression group and their corresponding NC groups was detected by CCK8 assay. *P< .05, **P< .01 compared to the LV3-NC group; #P < .05, ##P < .01 compared to the LV5-NC group. (B) The effect of GUSBP5-AS on EPC cycle was investigated by FCM cell cycle distribution test. (C) The effect of lncRNA GUSBP5-AS on cell apoptosis was measured with Annexin V-APC/7-AAD Apoptosis Detection Kit by FCM. Cell apoptosis was analysed. Data from 3 experiments are summarized. *P< .05, **P< .01 compared to the corresponding negative control group. Data are represented as mean ± SEM.

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  Figure 4. The up- and down- regulated mRNAs were filtrated using microarray analysis. (A) Heatmap analysis of dysregulated mRNAs in negative controls and GUSBP5-AS-overexpressing EPCs. Control group indicates LV5-NC group and treat for GUSBP5-AS-overexpressing EPC group. (B) Proportion of up- and down- regulated genes were filtered using microarray analysis in the GUSBP5-AS overexpression group compared with the LV5-NC group. Blue indicates up-regulated genes, and red for down-regulated genes. (C) Several biological process pathways were enriched. Up indicates the enrichment analysis of up-regulated genes, and down for down-regulated genes. (D) The regulatory network of lncRNA-miRNA-mRNA.

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  Figure 5. LncRNA GUSBP5-AS directly bound with miR-223-3p. (A) Bioinformatics analysis revealed GUSBP5-AS contains binding sequences complementary to the binding sites of miR-223-3p. (B) The luciferase reporter constructs containing WT-GUSBP5-AS or MU-GUSBP5-AS sequence. (C) Schematic graph of the constructed reporter plasmid containing putative. (D) Dual-luciferase reporter assay verified the targeting relationship between miR-223-3p and GUSBP5-AS. (E) The expression of miR-223-3p was measured by qRT-PCR after cells treated with GUSBP5-AS, GUSBP5-AS+miR-223-3p or miR-223-3p. (F) Effects of cotransfection of GUSBP5-AS and miR-223-3p on in vitro tube formation were analysed. Scale bar=100μm. (original magnification, ×100). (G) Effects of cotransfection of GUSBP5-AS and miR-223-3p on cell migration were detected by wound healing assay. Scale bar=100μm. (original magnification, ×100). (H) Effects of cotransfection of GUSBP5-AS and miR-223-3p on cell invasion were analysed by transwell cell invasion assay. Scale bar=100μm. (original magnification, ×100). **P < .01, ***P < .001 compared to the NC group, ###P < .001 compared to the GUABP5-AS treated group. Data are represented as mean ± SEM.

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  Figure 6. FOXO1 acted as the direct target of miR-223-3p. (A) Bioinformatics programs assay revealed complementary binding within miR-223-3p and FOXO1 3’UTR. (B) The luciferase reporter constructs containing WT-FOXO1 or MU-FOXO1 sequence. (C) Dual-luciferase reporter assay verified the targeting relationship between miR-223-3p and FOXO1. (D) Relative quantification of mRNA levels of FOXO1. Three independent experiments were performed. (E) Tube formation assay showing the angiogenesis ability of EPCs by FOXO1 knockdown and overexpression. Scale bar=200μm. (original magnification, ×100). FOXO1 knockdown and overexpression, respectively, repressed and enhanced capability of angiogenesis in EPCs. (F) Effects of FOXO1 on cell migration were analysed by wound healing assay. Scale bar=200μm. (original magnification, ×100). FOXO1 knockdown and overexpression, respectively, reduced and increased number of EPCs migrated. (G) Transwell invasion assay showed that FOXO1 knockdown and overexpression, respectively, inhibited and promoted the ability of cell invasion in EPCs. Scale bar=100μm. (original magnification, ×200). **P < .01, ***P < .001 compared to NC group. Data are represented as mean ± SEM.

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  Figure 7. The relationship between GUSBP5-AS, miR-223-3p and FOXO1 expression. (A–C) Spearman rank correlation analyses showed negative correlation with GUSBP5-AS and miR-223-3p in 15 cases of EPCs samples from DVT patients, and FOXO1 was negatively correlated with miR-223-3p expression, while GUSBP5-AS was positively correlated with FOXO1 expression. (D) miR-223-3p mimics and inhibitor, respectively, inhibited and enhanced the expression of GUSBP5-AS in EPCs. (E) GUSBP5-AS knockdown and overexpression, respectively, reduced and increased the mRNA expression levels of FOXO1 in EPCs. *P < .05, **P < .01 compared to the corresponding control group. Data are represented as mean ± SEM.

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  Figure 8. GUSBP5-AS regulates FGF2 and MMP2/9 expression through FOXO1/Akt pathway by sponging with miR-223-3p. (A) Effects of GUSBP5-AS on the regulation of the protein expression of FOXO1, p-Akt, MMP2, MMP9 and FGF2 in EPCs by western blot analysis. GUSBP5-AS knockdown and overexpression, respectively, reduced and increased these protein expression levels. (B) Western blot analysis of the protein expression levels of FOXO1, p-Akt, MMP2, MMP9 and FGF2 in EPCs after infected with NC, miR-223-3p inhibitor and mimics. The resulted showed that miR-223-3p inhibitor and mimics, respectively, promoted and inhibited these protein expression. *P < .05, **P < .01, ***P < .001 compared to the corresponding control group. Data are represented as mean ± SEM. (C) Schematic of the role of GUSBP5-AS-mediated effects in EPCs. Red arrow indicates inhibition and blue arrow indicates promotion.