LINC00659 exacerbates endothelial progenitor cell dysfunction in deep vein thrombosis of the lower extremities by activating DNMT3A-mediated FGF1 promoter methylation

Figures

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  Fig. 1. LINC00659 is upregulated in IVC tissues and peripheral blood of patients with LEDVT A Volcano plot of differentially expressed lncRNAs in 6 human varicose vein tissues and 6 normal adjacent vein tissues in GSE51260 dataset. B RT-qPCR was applied to check LINC00659 level in the IVC tissues of patients with LEDVT (n = 15) and healthy controls (n = 15). C RT-qPCR detection of LINC00659 level in peripheral blood of patients with LEDVT (n = 15) and healthy controls (n = 15). D EPCs showed a cobblestone like morphology on day 3 after the first passage (n = 6), Scale bar = 25 μm. E Dual fluorescence labeling method of Dil-Ac-LDL/FITC-UAE-1 evaluation of the physiological behaviors of the EPCs (n = 4), Scale bar = 25 μm. F Immunofluorescence evaluation of CD34 and CD133 expression in EPCs (n = 4), Scale bar = 25 μm. G: RT-qPCR detection of LINC00659 level in EPCs isolated from patients with LEDVT and healthy controls (n = 6). Each experiment was repeated for 3 times. **P < 0.01, **P < 0.01 vs Control group

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  Fig. 2. LINC00659 inhibits the proliferation, migration, and angiogenesis ability of EPCs. EPCs were transfected with pcDNA-LINC00659 or LINC00659 siRNA alone for 24 h. A RT-qPCR detection of the LINC00659 expression level of EPCs transfected with pcDNA-LINC00659 or LINC00659 siRNA (n = 6). B EPC, transfected with pcDNA-LINC00659 or LINC00659 siRNA, proliferation was checked by CCK-8 assay (n = 6). C Transwell assay was applied to test the effects of pcDNA-LINC00659 or LINC00659 siRNA transfected on EPC migration (n = 6). D Tube formation assay was applied to test the effects of pcDNA-LINC00659 or LINC00659 siRNA transfected on EPC angiogenesis (n = 6). E–F Effect of pcDNA-LINC00659 or LINC00659 siRNA transfected on actin cytoskeleton structure in cultured EPCs (n = 4). Each experiment was repeated for 3 times. **P < 0.01

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  Fig. 3. LINC00659 upregulated EIF4A3 expression. A The consensus sequence of EIF4A3 motif, and the possible binding sites of LINC00659 and EIF4A3. B The relationship between LINC00659 and EIF4A3 in EPCs (n = 6). C Luciferase reporter assay investigated whether LINC00659 could bind to the EIF4A3 promoter at the putative sites (n = 6). D The enrichment of LINC00659 in the EIF4A3 precipitate was determined by RIP (n = 6). E RT-qPCR displayed the EIF4A3 mRNA expression with pcDNA-LINC00659 or LINC00659 siRNA transfection (n = 6). F Western blotting displayed the EIF4A3 protein level with pcDNA-LINC00659 or LINC00659 siRNA transfection (n = 6). G Western blotting detection of EIF4A3 level in EPCs isolated from patients with LEDVT and healthy controls (n = 6). Each experiment was repeated for 3 times. ***P < 0.001, **P < 0.01.

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  Fig. 4. EIF4A3 overexpression reversed the promoting effect of LINC00659 interference on EPCs migration and angiogenesis. LINC00659 siRNA was transfected alone or together with pcDNA-EIF4A3 into EPCs for 24 h. A Western blotting was employed to determine the levels of EIF4A3 in EPCs transfected with LINC00659 siRNA or/and pcDNA-EIF4A3 (n = 6). B The cell proliferation of EPCs transfected with LINC00659 siRNA or/and pcDNA-EIF4A3 was checked by CCK-8 assay (n = 6). C Transwell detection of the cell migration of EPCs transfected with LINC00659 siRNA or/and pcDNA-EIF4A3 (n = 6). D Tube formation assay was applied to test the effects of LINC00659 siRNA or/and pcDNA-EIF4A3 on EPC angiogenesis (n = 6). E–F Effect of LINC00659 siRNA or/and pcDNA-EIF4A3 on actin cytoskeleton structure in cultured EPCs (n = 4). Each experiment was repeated for 3 times. *P < 0.05, **P < 0.01

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  Fig. 5. EIF4A3 recruits DNMT3A to the FGF1 promoter region and represses FGF1 expression. A Co-IP was employed to check the interaction between EIF4A3 and DNMT3A (n = 6). B The enrichment of DNMT3A in the FGF1 promoter region was tested by ChIP assay (n = 6). C Western blotting showed the FGF1 protein expression with LINC00659 siRNA or/and pcDNA-EIF4A3 transfection (n = 6). D Methylation levels of the FGF1 promoter in EPCs were checked by MSP-PCR (n = 6). E Western blotting was applied to test the protein level of FGF1 in EPCs (n = 6). F Western blotting detection of FGF1 level in EPCs isolated from patients with LEDVT and healthy controls (n = 6). SGI-1027: DNMT3A inhibitor (8 µM). Each experiment was repeated for 3 times. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 6. Interference with FGF1 reversed the facilitative effect of LINC00659 inhibition on endothelial progenitor cell migration and angiogenesis. LINC00659 siRNA and FGF1 siRNA were transfected individually or together into EPCs. A Western blotting was applied to determine the levels of EIF4A3 in EPCs transfected with FGF1 siRNA or/and LINC00659 siRNA (n = 6). B The cell proliferation of EPCs transfected with FGF1 siRNA or/and LINC00659 siRNA was tested by CCK-8 assay (n = 6). C Transwell detection of the cell migration of EPCs transfected with FGF1 siRNA or/and LINC00659 siRNA (n = 6). D Tube formation assay was employed to check the effects of LINC00659 siRNA or/and FGF1 siRNA on EPC angiogenesis (n = 6). E-F Effect of LINC00659 siRNA or/and FGF1 siRNA on actin cytoskeleton structure in cultured EPCs (n = 4). Each experiment was repeated for 3 times. **P < 0.01.

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  Fig. 7. Knockdown of LINC00659 alleviates LEDVT in mice. A In vivo angiogenesis was assessed on day 7 after subcutaneous injection of LINC00659 shRNA lentivirus into mice (n = 10). B HE staining was applied to check IVC thrombosis pathological changes (n = 10). C The ratio of thrombus tissue weight to length in mice of each group (n = 10). D Leukocyte numbers in thrombi of mice in each group. E The level of LINC00659 in IVC was checked by RT-qPCR (n = 10). F–H The protein levels of EIF4A3 and FGF1 in IVC were tested by Western blotting (n = 10). *P < 0.05, **P < 0.01