LncEGFL7OS regulates human angiogenesis by interacting with MAX at the EGFL7/miR-126 locus
- Tulane University, United States
- Boston Children’s Hospital, Harvard Medical School, United States
- Xavier University, United States
- University of Rochester School of Medicine and Dentistry, United States
- Oklahoma Medical Research Foundation, United States
Figures
Figure 1 with 2 supplements
lncRNA profiling in ECs.
(A) Hierarchy cluster analysis of lncRNA and mRNA expression data from five different cell lines. (B) Heatmap showing the top-50 enriched lncRNAs in three EC lines compared to the two non-EC lines. …
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Figure 1—source data 1
Figure 1D source data.
- https://doi.org/10.7554/eLife.40470.006
Figure 1—figure supplement 1
EC Marker staining of the EC cells used.
A (Upper panels). Uptake of DiI labeled Acetyl-LDL in the EC lines (HUVEC, HREC and HCEC); B (lower panels). Staining of the EC lines with antibody to EC marker vWF.
Figure 1—figure supplement 2
Functional encrichment analysis and tissue distribution of the EC-enriched lncRNAs.
(A) Functional enrichment analysis of EC-enriched lncRNAs and their associated genes. P-values are indicated. (B) Tissue distribution of the candidate lncRNAs from the top-50 EC-enriched lncRNA …
Figure 2 with 2 supplements
Expression, regulation and subcellular localization of lncEGFL7OS, as well as its regulation in DCM patients.
(A) Genomic organization of lncEGFL7OS and its host gene EGFL7/miR-126. Exons are shown in orange and the introns are shown in blue. Direction of gene transcription is indicated by arrows. Scale = 1 …
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Figure 2—source data 1
Figure 2 source data.
- https://doi.org/10.7554/eLife.40470.011
Figure 2—figure supplement 1
lncEGFL7OS RACE-PCR data and Real-time PCR data of ANP, PECAM1, EGFL7 and miR-126 in different tissues.
(A) Gel picture of RACE-PCR product of lncEGFL7OS from RACE-ready human placenta cDNA. Marker size was shown. (B) Expression of EGFL7B and EGFL7C by qRT-PCR in different human tissues. GAPDH served …
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Figure 2—figure supplement 1—source data 1
Figure 2—figure supplement 1 source data.
- https://doi.org/10.7554/eLife.40470.009
Figure 2—figure supplement 2
ChIP assay showing binding for the indicated factors to the promoter region (n = 3 each).
IgG was used as control. The positions for PCR primers are shown in Figure 2F. Size of the marker was shown.
Figure 3 with 2 supplements
Regulation of angiogenesis by lncEGFL7OS in vitro, ex vivo and in vivo.
(A) Decreased capillary tube formation at 7 days after lncEGFL7OS silencing in HUVECs in an EC-fibroblast co-culture assay. The capillaries are stained with PECAM-1 antibody. Scale bar equals to 500 …
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Figure 3—source data 1
Figure 3 source data.
- https://doi.org/10.7554/eLife.40470.016
Figure 3—figure supplement 1
Schemetics of lncEGFL7OS siRNA localization and siRNA knockdown efficiency.
(A) Schematics of the siRNA locations in lncEGFL7OS. (B) Silencing of lncEGFL7OS by siRNAs as shown by qRT-PCR (n = 3). ***, p<0.001.
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Figure 3—figure supplement 1—source data 1
Figure 3 source data.
- https://doi.org/10.7554/eLife.40470.014
Figure 3—figure supplement 2
Immunostaining of the Matrigel assay in Figure 3C.
(A) Single channel image of the Matrigel assay in Figure 3C. HUVEC cells were stained with human PECAM-1 antibody (Red) and mouse red blood cells were stained with mouse Ter-119 (Green) antibody. …
Figure 4 with 2 supplements
Regulation of EC proliferation and migration by lncEGFL7OS.
(A) Quantification of EC proliferation in response to VEGF-A as indicated by BrDU incorporation after lncEGFL7OS silencing (n = 3). (B) Representative ell cycle profile in ECs after lncEGFL7OS …
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Figure 4—source data 1
Figure 4 source data.
- https://doi.org/10.7554/eLife.40470.022
Figure 4—figure supplement 1
Quantification of TUNEL positive cells in ECs transfected siRNAs for lncEGFL7OS (n = 3).* p<0.05.
https://doi.org/10.7554/eLife.40470.018-
Figure 4—figure supplement 1—source data 1
Figure 4—figure supplement 1 source data.
- https://doi.org/10.7554/eLife.40470.019
Figure 4—figure supplement 2
Effect of lncEGFL7OS oeverexpression in angiogenesis.
(A) qRT-PCR showing overexpression of lncEGFL7OS in ECs infected with lncEGFL7OS expressing adenovirus. LacZ expression virus was used as control; (B) Representative images showing enhanced …
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Figure 4—figure supplement 2—source data 1
Figure 4—figure supplement 2 source data.
- https://doi.org/10.7554/eLife.40470.021
Figure 5 with 2 supplements
Regulation of EGFL7/miR-126 and angiogenic signaling by lncEGFL7OS.
(A) Expression of EGFL7 B and EGFL7C by qRT-PCR after lncEGFL7OS knockdown in ECs (n = 3). GAPDH served as normalization control. (B) Expression of miR-126 and miR-126* after lncEGFL7OS knockdown in …
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Figure 5—source data 1
Figure 5 source data.
- https://doi.org/10.7554/eLife.40470.027
Figure 5—figure supplement 1
Expression of EGFL7 protein by Western blot after lncEGFL7OS knockdown in ECs (n = 3).
β-Tubulin was used as a loading control.
Figure 5—figure supplement 2
Upregulation of miR-126 and EGFL7 by lncEGFL7OS oeverexpression.
(A) qRT-PCR showing upregulation of miR-126 expression in ECs infected with lncEGFL7OS expressing adenovirus. GFP expression virus was used as control (n = 3). ***p<0.001; (B) qRT-PCR showing …
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Figure 5—figure supplement 2—source data 1
Figure 5—figure supplement 2 source data.
- https://doi.org/10.7554/eLife.40470.026
Figure 6 with 1 supplement
lncEGFL7OS regulates EGFL7/miR-126 transcription by interaction with MAX transcription factor.
(A) Schematic EGFL7/miR-126 enhancer/promoter region. The boxed region is predicted by ENCODE to bind MAX and H3K27Ac. (B) RIP-PCR showing binding of MAX to lncEGFL7OS in ECs. Overexpression of …
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Figure 6—source data 1
Figure 6 source data.
- https://doi.org/10.7554/eLife.40470.031
Figure 6—figure supplement 1
Overexpression of lncEGFL7OS(F2+3) does not affect miR-126 and EGFL7B expression.
(A) ChIP-PCR showing no specific binding of MAX to a control region (primer1 two set in material and method section). Overexpression of lncEGFL7OS does not affect MAX binding to the region (n = 3). …
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Figure 6—figure supplement 1—source data 1
Figure 6—figure supplement 1 source data.
- https://doi.org/10.7554/eLife.40470.030
Figure 7
lncEGFL7OS-dependent MAX-regulated gene expression is locus dependent.
(A) ChIP-PCR showing specific binding of MAX to region 3 (as in Figure 6A). Silencing of lncEGFL7OS decreased MAX binding to the region (n = 3). *p<0.05; ***p<0.001. (B) ChIP-PCR showing specific …
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Figure 7—source data 1
Figure 7 source data.
- https://doi.org/10.7554/eLife.40470.033
Figure 8
Inhibition of angiogenesis by CRISPR-mediated targeting of the EGFL7/miR-126/lncEGFL7OS locus.
(A) Schematic locations of the sgRNAs in the EGFL7/miR-126/lncEGFL7OS genes. (B) Representative images showing sgRNA mediated repression of angiogenesis in an EC-fibroblast co-culture assay. The …
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Figure 8—source data 1
Figure 8 source data.
- https://doi.org/10.7554/eLife.40470.035
Figure 9
A model for lncEGFL7OS in human angiogenesis.
LncEGFL7OS is transcribed in the opposite strand of EGFL7/miR-126 gene under the control of an ETS transcription factors-regulated bidirectional promoter. In turn, lncEGFL7OS transcripts recruit …
Additional files
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Supplementary file 1
List of top-50 EC-enriched lncRNAs and their associated genes.
- https://doi.org/10.7554/eLife.40470.037
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Supplementary file 2
List of EC-enriched enhancer-like lncRNAs from the array.
- https://doi.org/10.7554/eLife.40470.038
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Supplementary file 3
List of the EC-enriched lncRNAs that have associated protein-coding genes within 10 kb, showing parallel or inverse expression pattern with their associated genes.
- https://doi.org/10.7554/eLife.40470.039
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Supplementary file 4
(A) CT values from the PCR using standard in vitro transcribed lncEGFL7OS RNA.
The RNA was harvested at 1.85*1011 copies per µl. After reverse transcription, 1 µl the cDNA was diluted at 103, 104, 105, 106 and 107 times, respectively, as templates to carry out Real-time PCR. The copy numbers were calculated based on the dilution folds. (B) The CT values and the log10 (Copy number) were used to establish the standard curve and formulation for copy number calculation. The Log10 (copy number) and CT value relation can be modeled as: Y = −0.4438*X + 16.15. R square is 0.9415. (C) The formulation in (B) was used to calculate the copy number per well of the HUVEC cell samples. Based on the calculation that each well has ~1600 cells, the copy number per cell was calculated.
- https://doi.org/10.7554/eLife.40470.040
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Supplementary file 5
LncEGFL7OS Stellaris FISH probes designed according to Stellaris FISH probe designer.
- https://doi.org/10.7554/eLife.40470.041
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Transparent reporting form
- https://doi.org/10.7554/eLife.40470.042
