Journal: Transplantation

Article Title: Renal Protection Mediated by Hypoxia Inducible Factor-1α Depends on Proangiogenesis Function of miR-21 by Targeting Thrombospondin 1

doi: 10.1097/TP.0000000000001501

Figure Lengend Snippet: miR-21 targeted TSP-1 and might contribute to hypoxia-induced tube formation in HUVECs. miR-21 was upregulated by 1% O 2 treatment for 24 hours in HUVECs (A), with decreased expression of TSP-1 mRNA (B) and protein (C). n = 3 per group, * P < 0.05 versus the normoxia group, ** P < 0.01 versus the normoxia group. D, Expression of miR-21 was suppressed by LNA anti-miR-21 effectively in hypoxic HUVEC. It was showed that LNA anti-miR-21 increased TSP-1 mRNA (E) and protein expression (F). G, Inhibition of miR-21 decreased capillary-like endothelial tube formation in hypoxic HUVECs, compared with the scrambled control. n = 4 per group, * P < 0.05 versus the scrambled control group. H, Luciferase reporter assays analysis confirmed an interaction between miR-21 and a 3′-UTR of TSP-1 mRNA. This interaction did not occur when action with negative control or TSP-1 3′-UTR mutant construct. n = 8 per group,* P < 0.05 versus control oligonucleotides or TSP-1 3′-UTR mutant construct.

Article Snippet: The membrane was blocked with 5% nonfat milk, followed by incubation with primary antibodies against HIF-1α (rabbit polyclonal, 1:1000, Novus, Littleton), TSP-1 (mouse monoclonal, 1:100, Pierce), GAPDH (rabbit polyclonal, 1:1000, Abcam), Histone H3 (mouse monoclonal, 1:1000, Cell Signaling Technology, Danvers, MA) overnight at 4°C.

Techniques: Expressing, Inhibition, Luciferase, Negative Control, Mutagenesis, Construct

Journal: Transplantation

Article Title: Renal Protection Mediated by Hypoxia Inducible Factor-1α Depends on Proangiogenesis Function of miR-21 by Targeting Thrombospondin 1

doi: 10.1097/TP.0000000000001501

Figure Lengend Snippet: The renal protective effect of HIF-1α induced by CoCl 2 was associated with improved angiogenesis, upregulation of VEGF and miR-21, and downregulation of TSP-1. A, Immunohistochemical staining of CD31 in the renal sections and the CD31 positive area was analysis. Representative photomicrographs, ×10 magnification, with brown color indicating positive staining. Bar = 50 μm. VEGF mRNA (B) and miR-21 (C) expression was increased by the CoCl 2 treatment, companied with decreased expression of TSP-1 mRNA (D) and protein (E) in the mice I/R kidneys. n = 6 per group, * P < 0.05 versus NS + I/R group, ** P < 0.01 versus Sham + I/R group.

Article Snippet: The membrane was blocked with 5% nonfat milk, followed by incubation with primary antibodies against HIF-1α (rabbit polyclonal, 1:1000, Novus, Littleton), TSP-1 (mouse monoclonal, 1:100, Pierce), GAPDH (rabbit polyclonal, 1:1000, Abcam), Histone H3 (mouse monoclonal, 1:1000, Cell Signaling Technology, Danvers, MA) overnight at 4°C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Transplantation

Article Title: Renal Protection Mediated by Hypoxia Inducible Factor-1α Depends on Proangiogenesis Function of miR-21 by Targeting Thrombospondin 1

doi: 10.1097/TP.0000000000001501

Figure Lengend Snippet: Knockdown of miR-21 attenuated HIF-1α protection against I/R, upregulated TSP-1, and decreased vascular density in mouse kidneys. A, LNA anti–miR-21, administered before CoCl 2 pretreatment inhibited miR-21 expression effectively in kidneys 24 hours after cobalt exposure. Knockdown of miR-21 in renal I/R mice exposed to CoCl 2 significantly increased serum creatinine (B) and worsened renal histological injury (C) 24 hours after I/R injury. Kidney sections were stained with periodic acid–Schiff and photographed at x20 magnification in the corticomedullary junction. D, E, TSP-1 real-time PCR and western blot showed that LNA anti-miR-21 increased TSP-1 mRNA and protein expression in mice kidneys 24 hours after I/R. F, As shown in representative images of miR-21 expression by ISH, TSP-1 and CD31 staining by immunohistochemistry in mouse kidney sections, increased miR-21 expression was significant in the vascular endothelial cells of anti–scrambled-treated mice besides in the renal tubular cells, accompanied with the decreased TSP-1 and CD31 positive staining. G, The semi-quantitative analysis shown anti-miR-21 treatment significantly restored renal TSP-1 abundance with reduced number of CD31-positive capillaries. H, TSP-1 expressed in the vascular endothelial cell which was marked by CD31 staining (the arrow). It was photographed at ×20 magnification with purple color indicating positive staining for miR-21 and brown color for TSP-1 and CD31.The arrow indicates vascular endothelial cells. n = 6 per group. Bar = 50 μm * P < 0.05 versus scrambled control group.

Article Snippet: The membrane was blocked with 5% nonfat milk, followed by incubation with primary antibodies against HIF-1α (rabbit polyclonal, 1:1000, Novus, Littleton), TSP-1 (mouse monoclonal, 1:100, Pierce), GAPDH (rabbit polyclonal, 1:1000, Abcam), Histone H3 (mouse monoclonal, 1:1000, Cell Signaling Technology, Danvers, MA) overnight at 4°C.

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry

Journal: Transplantation

Article Title: Renal Protection Mediated by Hypoxia Inducible Factor-1α Depends on Proangiogenesis Function of miR-21 by Targeting Thrombospondin 1

doi: 10.1097/TP.0000000000001501

Figure Lengend Snippet: Schema of the pathways underlying renal protection conferred by HIF-1α. HIF-1α could upregulate expression of hypoxia-related miRNAs including miR-21. MiR-21 further increased expression of HIF-1α in a positive feedback loop, reinforcing the protective effect of HIF-1α. HIF-1α induced angiogenesis by directly increasing VEGF or indirectly inhibiting antiangiogenesis target TSP-1 of miR-21. Other HIF-1α downstream genes, such as heme-oxygenase 1, erythropoietin, and other miR-21 target genes including programmed cell death protein 4 and PTEN may also contribute to renal protection. Vertical arrows: “↓” means decrease, “↑” means increase.

Article Snippet: The membrane was blocked with 5% nonfat milk, followed by incubation with primary antibodies against HIF-1α (rabbit polyclonal, 1:1000, Novus, Littleton), TSP-1 (mouse monoclonal, 1:100, Pierce), GAPDH (rabbit polyclonal, 1:1000, Abcam), Histone H3 (mouse monoclonal, 1:1000, Cell Signaling Technology, Danvers, MA) overnight at 4°C.

Techniques: Expressing