Differential microRNA (miRNA) and target gene expression in young and aged lineage-negative bone marrow cells (lin−BMCs). Unsupervised hierarchical clustering of differentially expressed miRNAs (A) in lin− BMCs from old apolipoprotein E–deficient (apoE−/−) (AO) and wild-type (WO) as well as young apoE−/−(AY) and wild-type (WY) mice. Red indicates higher expression; green denotes lower expression. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) validation of miR-10A* and miR-21 (B) and their target Hmga2 expression (C) reveals similar reciprocal expression changes in young and aged lin−BMCs between microarray and qRT-PCR analyses. Regulation of Hmga2 by miR-10A* (D) and miR-21 (E) was confirmed by luciferase reporter and mutagenesis assays. human embryonic kidney cell line 293T cells were cotransfected with precursors of miR-10A*, miR-21, or miR-Ctr, renilla luciferase reporter constructs containing WT or 3 mutated 3′untranslated regions (UTRs) of Hmga2, and a firefly luciferase reporter control vector to normalize the transfection efficiency. Luciferase was measured 48 hours after transfection. The luciferase activities represent the Renilla/firefly luciferase ratios (mean±SD, n≥6).**P<0.01. WT indicates wild-type.
MicroRNA (miR)-10A* and miR-21 repress endogenous and exogenous Hmga2 expression in lineage-negative bone marrow cells (lin− BMCs). Young wild-type (WT) lin− BMCs were infected with lentivirus encoding miR-10A*, miR-21, or miR-Ctr alone or in combination with WT Hmga2 3′untranslated region (UTR) (WT 3′UTR) or mutant Hmga2 3′UTR (M3′UTR). The Hmga2 mRNA and protein expression were detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) (A) and Western blotting (B) and quantified by densitometry (C) after 48 hours and 72 hours, respectively. Aged WT lin− BMCs were infected with lentivirus encoding anti-miR-10A*, anti-miR-21, and miR-Ctr. The effects of miR-10A* and miR-21 on endogenous Hmga2 mRNA and protein expression were detected by qRT-PCR (D) and Western blotting (E and F). The blots are representatives of 3 independent experiments. Mutant 10A*-2 is mutant Hmga2 3′UTR disrupting binding for miR-10A* at binding sites 860 and 1548; mutant miR-10A* is mutant Hmga2 3′UTR for miR-10A* at binding sites 860 and 1504. qRT-PCR and densitometry data are presented as means±SD. *P<0.05, **P<0.01 vs corresponding controls (n≥3).
MicroRNA (MiR)-10A* and miR-21 regulate lineage negative bone marrow cell (lin−BMC) senescence, proliferation, and self-renewal through Hmga2 and p16Ink4a/p19Arf. Young and aged wild-type (WT) lin− BMCs were infected with lentivirus encoding miR-10A* and miR-21 sense sequences or their anti-miRs, respectively. The effects of miR-10A* and miR-21 overexpression and inhibition on the expression of Hmga2 and p16Ink4a/p19Arf (A), cell senescence as evidenced by senescence-associated β-galactosidase (SA-β-gal) staining (B), self-renewal potential as evaluated by the secondary(2°) colony forming ability in methylcellulose-based media (C, D), and cell proliferation as assessed by proliferating cell nuclear antigen (PCNA) staining and confirmed by 3-[4,5-dimethylthiazol-zyl]-2,5-dipheny-tetrazolium bromide (MTT) cell proliferation assay (E, F) are shown.*P<0.05, **P<0.01 (n≥3) relative to miR-Ctr. Western blots are representative of experiments performed at least in triplicate. Densitometry data for A are presented in Online Figure IVA–IVC.
MicroRNA (miR)-10A* and miR-21 expression regulates wound healing and angiogenesis in vitro. Young wild-type (WT) lineage-negative bone marrow cells (lin− BMCs) were infected with lentivirus encoding miR-Ctr, miR-10A*, or miR-21 (A, top), or aged WT lin− BMCs were infected with lentivirus encoding miR-Ctr, anti-miR-10A*, and anti-miR-21(A, bottom). Wound closure was assessed and quantified by the relative migration distance 24 hours after performing a scratch in confluent monolayer of cells (n=3; B). The effects of miR-10A* and miR-21 in young (C, top) and aged (C, bottom) lin− BMCs to form capillary tubes in Matrigel assay were determined 10 hours after seeding. The images are representatives of at least 3 independent experiments. Relative tube lengths from 5 microscopic fields are shown in (D). **P<0.01 relative to miR-Ctr.
Hmga2 regulates lineage negative bone marrow cell (lin− BMC) senescence, self-renewal potential, proliferation, and vascular tube formation via activation of p16Ink4a/p19Arf. ShRNA-induced knockdown and wild-type (WT) Hmga2 cDNA or Hmga2 open reading frame (ORF) with 3′ untranslated region (UTR) deletion (Hmga2-3′del)-induced overexpression of Hmga2 result in opposite changes in Hmga2 and p16Ink4a/p19Arf mRNA and protein expression in young and aged lin− BMCs, respectively (A). ShRNA-induced Hmga2 knockdown in young lin− BMCs results in increased senescence-associated β-galactosidase (SA-β-gal) staining (B); decreased self-renewal potential by 2° colony forming units (C); decreased proliferation by proliferating cell nuclear antigen staining and 3-[4,5-dimethylthiazol-zyl]-2,5-dipheny-tetrazolium bromide (MTT) assay (D) and decreased capillary tube formation (E) as compared with shRNA control. Overexpression of Hmga2 by Hmga2-3′del transfection decreases SA-β-gal staining and enhances self-renewal potential (F), increases cell proliferation by MTT assay (G), and improves capillary tube formation (H) in aged lin− BMCs, whereas WT Hmga2 overexpression only transiently improved self-renewal potential and cell proliferation. Data are presented as mean±SD (n=3). *P<0.05, **P<0.01 (n≥3). Western blots are representative of experiments performed at least in triplicate. Densitometry data for western blotting (B) are shown in Online Figure VI.
Overexpression of Hmga2 lacking a 3′ untranslated region (UTR) rescues the effects of microRNA (miR)-10A* and miR-21 overexpression. Coinfection of the lentivirus encoding miR-10A*, miR-21, or miR-Ctr together with Hmga2 open reading frame (ORF) with 3′ UTR deletion (Hmga2-3′del), but not wild-type (WT) Hmga2, in young lineage-negative bone marrow cells (lin− BMCs) results in upregulation of Hmga2 and downregulation of p16Ink4a/p19Arf (A–D). Cotransfection of Hmga2-3′del with miR-10A* or miR-21, but not WT Hmga2, rescues the effects of miR-10A* or miR-21 on senescence-associated β-galactosidase (SA-β-gal) staining (E) and vascular tube formation as shown by relative tube length (F).*P<0.05, **P<0.01 for Hmga2 or Hmga2-3′del-treated groups relative to control. Western blots are representative of experiments performed at least in triplicate. Relative densitometry data (mean±SD) are from 3 independent experiments normalized with GAPDH.
MicroRNA (miR)-10A* and miR-21 regulate angiogenesis in vivo. Matrigel (250 μL per plug) was mixed with 1.3×106 young or aged lineage negative bone marrow cells (lin− BMCs) transduced with lentivirus coding for miR-10A*, miR-21, and ShRNA-mediated Hmga2 silencing (shRNA-Hmga2), or their anti-miRs, and Hmga2 open reading frame (ORF) with 3′ untranslated region (UTR) deletion (Hmga2-3′del) miR-Ctr, respectively, and was injected subcutaneously into C57BL/6J mice (n=3). Noninfected or control infected lin− BMCs from young and aged mice were also included. After 11 days, Matrigel plugs were removed, and capillary tube formation was detected by confocal microscopy. Representative pictures of indicated groups are shown in (A) and (B) (×100 magnification). Quantitative analysis of tube formation in Matrigel plugs is shown in (C–E). Data represent means±SD.*P<0.05 and**P<0.01 vs control group. The capillaries in green and red are newly formed vessels. Red fluorescence indicates blood vessels stained by DiL, green fluorescence indicates green fluorescent protein (GFP)-positive infected cells, double-labeling indicates capillaries derived from the injection of GFP-positive lentiviral transduced lin− BMCs.
MicroRNA (miR)-21 regulates neovascularization in hindlimb ischemia model. Reperfusion in the ischemic hindlimb (A and B) and neovascularization (C) were improved when young endothelial progenitor cells (EPCs) infected with lentivirus coding for miR-Ctr or aged EPCs transduced with scrambled anti-miRNA control (anti-miR-Ctr), anti-miR-21 or Hmga2-3′ deletion (Hmga2 3′del) were injected, as compared with young EPCs infected with lentivirus coding for miR-21 or shRNA for Hmga2 or aged cells transduced with anti-miR-Ctr. Laser Doppler perfusion imaging was obtained at 0, 7, and 21 days after operation, and recovery of perfusion was assessed. **P<0.01 vs miR-Ctr (n=4 for each group).
Differential microRNA (miRNA) and target gene expression in young and aged lineage-negative bone marrow cells (lin−BMCs). Unsupervised hierarchical clustering of differentially expressed miRNAs (A) in lin− BMCs from old apolipoprotein E–deficient (apoE−/−) (AO) and wild-type (WO) as well as young apoE−/−(AY) and wild-type (WY) mice. Red indicates higher expression; green denotes lower expression. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) validation of miR-10A* and miR-21 (B) and their target Hmga2 expression (C) reveals similar reciprocal expression changes in young and aged lin−BMCs between microarray and qRT-PCR analyses. Regulation of Hmga2 by miR-10A* (D) and miR-21 (E) was confirmed by luciferase reporter and mutagenesis assays. human embryonic kidney cell line 293T cells were cotransfected with precursors of miR-10A*, miR-21, or miR-Ctr, renilla luciferase reporter constructs containing WT or 3 mutated 3′untranslated regions (UTRs) of Hmga2, and a firefly luciferase reporter control vector to normalize the transfection efficiency. Luciferase was measured 48 hours after transfection. The luciferase activities represent the Renilla/firefly luciferase ratios (mean±SD, n≥6).**P<0.01. WT indicates wild-type.
