Gradual Rarefaction of Hematopoietic Precursors and Atrophy in a Depleted microRNA 29a, b and c Environment

<div><p>Background</p><p>The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood.</p><p>Results and Significance</p><p>Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out – miR29ab1b2c – has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow.</p></div

Histology of the spleen showing reduction of the myeloid lineage in the knockout versus the wild type.

<p>Histology of the spleen showing reduction of the myeloid lineage in the knockout versus the wild type.</p

Colony forming assay on 10 months old miR29ab1 knockout bone marrows (n = 8; 4 ko + 4 wt; p = 0.006).

<p>Colony forming assay on 10 months old miR29ab1 knockout bone marrows (n = 8; 4 ko + 4 wt; p = 0.006).</p

Colony forming assay on 2 months old miR29ab1 knockout bone marrows (n = 4; 2 WT and 2 KO; p = 0.04).

<p>Colony forming assay on 2 months old miR29ab1 knockout bone marrows (n = 4; 2 WT and 2 KO; p = 0.04).</p

Colony forming assay on 6 months old miR29ab1 knockout bone marrows (n = 8; 4 ko + 4 wt; p = 0.01).

<p>Colony forming assay on 6 months old miR29ab1 knockout bone marrows (n = 8; 4 ko + 4 wt; p = 0.01).</p

Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).

<p>Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).</p

Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).

<p>Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).</p

Kaplan-Meyer survival chart for 29ab1 ko mice.

<p>Kaplan-Meyer survival chart for 29ab1 ko mice.</p

Upregulation of KLF4 in miR-29 null lungs.

<p><b>(A&B)</b> Double IF staining of KLF4 and α-SMA; reduced vasculature α-SMA staining (green) is associated with increased KLF4 staining in the nucleus of cells associated with vessel walls (white arrowheads in <b>D</b>). <b>(C&D)</b> are enlarged images of highlighted areas of <b>A&B</b>. Images are representative of four littermate-matched WT/DKO pairs. <b>(E)</b> Levels of Klf4 mRNA in lungs of wild type and DKO mice (n = 3). <b>(F)</b> Western blot of KLF4 of whole lung protein extracts from three pairs of littermate-matched WT and DKO mice. <b>(G)</b> Densitometric analyses of the blot are presented as relative ratios of KLF4 /GAPDH. Ratio of WT lungs is arbitrarily presented as 1. Data are mean ± SEM; Student’s <i>t</i> test, ** P<0.01.</p

miR-29 promotes the expression of contractile SMC markers.

<p><b>(A)</b> qRT-PCR results of α-SMA and CNN1 mRNAs in PASMCs in which the level of miR-29 is either elevated by miR-29 mimic or knocked down by miR-29 antisense LNA oligos (n = 3); <b>(B)</b> mRNA levels of contractile SMC markers in IMR-90 cells, in which miR-29 is knocked down (n = 3, Affymetrix array data); <b>(C)</b> Levels of α-SMA protein in IMR-90 cells, in which endogenous miR-29 is knocked down. Densitometric analyses of the blot are presented as relative ratios of α-SMA /GAPDH. Ratio of the control is arbitrarily presented as 1. Fold changes of ratio are indicated beneath the corresponding bands. Data are mean ± SEM; Student’s <i>t</i> test, * P<0.05; ** P<0.01.</p