16 research outputs found

    Successful overexpression of hsa-miR-146b-3p by plvx-hs-146b-3p with no detectable increase of hsa-miR-146b-5p.

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    <p>HeLa cells were transfected with the indicated plasmids (400 ng per well) and RNA was collected and extracted 24 h later. The expression of hsa-miR-146b-3p (A) and hsa-miR-146b-5p (B) was detected using qRT–PCR. The same experiments were done in Hep G2 cells (C) for hsa-miR-146b-3p; (D) for hsa-miR-146b-5p and HEK 293T cells (E) for hsa-miR-146b-3p; (F) for hsa-miR-146b-5p. Each graph shows the mean of three independent experiments that measured the relative expression levels (2<sup>−deltaCT</sup>) of the two miRNAs to the reference gene RNU48. Error bars represent SEMs. * means p value ≤0.05; ** means p value ≤0.01; *** means p value ≤0.001; ns means no significance.</p

    Design and generation of plasmids for the overexpression of microRNA* species.

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    <p>(A) Design of the stem-loop miRNA precursor. The mature sequences of miRNA* species were incorporated into the 3′ arm of the precursor, whereas the complementary sequences of the miRNA* species were incorporated into the 5′ arm. Note that the complementary sequences could either be completely complementary to the miRNA* species or could be modified by mutating several bases in its seed region, which were not complementary to the miRNA* species. (B) The designed hsa-miR-146b-3p stem-loop precursor. The red characters in the 3′ arm correspond with the sequences of hsa-miR-146b-3p. The 5′ arm sequences are the modified complementary sequences of hsa-miR-146b-3p, and the sequences above are hsa-miR-146b-5p, with blue characters denoting seed sequences and solid lines between them indicating the similarity of the two sequences. The dotted lines show the modified sites in the 5′ arm sequences and their corresponding nucleotides in hsa-miR-146b-3p sequences. (C) The uppermost oligo is the DNA insert of hsa-miR-146b-3p precursor obtained by annealing two single stranded DNA oligos with <i>Bam</i>H1 and <i>Eco</i>R1 sticky ends. The DNA insert was incorporated into plvx-shRNA2 between the recognition sites for the restriction enzymes <i>Bam</i>H1 and <i>Eco</i>R1. The representation of the vector was modified from an illustration in the instructions provided with the product.</p

    A Novel Vector-Based Method for Exclusive Overexpression of Star-Form MicroRNAs

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    <div><p>The roles of microRNAs (miRNAs) as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*), which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA) overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like natural miRNAs, the overexpressed hsa-miR-146b-3p inhibited target gene expression by 3′UTR seed pairing. Our demonstration that this method could overexpress two other miRNAs suggests that the approach should be broadly applicable. Our novel strategy opens the way for exclusively stable overexpression of miRNA* species and analyzing their unique functions both <em>in vitro</em> and <em>in vivo</em>.</p> </div

    plvx-hs-146b-3p overexpressed functional hsa-miR-146b-3p with no detectable activity change of hsa-miR-146b-5p.

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    <p>(A) Transcription products from plvx-hs-146b-3p had hsa-miR-146b-3p activities, without changing the activity of hsa-miR-146b-5p. HeLa cells were transfected with psicheck-ctrl (20 ng per well), psicheck-146b-3psensor (20 ng per well), or psicheck-146b-5psensor (20 ng per well), in combination with either plvx-ctrl (100 ng per well) or plvx-hs-146b-3p (100 ng per well), as indicated in the graph. (B) The repression of psicheck-146b-3psensor activity could be rescued using hsa-miR-146b-3p inhibitors. HeLa cells were transfected with psicheck-146b-3psensor (20 ng per well) in combination of plvx-ctrl (100 ng per well) or plvx-hs-146b-3p (100 ng per well) under the presentation of hsa-miR-146b-3p inhibitors (100 nM) or inhibitor negative controls (100 nM), as indicated in the graph. (C) Designed modified complementary sequences (designed anti-hsa-miR-146b-3p) could not repress psicheck-146b-3psensor activity as effectively as hsa-miR-146b-3p. HeLa cells were transfected with psicheck-146b-3psensor (20 ng per well) in combination with single stranded RNA mimics of negative control sequences (100 nM), designed anti-hsa-miR-146b-3p (100 nM), hsa-miR-146b-3p (100 nM), and hsa-miR-410 (100 nM), as indicated in the graph. The miRNA hsa-miR-410, which had no ability to repress psicheck-146b-3psensor activity, was used as a negative control miRNA. (D) The miRNA hsa-miR-146b-3p overexpressed by plvx-hs-146b-3p could repress the activity of bulged sensor of hsa-miR-146b-3p (pMIR-hs-146b-3p) and this repression was abolished by mutation of the seed sequences of the bulged sensor. HeLa cells were transfected with pMIR-hs-146b-3p (2 ng per well) or pMIR-hs-146b-3p-mut (2 ng per well), together with plvx-ctrl (400 ng per well) or plvx-hs-146b-3p (400 ng per well), as indicated in the graph. Renilla luciferase vector (5 ng per well) was delivered simultaneously as a transfection control. For all assays, protein was collected 24 h after transfection. Luciferase activity was quantified and expressed as relative luciferase activity. The data represent one of at least three independent experiments, each of which involved four replicates. Error bars represent SEMs. ** means p value ≤0.01; *** means p value ≤0.001; ns means no significance.</p

    MiR-125a Is a critical modulator for neutrophil development

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    <div><p>MicroRNAs are universal post-transcriptional regulators in genomes. They have the ability of buffering gene expressional programs, contributing to robustness of biological systems and playing important roles in development, physiology and diseases. Here, we identified a microRNA, miR-125a, as a positive regulator of granulopoiesis. <i>MiR125a</i> knockout mice show reduced infiltration of neutrophils in the lung and alleviated tissue destruction after endotoxin challenge as a consequence of decreased neutrophil numbers. Furthermore, we demonstrated that this significant reduction of neutrophils was due to impaired development of granulocyte precursors to mature neutrophils in an intrinsic manner. We showed that <i>Socs3</i>, a critical repressor for granulopoiesis, was a target of miR-125a. Overall, our study revealed a new microRNA regulating granulocyte development and supported a model in which miR-125a acted as a fine-tuner of granulopoiesis.</p></div

    MiR-125a regulates maturation of neutrophils by targeting <i>Socs3 in vivo</i>.

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    <p>(A) Flow cytometry analysis of GFP<sup>+</sup> bone marrow neutrophils after bone marrow transplantation of miR-125a<sup>-/-</sup> ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Bar graphs indicated numbers of GFP<sup>+</sup> neutrophils per femur and tibia. (B) Flow cytometry analysis of GFP<sup>+</sup> myeloid precursor cell populations after bone marrow transplantation of miR-125a<sup>-/-</sup> ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Plots shown here were previously gated on Lin<sup>-</sup>Sca-1<sup>-</sup>c-Kit<sup>+</sup> cells. Bar graphs indicated numbers of GFP<sup>+</sup> GMPs (upper) or CMPs (lower) per femur and tibia. (C) Flow cytometry analysis of neutrophils incorporating BrdU in bone marrow GFP<sup>+</sup> GMPs (upper) or CMPs (lower). Bar graphs indicate the mean fluorescence intencities of BrdU-incorporating GMPs (upper) or CMPs (lower). ns, none significant difference, *<i>P</i><0.05(Student’s <i>t</i>-test).</p

    <i>Socs3</i> ia a target of miR-125a.

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    <p>(A) Protein expression of SOCS3 in bone marrow neutrophils from <i>MiR125a</i><sup><i>+/+</i></sup> and <i>MiR125a</i><sup><i>-/-</i></sup> mice. Cell lysates were analyzed by immunoblot using SOCS3 antibody. (B) Schematic presentation of a potential miR-125a binding sites in the 3’UTR regions of Socs3. Sequences below indicate the mutant form of this site. (C) Luciferase reporter gene assay performed on 293T cells transfected with plasmids on which the luciferase reporter gene fused to the fragment of wild-type or mutant 3’UTRs of Socs3. Values were normalized to a firefly gene’s activity on the same construct (mean±s.d., n = 3). (D) The mRNA expression of Socs3 in sorted GFP<sup>+</sup> GMPs which were transduced with retrovirus of Socs3 shRNA or a control(Ctrl) shRNA. (E-G) 1000 GMPs were sorted from <i>MiR125a</i><sup><i>-/-</i></sup> bone marrow lin<sup>-</sup> cells which were transduced with retrovirus of Socs3 shRNA or a Ctrl shRNA and then cultivated in G-CSF containing methylcellulose media. Photographed CFUs (E), colony numbers (F) and cell number per CFUs (G) were shown. Representative data were from three independent experiments. <i>**P</i><0.01 (Student’s <i>t</i>-test).</p

    Decreased neutrophils in <i>MiR125a</i><sup><i>-/-</i></sup> mice.

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    <p>(A) Flow cytometry analysis of bone marrow (upper panel) and peripheral blood (lower panel). Neutrophils were stained with CD11b-Percp cy5.5 and Ly6G-APC. Bar graphs indicated numbers of neutrophils per femur. Values were represented as mean±s.d., n = 5 mice of each genotype. (B) Flow cytometry analysis of bone marrow neutrophils after bone marrow transplantation for 6 weeks. Bar graphs indicated total numbers of neutrophils. Values were represented as mean±s.d.,n = 5 mice of each genotype. (C) Morphological character of neutrophils in <i>MiR125a</i><sup><i>+/+</i></sup>and <i>MiR125a</i><sup><i>-/-</i></sup> mice. Peripheral blood (original magnification, x1000) of control and knockout mice were stained with Giemsa. (D) Expression of miR-125a during myeloid development (mean±s.d.,n = 3). HSC, hematopoietic stem cells; CMP, common myeloid progenitors; GMP, granulocyte–monocyte progenitors; MEP, megakaryocyte erythroid progenitors; BM-N, bonemarrow neutrophils. **<i>P</i><0.01, *<i>P</i><0.05(Student’s <i>t</i>-test).</p

    Decreased proliferation of immature neutrophils in <i>MiR125a</i><sup><i>-/-</i></sup> mice.

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    <p>(A) Flow cytometry analysis of three subpopulations in CD11b<sup>+</sup>Gr-1<sup>+</sup> neutrophils in bone marrow. Mature neutrophils (mNeu) indicate CD11b<sup>hi</sup> Gr-1<sup>hi</sup> cells. Immature neutrophils (imNeu) indicate CD11b<sup>low</sup>Gr-1<sup>hi</sup> cells and promyelocytes/myelocytes (pro/mye) indicate CD11b<sup>int</sup>Gr-1<sup>int</sup> cells. The bar graph shows the average numbers of these subpopulations in <i>MiR125a</i><sup><i>+/+</i></sup> and <i>MiR125a</i><sup><i>-/-</i></sup> mice. Values were represented as mean±s.d., n = 5 mice of each genotype. (B) Flow cytometry analysis of three populations of CD11b<sup>+</sup>Gr-1<sup>+</sup> neutrophils incorporating BrdU in bone marrow after in vivo pulsing BrdU for 72 hours. The bar graph indicates the average percentage of intensities of BrdU-incorporating cells (mean±s.d., n = 5). Ns, none significant difference, *<i>P</i><0.05, ***<i>P</i><0.001 (Student’s <i>t</i>-test).</p
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