Characterizing miRNA mediated regulation of proliferation

textCell proliferation is a fundamental biological process, and the ability of human cells to transition from a quiescent to proliferative state is essential for tissue homeostasis. Most cells in eukaryotic organisms are in a quiescent state, but on appropriate physiological or pathological stimuli, many types of somatic cells may exit quiescence, re-enter the cell cycle and begin to proliferate. The ability of cells to remain viable while quiescent, exit quiescence and re-enter into the cell cycle is the basis for varied physiological processes such as wound healing, lymphocyte activation and hepatocyte regeneration, but is also a hallmark of cancer. The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. Our understanding of miRNA biology has significantly improved, but the miRNA regulatory networks that govern cell proliferation are still largely unknown. We characterized a miR-22 Myc network that mediates proliferation through regulation of the interferon response and multiple cell cycle arrest genes. We identified several cell cycle arrest genes that regulate the effects of the tumor suppressor p53 as direct targets of miR-22, and discovered that miR-22 suppresses interferon gene expression. We go on to show that miR-22 is activated by the transcription factor Myc as quiescent cells enter proliferation, and that miR-22 represses the Myc transcriptional repressor MXD4, mediating a feed forward loop to elevate Myc expression levels. To more effectively determine miRNA targets, we utilized a combination of RNA-induced silencing complex immunoprecipitations and gene expression profiling. Using this approach for miR-191, we constructed an extensive transcriptome wide miR-191 target set. We show that miR-191 regulates proliferation, and targets multiple proto-oncogenes, including CDK9, NOTCH2, and RPS6KA3. Recent advances in determining miRNA targetomes have revealed widespread non-canonical miRNA-target pairing. We experimentally identified the transcriptome wide targets of miR-503, miR-103, and miR-494, and observed evidence of non-canonical target pairing for these miRNAs. We went on to confirm that miR-503 requires pairing outside of the canonical 5' seed region to directly target the oncogene DDHD2. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis.Cellular and Molecular Biolog

miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing

The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation. Results: Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5' seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis. Conclusions: Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2.National Institutes of Health CA130075Cancer Prevention and Research Institute of Texas RP120194Cellular and Molecular Biolog

Author Correction: Transcriptomic and cellular decoding of regional brain vulnerability to neurogenetic disorders

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Transcriptomic and cellular decoding of regional brain vulnerability to neurogenetic disorders

Abstract: Neurodevelopmental disorders have a heritable component and are associated with region specific alterations in brain anatomy. However, it is unclear how genetic risks for neurodevelopmental disorders are translated into spatially patterned brain vulnerabilities. Here, we integrated cortical neuroimaging data from patients with neurodevelopmental disorders caused by genomic copy number variations (CNVs) and gene expression data from healthy subjects. For each of the six investigated disorders, we show that spatial patterns of cortical anatomy changes in youth are correlated with cortical spatial expression of CNV genes in neurotypical adults. By transforming normative bulk-tissue cortical expression data into cell-type expression maps, we link anatomical change maps in each analysed disorder to specific cell classes as well as the CNV-region genes they express. Our findings reveal organizing principles that regulate the mapping of genetic risks onto regional brain changes in neurogenetic disorders. Our findings will enable screening for candidate molecular mechanisms from readily available neuroimaging data

Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map.

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.The study was supported by the Paul G. Allen Foundation Distinguished Investigator Program (E.M.U. and D.H.R.), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (D.H.R., D.G. and G. C.), BRAIN initiative (1U01 MH105991 to D.G.) and National Institute of Health (1R01 MH109912 to D.G.; P01NS08351 to D.H.R.), National Institute of Health Research and the European Union Seventh Framework (to P.H.), NINDS Informatics Center for Neurogenetics and Neurogenomics (P30 NS062691 to G.C.), Wellcome Trust core support (M.H., O.A.B.), European Research Council (281961 to M.G.H.), Fonds Wetenschappelijk Onderzoek (G066715N and 1523014N to M.G.H.), Stichting Alzheimer Onderzoek (S#16025 to M.G.H.) and VIB Institutional Support and Tech Watch funding (to M.G.H.), Howard Hughes Medical Institute and the Wellcome Trust (to D.H.R.)

Experimental setup used to profile RISC association and repression of gene expression.

<p>Experimental setup used to profile RISC association and repression of gene expression.</p

MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes

<div><p>miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191’s regulation of primary human fibroblast proliferation.</p></div

miR-191 directly targets multiple proto-oncogenes.

<p>(A) Enriched Gene Ontology terms for the experimentally identified set of miR-191 targets. BP: Biological process. Numbers indicate gene counts. (B) Luciferase reporter assays showed miR-191 directly targets the 3’ UTRs of the genes indicated on the X-axis. The Y-axis denotes relative luciferase units from miR-191 transfected HEK293 cells normalized to Control siRNA transfected cells. Purple bars: 3’ UTRs with the putative miR-191 target site entirely deleted. Green bars: Intact 3’ UTRs. P-values were estimated by Student’s one tailed t-test comparing miR-191 normalized to Control siRNA luciferase activity with intact 3’ UTRs to normalized luciferase activity with miR-191 target site deleted 3’ UTRs. (C) miR-191 transfection significantly decreased <i>AGO2</i>, <i>BCL2</i>, <i>CDK6</i>, <i>CDK9</i>, <i>NOTCH2</i>, and <i>RPS6KA3</i> transcript levels in fibroblasts. Fold changes are indicated on the Y-axis relative to the Control siRNA transfection. GAPDH was used to normalize input RNA levels. P-values were estimated by Student’s one tailed t-test comparing normalized transcript levels following miR-191 transfection to normalized transcript levels following Control siRNA transfection. (D) miR-191 transfection in fibroblasts decreased protein expression of CDK9, NOTCH2, and RPS6KA3 compared to Control siRNA transfection. Band intensities were quantified, normalized to GAPDH, and shown relative to the Control siRNA. For B and C, bars indicate the mean, and error bars denote ± SD, n = 6 and 3 respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p

miR-191 represses proliferation.

<p>(A) miR-191 transfection reduces the rate of cell growth. Average cell number relative to 0 hr following miR-191 or control siRNA transfection is shown for each time point indicated. Error bars denote ± SD, n = 4 (independent biological replicates). P-values were calculated by Student’s t-test comparing cell numbers following miR-191 transfection to cell numbers following Control siRNA transfection at each time point. (B) miR-191 transfection represses proliferation. The Y-axis indicates the relative percentage of cells expressing Ki67. Bars are the mean percentage of cells expressing Ki67 relative to Control RNA 2, and error bars denote ± SD, n = 3. (C) miR-191 transfection slows progression through the cell cycle. Cell-cycle profiles of transiently transfected fibroblasts following treatment with nocodazole. Representative histograms shown are the median for each treatment of 3 biological replicates. The Y-axis denotes cell number and the X-axis DNA content. Numbers in each histogram indicate percentage of cells in G1 or G2. For the bar graph, the Y-axis denotes the percentage of cells found in each stage of the cell cycle. Error bars indicate ± SD, n = 3. (D) Inhibition of miR-191 increases cell growth in fibroblasts serum starved into quiescence. Average cell number relative to 0 hours post transfection of an LNA targeting miR-191 or a LNA negative control is shown for each time point indicated. Error bars denote ± SD, n = 6. P-values were estimated by Student’s one tailed t-test comparing cell numbers following miR-191 inhibitor transfection to cell numbers following the Control inhibitor transfection at each time point. For B, and C, P-values were calculated by Student’s one tailed t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p