Playing a central role in the maintenance of hemostasis as well as in thrombotic disorders, platelets contain a relatively diverse messenger RNA (mRNA) transcriptome as well as functional ...mRNA-regulatory microRNAs, suggesting that platelet mRNAs may be regulated by microRNAs. Here, we elucidated the complete repertoire and features of human platelet microRNAs by high-throughput sequencing. More than 492 different mature microRNAs were detected in human platelets, whereas the list of known human microRNAs was expanded further by the discovery of 40 novel microRNA sequences. As in nucleated cells, platelet microRNAs bear signs of post-transcriptional modifications, mainly terminal adenylation and uridylation. In vitro enzymatic assays demonstrated the ability of human platelets to uridylate microRNAs, which correlated with the presence of the uridyltransferase enzyme TUT4. We also detected numerous microRNA isoforms (isomiRs) resulting from imprecise Drosha and/or Dicer processing, in some cases more frequently than the reference microRNA sequence, including 5' shifted isomiRs with redirected mRNA targeting abilities. This study unveils the existence of a relatively diverse and complex microRNA repertoire in human platelets, and represents a mandatory step towards elucidating the intraplatelet and extraplatelet role, function and importance of platelet microRNAs.
While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning ...transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.
The osteoblastic and adipocytic lineages arise from mesenchymal stem cells (MSCs), but few regulators of self-renewal and early cell-fate decisions are known. Here, we show that the Hippo pathway ...effector YAP1 is a direct target of SOX2 and can compensate for the self-renewal defect caused by SOX2 inactivation in osteoprogenitors and MSCs. Osteogenesis is blocked by high SOX2 or YAP1, accelerated by depletion of either one, and the inhibition of osteogenesis by SOX2 requires YAP1. SOX2 favors adipogenesis and induces PPARγ, but adipogenesis can only occur with moderate levels of YAP1. YAP1 induction by SOX2 is restrained in adipogenesis, and both YAP1 overexpression and depletion inhibit the process. YAP1 binds β-catenin and directly induces the Wnt antagonist Dkk1 to dampen pro-osteogenic Wnt signals. We demonstrate a Hippo-independent regulation of YAP1 by SOX2 that cooperatively antagonizes Wnt/β-catenin signals and regulates PPARγ to determine osteogenic or adipocytic fates.
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•SOX2 induces YAP1, the Hippo effector, in osteoprogenitors and MSCs•Loss of self-renewal caused by SOX2 depletion can be rescued by transgenic YAP1•SOX2 and YAP1 inhibit osteogenic differentiation by antagonizing Wnt signaling•High SOX2 and moderate YAP1 expression favors adipogenesis by induction of PPARγ
Little is known about the regulators of self-renewal in mesenchymal stem cells and early commitment to the reciprocal bone or fat lineages. Lim, Mansukhani, and colleagues demonstrate that the stem cell factor SOX2 directly regulates YAP1 to maintain self-renewal and block formation of bone lineage cells by inhibiting the Wnt pathway. SOX2 tips the balance toward adipogenesis at the expense of bone-forming cells. YAP1 also affects adipogenesis, and its depletion derepresses Wnt signaling to prevent adipogenesis while enhancing osteogenesis.
Abstract Lack of affordable technologies for delivering microRNAs and siRNAs into cells on a large scale has hindered our efforts to rapidly parse through hundreds of dysregulated genes/microRNAs in ...order to identify drivers of complex diseases. The instability and polyanionic nature of naked microRNAs impede efficient cellular uptake and reduce half-life. Viral delivery requires cloning, microRNA mimics/inhibitors require costly modifications, and both require toxic lipofection or electroporation. To address these challenges, we developed a robust method for delivering unmodified microRNAs into cells on cysteamine-functionalized gold nanoparticles (AuNPs). We validated our method in two different tumor models and found that the best formulation of miR 1 -AuNP 10 -S-PEG 0.5 had the highest payload (10–20 fold higher than lipofection), lowest toxicity (98% of cell viability following treatment), efficient uptake (96% of cells took it), fastest endosomal escape and increased half-lives (at least 5 days) impacting cell proliferation and patterns of target gene expression.
Annexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of ...breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s).
By mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways.
Our in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1.
Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.
Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures ...implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene-pachytene spermatocyte block and male sterility defect observed in MILI null mice. In Gasz null testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI null testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.
Significance Modulation of X-linked gene expression is essential in organisms with XX females and XY males. Various strategies for global regulation of X chromosomes have been proposed, but all ...require highly selective recognition of X chromatin. How this is achieved is not understood. The siRNA pathway contributes to X recognition in a well-studied Drosophila model. We now show that ectopic production of siRNA from a repetitive sequence that is limited to the X chromosome also promotes X recognition. Differential activities of X-linked repeats suggest a control region model, in which siRNA produced by a few repeats acts on widely distributed X-linked target sequences to promote selective recognition, and modification, of a single chromosome.
Highly differentiated sex chromosomes create a lethal imbalance in gene expression in one sex. To accommodate hemizygosity of the X chromosome in male fruit flies, expression of X-linked genes increases twofold. This is achieved by the male- specific lethal (MSL) complex, which modifies chromatin to increase expression. Mutations that disrupt the X localization of this complex decrease the expression of X-linked genes and reduce male survival. The mechanism that restricts the MSL complex to X chromatin is not understood. We recently reported that the siRNA pathway contributes to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm ³ satellite related repeats (1.688 X repeats) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. We tested RNA from these repeats for a role in dosage compensation and found that ectopic expression of single-stranded RNAs from 1.688 X repeats enhanced the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688 X repeat generated abundant siRNA and dramatically increased male survival. Consistent with improved survival, X localization of the MSL complex was largely restored in these males. The striking distribution of 1.688 X repeats, which are nearly exclusive to the X chromosome, suggests that these are cis -acting elements contributing to identification of X chromatin.
According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells ...develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34−CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34−CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
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•Human NK cells, ILC2s, and ILC3s develop from tonsil CD34−CD117+ ILC precursors•A CD56+ subset of CD34−CD117+ precursors generates NK cells and ILC3s but not ILC2s•Human ILC development is distinct from the established murine model
Human innate lymphoid cells (ILCs) develop from tissue-resident ILC precursors (ILCPs) in secondary lymphoid tissues. Here, Chen et al. describe a model of human ILC development in tonsils in which NK cells and group 3 ILCs derive from a CD56+ subset of ILCPs that cannot differentiate into group 2 ILCs.
Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system ...to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.
MicroRNAs (miRNA) regulate complex patterns of gene expression, and the relevance of altered miRNA expression to ovarian cancer remains to be elucidated. By comprehensively profiling expression of ...miRNAs and mRNAs in serous ovarian tumors and cell lines and normal ovarian surface epithelium, we identified hundreds of potential miRNA-mRNA targeting associations underlying cancer. Functional overexpression of miR-31, the most underexpressed miRNA in serous ovarian cancer, repressed predicted miR-31 gene targets including the cell cycle regulator E2F2. MIR31 and CDKN2A, which encode p14(ARF) and p16(INK4A), are located at 9p21.3, a genomic region commonly deleted in ovarian and other cancers. p14(ARF) promotes p53 activity, and E2F2 overexpression in p53 wild-type cells normally leads via p14(ARF) to an induction of p53-dependent apoptosis. In a number of serous cancer cell lines with a dysfunctional p53 pathway (i.e., OVCAR8, OVCA433, and SKOV3), miR-31 overexpression inhibited proliferation and induced apoptosis; however, in other lines (i.e., HEY and OVSAYO) with functional p53, miR-31 had no effect. Additionally, the osteosarcoma cell line U2OS and the prostate cancer cell line PC3 (p14(ARF)-deficient and p53-deficient, respectively) were also sensitive to miR-31. Furthermore, miR-31 overexpression induced a global gene expression pattern in OVCAR8 associated with better prognosis in tumors from patients with advanced stage serous ovarian cancer, potentially affecting many genes underlying disease progression. Our findings reveal that loss of miR-31 is associated with defects in the p53 pathway and functions in serous ovarian cancer and other cancers, suggesting that patients with cancers deficient in p53 activity might benefit from therapeutic delivery of miR-31.
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