An RNAi screen probing signaling control of human embryonic stem cells establishes the cell cycle-regulated restriction of the exit from pluripotency (CCNB1 overexpression)
ABSTRACT: Analysis of effect of Cyclin B1 overexpression on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that Cyclin B1 overexpression prevents the exit from pluripotency in hESCs. Results provide important information on the effect of Cyclin B1 on hESCs, such as changes in expression of TGFb-related genes and pluripotency markers, as well as mesodermic and neuroectodermic genes. Total RNA obtained from CCNB1 overexpressing hESCs incubated in medium without bFGF and TGF-beta for 0, 48 and 96 hours.
Project description:Analysis of effect of S-phase arrest and replication checkpoint activation on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that replication checkpoint activation prevents the exit from pluripotency in hESCs. Results provide important information of the response of hESCs to replication arrest, such as upregulation of genes involved in the TGF-beta signaling pathway, and subsequent maintenance of pluripotency marker expression. Total RNA obtained from hESCs incubated in medium without bFGF and TGF-beta supplemented with either DMSO, Aphidicolin or Aphidicolin+AZD7762 for 0, 48 and 96 hours.
Project description:We studied the expression levels of cyclins B1, D1, and E1 and the implications of cyclin overexpression for patient outcomes in distinct breast cancer subtypes defined by clinical variables and transcriptional profiling.The expression levels of cyclins B1, D1, and E1 were quantified in 779 breast tumors and 53 cell lines using reverse phase protein arrays and/or transcriptional profiling.Whereas cyclin E1 overexpression was a specific marker of triple-negative and basal-like tumors, cyclin B1 overexpression occurred in poor prognosis hormone receptor-positive, luminal B and basal-like breast cancers. Cyclin D1 overexpression occurred in luminal and normal-like cancers. Breast cancer subgroups defined by integrated expression of cyclins B1, D1, and E1 correlated significantly (P < 0.000001) with tumor subtypes defined by transcriptional profiling and clinical criteria. Across three hormone receptor-positive data sets, cyclin B1 was the dominant cyclin associated with poor prognosis in univariate and multivariate analyses. Although CCNE1 was present in significantly higher copy numbers in basal-like versus other subtypes (ANOVA P < 0.001), CCNB1 gene copy number did not show gain in breast cancer. Instead, cyclin B1 expression was increased in tumors with co-occurrence of TP53 mutations and MYC amplification, a combination that seems to characterize basal-like and luminal B tumors. CCNB1 gene expression was significantly correlated with PLK, CENPE, and AURKB gene expression.Cyclins B1, D1, and E1 have distinct expressions in different breast cancer subtypes. Novel PLK, CENPE, and AURKB inhibitors should be assessed for therapeutic utility in poor prognosis cyclin B1-overexpressing breast cancers.
Project description:The mechanisms of how signaling pathways are coordinated and integrated for the maintenance of the self-renewal of human embryonic stem cells (hESCs) and the acquisition of pluripotency in reprogramming are still only partly understood. CDK1 is a key regulator of mitosis. Recently, CDK1 has been shown to be involved in regulating self-renewal of stem cells, even though the mechanistic role of how CDK1 regulates pluripotency is unknown. In this report, we aim to understand how CDK1 can control pluripotency by reducing CDK1 activity to a level that has no effect on cell cycle progression. We demonstrated that high levels of CDK1 is associated with the pluripotency stage of hESCs; and decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation. CDK1 specifically targets the phosphorylation of PDK1 and consequently the activity of PI3K/Akt and its effectors ERK and GSK3?. Evidence of the reversion of inactive CDK1-mediated differentiation by the inhibition of Akt signaling effectors suggests that the CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the pluripotency of hESCs. Moreover, cyclin B1-CDK1 complexes promote somatic reprogramming efficiency, probably by regulating the maturation of induced pluripotent stem cells (iPSCs), as cyclin B1 stimulates a higher cellular level of LIN28A, suggesting that monitoring iPSC factors could be a new path for the enhancement of reprogramming efficiency. Together, we demonstrate an essential role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation of self-renewal, differentiation, and somatic reprogramming, which provides a novel kinase cascade mechanism for pluripotency control and acquisition.
Project description:Mammalian oocytes are arrested at the prophase of the first meiotic division for months and even years, depending on species. Meiotic resumption of fully grown oocytes requires activation of M-phase-promoting factor (MPF), which is composed of Cyclin B1 and cyclin-dependent kinase 1 (CDK1). It has long been believed that Cyclin B1 synthesis/accumulation and its interaction with CDK1 is a prerequisite for MPF activation in oocytes. In this study, we revealed that oocyte meiotic resumption occurred in the absence of Cyclin B1. Ccnb1-null oocytes resumed meiosis and extruded the first polar body. Without Cyclin B1, CDK1 could be activated by up-regulated Cyclin B2. Ccnb1 and Ccnb2 double knockout permanently arrested the oocytes at the prophase of the first meiotic division. Oocyte-specific Ccnb1-null female mice were infertile due to failed MPF activity elevation and thus premature interphase-like stage entry in the second meiotic division. These results have revealed a hidden compensatory mechanism between Cyclin B1 and Cyclin B2 in regulating MPF and oocyte meiotic resumption.
Project description:Control of protein turnover is critical for meiotic progression. Using RiboTag immunoprecipitation, RNA binding protein immunoprecipitation, and luciferase reporter assay, we investigated how rates of mRNA translation, protein synthesis and degradation contribute to the steady state level of Cyclin B1 and B2 in mouse oocytes. Ribosome loading onto Ccnb1 and Mos mRNAs increases during cell cycle reentry, well after germinal vesicle breakdown (GVBD). This is followed by the translation of reporters containing 3' untranslated region of Mos or Ccnb1 and the accumulation of Mos and Cyclin B1 proteins. Conversely, ribosome loading onto Ccnb2 mRNA and Cyclin B2 protein level undergo minimal changes during meiotic reentry. Degradation rates of Cyclin B1 or B2 protein at the GV stage are comparable. The translational activation of Mos and Ccnb1, but not Ccnb2, mRNAs is dependent on the RNA binding protein CPEB1. Inhibition of Cdk1 activity, but not Aurora A kinase activity, prevents the translation of Mos or Ccnb1 reporters, suggesting that MPF is required for their translation in mouse oocytes. Conversely, Ccnb2 translation is insensitive to Cdk1 inhibition. Thus, the poised state that allows rapid meiotic reentry in mouse GV oocytes may be determined by the differential translational control of two Cyclins.
Project description:It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3'UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.
Project description:Human induced pluripotent stem (iPS) cells are capable of differentiating into derivatives of the three embryonic germ layers both in vitro and in vivo. To date the the molecular differences between teratoma-forming cells and non-teratoma-forming cells has not been analysed. A cell line, B1, bears typical ES cell-like morphology, expression of pluripotency-associated genes, and in vitro pluripotency capacity, but fails to form teratomas after subcutaneously injected into immune-deficient mice based on histological analysis. Besides histological analysis, we characterized the tumors derived from line B1, and teratomas derived from bona fida iPS and ES (line H1) cells respectively, using microarray-based gene expression analysis. The expression levels of pluripotency-associated markers in B1 cells were comparable to that in iPS and ES cells, while the complexity of tissue expression commitment was decreased upon spontaneous differentiation of B1 cells as compared to iPS and ES cells. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, line B1, iPS-A4, iPS-B4 and ES (line H1) cells, and their derived tumors in immune-deficient mice.
Project description:Background:Hepatocellular carcinoma (HCC) is one of the most common malignancies with a high morbidity and mortality worldwide. MicroRNAs are key regulators of HCC genesis. However, the regulatory role and underlying mechanisms of microRNA in HCC is still limited. Methods:Cyclin B1 (CCNB1) mRNA levels were examined in non-tumor and liver cancer of The Cancer Genome Atlas (TCGA) cohort. CCNB1 was knockdown to evaluate the HCC cell proliferation, migration and invasion. MicroRNA-144 targeting CCNB1 was identified with TargetScan analysis and confirmed with reporter assay. Overexpression of MicroRNA-144 was achieved using microRNA mimics and function of microRNA-144 was tested in vitro HCC cell line proliferation and in vivo tumor formation experiments. Results:Here, we found that the high level expression of CCNB1 was closely associated with poor prognosis in HCC patients. Knockdown of CCNB1 by RNA interference significantly inhibited cell proliferation, migration and invasion in HCC. Furthermore, we found that miR-144 directly targeted CCNB1 and inhibited CCNB1 expression. Moreover, in vivo experiments of subcutaneous tumor formation further demonstrated that miR-144 delayed tumor formation by negative regulation of CCNB1. Conclusion:Therefore, we conclude that microRNA-144/CCNB1 axis plays an important role in human HCC. Therapies targeting microRNA-144 could potentially improve HCC treatment.
Project description:The unique cellular state of embryonic stem cells is maintained by pluripotency-associated transcription factors such as OCT4. The transcriptional regulatory circuitries between human and mouse pluripotent stem cells exhibit notable differences. Transposable elements have altered the pluripotency network by contributing new cis-regulatory elements to mammalian genomes. In our study, we found that the expression of HERVH is enriched in human embryonic stem cells (hESCs) and depletion of HERVH in hESCs induced differentiation. To confirm the differentiation phenotype and identify potential downstream target genes of HERVH, we conducted the microarray analysis on hESCs treated with control shRNA against Luciferase and shRNA against HERVH. Total RNA was extracted from cells treated with control shRNA or HERVH shRNA for 5 days. 3 replicates each.
Project description:MicroRNA (miRNA) are a class of single-stranded, small non-coding RNA that regulate various biological processes, including skin and hair cycle regulation, by modulating the expression of specific genes at the post-transcriptional level. Recently, several studies reported that miRNA directly or indirectly up-regulate target genes. Previously, we performed microarray analysis to identify the target genes of miR-199a-5p in a mouse skin keratinocyte cell line and detected more than 200 genes whose expression was significantly increased by miR-199a-5p overexpression (> 1.5-fold). In this study, we further investigated these genes and found that cyclin B1 (Ccnb1) expression was positively regulated by miR-199a-5p in keratinocyte. Moreover, Ccnb1 expression was inversely correlated with miR-199a-5p expression during the mouse hair cycle. Cell cycle analysis showed that the proportion of cells in S phase was slightly increased, while the proportion of cells in G2/M phase decreased by miR-199-5p. Using luciferase assay, we found that the 3' untranslated region of Ccnb1 was a direct target of miR-199a-5p. We also found that the regulation of Ccnb1 expression by miR-199a-5p is mouse specific. CCNB1 expression was not affected in the human and monkey cell lines. These results provide a new relationship between Ccnb1 and miR-199a-5p in both mouse keratinocyte and miRNA biology.