Functional screen reveals essential roles of miR‐27a/24 in differentiation of embryonic stem cells
2014; Springer Nature; Volume: 34; Issue: 3 Linguagem: Inglês
10.15252/embj.201489957
ISSN1460-2075
AutoresYanni Ma, Nan Yao, Guang Liu, Lei Dong, Yufang Liu, Meili Zhang, Fang Wang, Bin Wang, Xueju Wei, He Dong, Lanlan Wang, Shaowei Ji, Ruoyu Zhang, Yangming Wang, Yue Huang, Jia Yu,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle17 December 2014free access Source Data Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells Yanni Ma Yanni Ma State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Nan Yao Nan Yao State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Guang Liu Guang Liu State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Lei Dong Lei Dong State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Yufang Liu Yufang Liu State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Meili Zhang Meili Zhang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Fang Wang Fang Wang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Bin Wang Bin Wang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Xueju Wei Xueju Wei State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author He Dong He Dong State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Lanlan Wang Lanlan Wang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Shaowei Ji Shaowei Ji State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Junwu Zhang Junwu Zhang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Yangming Wang Yangming Wang Peking-Tsinghua Joint Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Yue Huang Corresponding Author Yue Huang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Jia Yu Corresponding Author Jia Yu State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Yanni Ma Yanni Ma State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Nan Yao Nan Yao State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Guang Liu Guang Liu State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Lei Dong Lei Dong State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Yufang Liu Yufang Liu State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Meili Zhang Meili Zhang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Fang Wang Fang Wang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Bin Wang Bin Wang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Xueju Wei Xueju Wei State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author He Dong He Dong State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Lanlan Wang Lanlan Wang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Shaowei Ji Shaowei Ji State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Junwu Zhang Junwu Zhang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Yangming Wang Yangming Wang Peking-Tsinghua Joint Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Yue Huang Corresponding Author Yue Huang State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Jia Yu Corresponding Author Jia Yu State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Search for more papers by this author Author Information Yanni Ma1,2,‡, Nan Yao1,3,‡, Guang Liu1,3,‡, Lei Dong1,2, Yufang Liu1,3, Meili Zhang1,3, Fang Wang1,2, Bin Wang1,2, Xueju Wei1,2, He Dong1,2, Lanlan Wang1,2, Shaowei Ji1,3, Junwu Zhang1,2, Yangming Wang4, Yue Huang 1,3 and Jia Yu 1,2 1State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China 2Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China 3Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China 4Peking-Tsinghua Joint Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 10 69156462; E-mail: [email protected] *Corresponding author. Tel: +86 10 69156423; E-mail: [email protected] The EMBO Journal (2015)34:361-378https://doi.org/10.15252/embj.201489957 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract MicroRNAs play important roles in controlling the embryonic stem cell (ESC) state. Although much is known about microRNAs maintaining ESC state, microRNAs that are responsible for promoting ESC differentiation are less reported. Here, by screening 40 microRNAs pre-selected by their expression patterns and predicted targets in Dgcr8-null ESCs, we identify 14 novel differentiation-associated microRNAs. Among them, miR-27a and miR-24, restrained by c-Myc in ESC, exert their roles of silencing self-renewal through directly targeting several important pluripotency-associated factors, such as Oct4, Foxo1 and Smads. CRISPR/Cas9-mediated knockout of all miR-27/24 in ESCs leads to serious deficiency in ESC differentiation in vitro and in vivo. Moreover, depleting of them in mouse embryonic fibroblasts can evidently promote somatic cell reprogramming. Altogether, our findings uncover the essential role of miR-27 and miR-24 in ESC differentiation and also demonstrate novel microRNAs responsible for ESC differentiation. Synopsis A focused screen, followed by CRISPR/Cas9 functional genome editing establishes the miR-23~27~24 cluster as essential regulator of self-renewal and ESC-differentiation. A functional screen annotates 14 miRNAs as new regulators of ESC differentiation. miR-27a and miR-24 operate as suppressors of ESC self-renewal. The inhibition of miR-27a and miR-24 promotes somatic cell reprogramming. The miR-23~27~24 cluster is shown to be essential for mesoderm differentiation. Introduction Embryonic stem cells (ESCs) retain unlimited self-renewal potential in tissue culture and their propagation is characterized by a short cell cycle (White & Dalton, 2005). Under appropriate conditions in vitro, ESCs can be induced to differentiate into all somatic cell types. These lineage commitments involve the silencing of self-renewal program and the activation of lineage-specific programs (Jaenisch & Young, 2008; Silva & Smith, 2008). In contrast, reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is achieved by the re-establishment of the pluripotent state (Jaenisch & Young, 2008; Hochedlinger & Plath, 2009; Young, 2011). Accumulating evidence reveals that microRNAs (miRNAs) play important roles in control of pluripotent stem cell state. ESCs lacking of key enzymes in the miRNA biogenesis pathway, such as Dicer or Dgcr8, show deficiency in self-renewal and differentiation (Kanellopoulou et al, 2005; Murchison et al, 2005; Wang et al, 2007). A number of miRNAs have been reported to participate in regulating ESC self-renewal and differentiation (Barroso-delJesus et al, 2008; Wang et al, 2008; Ren et al, 2009; Sengupta et al, 2009). For example, ES cell-specific cell cycle (ESCC)-regulating miRNAs (Wang et al, 2008), miR-520 cluster (Ren et al, 2009), miR-302-367 cluster (Barroso-delJesus et al, 2008) and miR-92b (Sengupta et al, 2009) are important for the maintenance of ESC self-renewal. Contrarily, miR-134, miR-296, miR-470, miR-145 and let-7 family are involved in silencing of self-renewal program and/or promoting differentiation of ESCs (Tay et al, 2008a,b; Xu et al, 2009; Melton et al, 2010). Introduction of the ESCC-like miRNAs and/or suppression of those lineage commitment-related miRNAs can promote the reprogramming of somatic cells to iPSCs (Judson et al, 2009, 2013; Anokye-Danso et al, 2011; Choi et al, 2011; Li et al, 2011; Subramanyam et al, 2011; Yang et al, 2011). ESCC miRNAs were identified by screening a comprehensive miRNA mimic library in Dgcr8-deficient ESC, which overcomes issues of redundancy and saturation that are inherent to the miRNA system (Wang et al, 2008). However, miRNAs, which can suppress self-renewal and may contribute to lineage commitment of ESCs, have not been fully addressed. So, we aim to uncover novel miRNAs which can silence ESC self-renewal and to better understand the intricate regulatory mechanisms of ESC differentiation. Here, we report the identification of a novel class of differentiation-associated miRNAs by functional screening a collection of 40 miRNAs pre-selected by their expression patterns and predicted targets. miR-27a-3p and miR-24-3p, two representatives of these miRNAs, whose expressions are relatively low and restrained by c-Myc in ESCs, directly target the critical pluripotency transcription factors (Oct4, Foxo1) and signal transducers (gp130, Smads) to suppress ESC self-renewal program. Moreover, CRISPR/Cas9 technology-mediated bi-allelic double knockout of two miR-23~27~24 clusters in ESCs leads to serious defects in mesoderm differentiation of ESCs in vitro and in vivo. In addition, the efficiency of iPSC generation is improved evidently when miR-27a-3p or miR-24-3p is suppressed in mouse embryonic fibroblasts (MEFs). Results Bioinformatic prediction of miRNAs silencing ESC self-renewal The expression profile of miRNAs in ESCs or during ESC differentiation could, to some extent, hint their roles in regulating ESC self-renewal or differentiation. We used a newly designed strategy that combined miRNA expression pattern and miRNA with predicted targets to be pluripotency factors to identify candidate miRNAs responsible for ESC differentiation (Fig 1A). Specifically, by analyzing the published small RNA sequencing data of ESCs and MEFs (Marson et al, 2008), we found that there were 44 miRNAs enriched in MEFs compared with ESCs (Supplementary Table S1). From the sequencing data of ESCs and day 5 embryoid body (EB) derivatives (Ciaudo et al, 2009), 53 miRNAs were found to be up-regulated during EB formation, which recapitulates early events of embryogenesis (Supplementary Table S2). Using TargetScan (Lewis et al, 2003), we identified 454 miRNAs (Supplementary Table S3) that were predicted to target nine pluripotency-associated transcription factors, including Oct4 (also known as Pou5f1), Sox2, Nanog, Klf4, c-Myc, Lin28a, Sall4, Rex1 and Stella (Heo et al, 2008; Ng & Surani, 2011). By intersecting the two lists, we selected 52 miRNA candidates, which were not only up-regulated in MEFs or during EB differentiation but also potentially targeting the key ESC identity factors (Fig 1B and C). Among them, there were respective 30 and 25 miRNAs enriched in MEFs or up-regulated during EB differentiation, 3 miRNAs which were up-regulated both in MEFs and during EB differentiation, 14 miRNAs (including let-7 family) which had been reported to play important roles in suppressing mouse ESC self-renewal (Fig 1C). We focused on 40 miRNAs, excluding 12 well-studied miRNAs from the predicted 52 candidate miRNAs, in controlling ESC pluripotency and let-7c was set as a control. Figure 1. Screening of target miRNAs silencing ESC self-renewal Screening strategy. miRNAs potentially targeting important ESC pluripotency factors meanwhile enriched in MEFs or up-regulated during EB differentiation were selected as candidate miRNAs. Candidate miRNAs silencing ESC self-renewal were identified by the function analyses in Dgcr8−/− ESCs. Distribution of candidate miRNAs. There were 44 miRNAs enriched in MEFs (red region), 53 miRNAs up-regulated during EB differentiation (blue region) and 454 miRNAs with binding sites to the 3′ UTR of pluripotency factors (yellow region). The gray region represents 52 selected candidate miRNAs in silencing self-renewal. List of candidate miRNAs. Left panel shows candidate miRNAs enriched in MEFs comparing with ESCs. Right panel shows candidate miRNAs up-regulated during EB differentiation. Gray highlights the known miRNAs suppressing ESC self-renewal. Red marks the same miRNAs present in both panels. XY and XX indicate ESCs derived from male or female mice respectively. NA means D5/D0 was extremely high as the expression value in D0 was zero. Colony formation assay of miRNA mimic-transfected Dgcr8−/− ESCs. There were 17 miRNAs that decreased the colony-forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are available in Supplementary Fig S1. Download figure Download PowerPoint Validation of the functions of candidate miRNAs in Dgcr8−/− ESCs To uncover the roles of these miRNAs, we re-introduced miRNA mimics individually into Dgcr8−/− ESCs which exhibit a cell cycle defect and are incapable to silence the self-renewal program upon differentiation induction. We firstly investigated the colony-forming ability of transfected ESCs. Replating assays showed that 17 miRNAs decreased the capacity of ESCs to reform colonies. Of them, miR-9-5p, 200c-3p, 96-5p, 218-5p, 300-3p, 124-3p, 377-3p, 129-5p, 24-3p and 27a-3p, as well as let-7c, notably produced less and grossly differentiated colonies and possessed higher percentage of differentiated colonies than scramble control. There were 15 miRNAs which did not affect the colony-forming ability of ESCs, 8 miRNAs which enhanced the colony formation and yielded more compact and undifferentiated colonies (Fig 1D, Supplementary Fig S1 and Supplementary Table S4). Furthermore, we also detected the alkaline phosphatase (AP) activity in mimic-transfected ESCs without replating. To quantitatively analyze the AP activity, we scored the degree of AP staining on a scale from −3 to 3, with −3 being maximal loss of staining and with 3 being maximal boost of staining (Supplementary Fig S2 and Supplementary Table S5). We discovered that 21 miRNAs decreased the AP activity, 11 miRNAs mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs (Fig 2A). Of them, miR-129-5p, 200c-3p, 218-5p, 145a-5p, 9-5p, 300-3p, 31-5p, 24-3p markedly caused loss of AP activity and yielded flat and differentiated cell colonies even when the cells were maintained in ESC culture conditions. Figure 2. Functional analyses of candidate miRNAs in controlling ESC self-renewal Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are available in Supplementary Fig S2. Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic-transfected Dgcr8−/− ESCs. Error bars indicate SD (n = 3). Full data are shown in Supplementary Fig S3A and B. The heatmap representation of gene expression pattern in miRNA mimic-transfected Dgcr8−/− ESCs. Immunofluorescence staining of Oct4 in miRNA mimic-transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are available in Supplementary Fig S3C. Ranking of the miRNAs based on their ability to silence ESC self-renewal program. The color of the circles represents the fold change of these subjects compared to scramble control; the size of the circles represents the magnitude of P-values as determined by Student's t-test. Full list is available in Supplementary Fig S4A. AP staining of V6.5 ESCs after mimic transfection (full data are available in Supplementary Fig S4B). Download figure Download PowerPoint Another feature of self-renewing ESCs is their shortened G1 phase in cell cycle compared to differentiated cells (White & Dalton, 2005). Propidium iodide staining followed by flow cytometry analysis was performed in miRNA mimic-treated ESCs and revealed that 18 miRNAs could increase the fraction of cells in G1 phases, 12 miRNAs could reduce the fraction of cells in G1 phases and 10 miRNAs mildly affected cell cycle (Supplementary Fig S3A and B, and Supplementary Table S6). Similar to let-7c, miR-9-5p, 24-3p, 124-3p, 96-5p, 27a-3p, etc., led to G1 phase accumulation significantly (Fig 2B). We further analyzed the expression profiles of critical pluripotency factors and several differentiation markers through q-PCR. The results showed that most of the miRNAs decreasing AP activity in ESCs suppressed the expression of multiple pluripotency factors and obviously promoted cell differentiation to a certain extent. Conversely, some miRNAs played opposite roles in regulating ESC pluripotency (Fig 2C and Supplementary Table S7). The master transcription regulator of pluripotency maintenance, Oct4, was simultaneously analyzed in miRNA mimic-treated ESCs using immunofluorescence (Fig 2D). The results of Oct4 staining were consistent with that of AP staining. miR-218-5p, 200c-3p, 129-5p, 135b-5p, 24-3p, 9-5p, 32-5p and 27a-3p notably decreased Oct4 expression. Conversely, several miRNAs, such as miR-30a-5p, 541-5p, 152-3p and 141-3p, improved Oct4 expression (Fig 2D and Supplementary Fig S3C). Grading and scoring of screening results Systematically analyzing the data from colony formation assay, AP staining, cell cycle, gene expression pattern and Oct4 staining mentioned above, we ranked the miRNAs mainly based on their ability to silence ESC self-renewal (Fig 2E, Supplementary Fig S4A and Supplementary Table S8). The top 15 miRNAs were considered to be important differentiation-associated miRNAs, which can silence ESC self-renewal evidently. Most of them can inhibit the expression of pluripotency factors, decrease AP activity and also repress cell proliferation, but there were several miRNAs which can inhibit ESC self-renewal but had no influence or inverse effect on cell proliferation, hinting their distinct molecular mechanisms in regulating ESC pluripotency. Moreover, up-regulation of several lineage markers was observed in these miRNA mimics treated ESCs. Evaluation of the roles of the differentiation-associated miRNAs in wild-type ESCs We further evaluated the roles of the top 14 miRNAs by transfecting miRNA mimics to wild-type V6.5 ESCs. Among them, seven miRNAs (let-7c, miR-300-5p, 24-3p, 27a-3p, 124-3p, 27b-3p and 129-5p) can decrease the AP activity and produced smaller or flat cell colonies although not as dramatically as that in Dgcr8−/− ESCs (Fig 2F). However, the other seven miRNAs had no influence on the AP activity of V6.5 ESCs (Supplementary Fig S4B). These results agree with recent studies that the ESC-specific or enriched miRNAs can antagonize the effects of these differentiation-associated miRNAs in wild-type ESCs (Melton et al, 2010; Wang et al, 2013b). This does not indicate that these miRNAs are nonfunctional in ESCs and just suggests that it cannot be observed in wild-type ESC through miRNA mi
Referência(s)