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miR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

Fuchs H, Theuser M, Wruck W, Adjaye J - PLoS ONE (2014)

Bottom Line: Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells.Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells.Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Stem Cell Research and Regenerative Medicine, Faculty of Medicine, Heinrich Heine University, Duesseldorf, Germany.

ABSTRACT
Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8) axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

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miR-27 directly inhibits a number of genes reported to sustain self-renewal in embryonic stem cells.(A) Table showing three putative miR-27 target genes predicted by Diana Micro-T (DT), MiRanda (miRa), MirWalk (miRW) and TargetScan that maintain self-renewal. (B) Normalized GFP expression (48 hours post transfection) of HEK293 cells co-transfected with EGFP-sensors bearing parts of the 3′-UTR of LIN28B, NR5A2, POLR3G, NANOG, SOX2 or OCT4 together with either miR-27 mimics or a scrambled negative control mimic (neg. con.). All transfections were performed twice in biological triplicates (n = 6) and GFP expression measured by flow cytometry. An unpaired two tailed t-test was performed to reveal significant differences (** p<0.01). (C) Upper row: Schematic timeline of hESC (H1) undergoing hepatic differentiation. Total RNA was isolated at day zero (undifferentiated H1), three days after endoderm priming (DE) and 14 days after hepatocyte differentiation (HE). Lower row: miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples from the above described stages, DE and HE and normalized to the untreated/undifferentiated H1 control.
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pone-0111637-g002: miR-27 directly inhibits a number of genes reported to sustain self-renewal in embryonic stem cells.(A) Table showing three putative miR-27 target genes predicted by Diana Micro-T (DT), MiRanda (miRa), MirWalk (miRW) and TargetScan that maintain self-renewal. (B) Normalized GFP expression (48 hours post transfection) of HEK293 cells co-transfected with EGFP-sensors bearing parts of the 3′-UTR of LIN28B, NR5A2, POLR3G, NANOG, SOX2 or OCT4 together with either miR-27 mimics or a scrambled negative control mimic (neg. con.). All transfections were performed twice in biological triplicates (n = 6) and GFP expression measured by flow cytometry. An unpaired two tailed t-test was performed to reveal significant differences (** p<0.01). (C) Upper row: Schematic timeline of hESC (H1) undergoing hepatic differentiation. Total RNA was isolated at day zero (undifferentiated H1), three days after endoderm priming (DE) and 14 days after hepatocyte differentiation (HE). Lower row: miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples from the above described stages, DE and HE and normalized to the untreated/undifferentiated H1 control.

Mentions: The fact that miR-27 regulates the SMAD2/3 branch prompted us to search for additional pluripotency-associated genes that might be regulated by miR-27. Screening with TargetScan we identified LIN28B as a putative miR-27 target gene (Figure 2A). Two isoforms of LIN28 have been described, the predominantly cytoplasmic expressed LIN28A and the nucleus-enriched LIN28B. Both isoforms have been reported to inhibit processing of miRNA let-7, a well-studied miRNA that promotes differentiation. Within the nucleus, LIN28A blocks the cleavage of the primary let-7 transcript by the microprocessor complex into the precursor forms, whilst LIN28B binds the precursor forms of let-7 and prevents let-7 maturation by the ribonuclease DICER [16], [17], [38]. By using the above described GFP-sensor assay, we observed a significant (16%) reduction in GFP expression (p = 0.008) in the presence of exogenous miR-27 compared to the negative control, thus suggesting that LIN28B is a direct target of miR-27 (Figure 2B).


miR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

Fuchs H, Theuser M, Wruck W, Adjaye J - PLoS ONE (2014)

miR-27 directly inhibits a number of genes reported to sustain self-renewal in embryonic stem cells.(A) Table showing three putative miR-27 target genes predicted by Diana Micro-T (DT), MiRanda (miRa), MirWalk (miRW) and TargetScan that maintain self-renewal. (B) Normalized GFP expression (48 hours post transfection) of HEK293 cells co-transfected with EGFP-sensors bearing parts of the 3′-UTR of LIN28B, NR5A2, POLR3G, NANOG, SOX2 or OCT4 together with either miR-27 mimics or a scrambled negative control mimic (neg. con.). All transfections were performed twice in biological triplicates (n = 6) and GFP expression measured by flow cytometry. An unpaired two tailed t-test was performed to reveal significant differences (** p<0.01). (C) Upper row: Schematic timeline of hESC (H1) undergoing hepatic differentiation. Total RNA was isolated at day zero (undifferentiated H1), three days after endoderm priming (DE) and 14 days after hepatocyte differentiation (HE). Lower row: miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples from the above described stages, DE and HE and normalized to the untreated/undifferentiated H1 control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219743&req=5

pone-0111637-g002: miR-27 directly inhibits a number of genes reported to sustain self-renewal in embryonic stem cells.(A) Table showing three putative miR-27 target genes predicted by Diana Micro-T (DT), MiRanda (miRa), MirWalk (miRW) and TargetScan that maintain self-renewal. (B) Normalized GFP expression (48 hours post transfection) of HEK293 cells co-transfected with EGFP-sensors bearing parts of the 3′-UTR of LIN28B, NR5A2, POLR3G, NANOG, SOX2 or OCT4 together with either miR-27 mimics or a scrambled negative control mimic (neg. con.). All transfections were performed twice in biological triplicates (n = 6) and GFP expression measured by flow cytometry. An unpaired two tailed t-test was performed to reveal significant differences (** p<0.01). (C) Upper row: Schematic timeline of hESC (H1) undergoing hepatic differentiation. Total RNA was isolated at day zero (undifferentiated H1), three days after endoderm priming (DE) and 14 days after hepatocyte differentiation (HE). Lower row: miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples from the above described stages, DE and HE and normalized to the untreated/undifferentiated H1 control.
Mentions: The fact that miR-27 regulates the SMAD2/3 branch prompted us to search for additional pluripotency-associated genes that might be regulated by miR-27. Screening with TargetScan we identified LIN28B as a putative miR-27 target gene (Figure 2A). Two isoforms of LIN28 have been described, the predominantly cytoplasmic expressed LIN28A and the nucleus-enriched LIN28B. Both isoforms have been reported to inhibit processing of miRNA let-7, a well-studied miRNA that promotes differentiation. Within the nucleus, LIN28A blocks the cleavage of the primary let-7 transcript by the microprocessor complex into the precursor forms, whilst LIN28B binds the precursor forms of let-7 and prevents let-7 maturation by the ribonuclease DICER [16], [17], [38]. By using the above described GFP-sensor assay, we observed a significant (16%) reduction in GFP expression (p = 0.008) in the presence of exogenous miR-27 compared to the negative control, thus suggesting that LIN28B is a direct target of miR-27 (Figure 2B).

Bottom Line: Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells.Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells.Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Stem Cell Research and Regenerative Medicine, Faculty of Medicine, Heinrich Heine University, Duesseldorf, Germany.

ABSTRACT
Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8) axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

Show MeSH
Related in: MedlinePlus