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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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OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression.Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative OCT4 and NANOG expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.
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pone-0111637-g003: OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression.Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative OCT4 and NANOG expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.

Mentions: Ablating the function of OCT4 in hESC leads to reduced expression of pluripotency-associated genes such as NANOG, LEFTY1, LEFTY2 and NODAL. A consequence of this is the up-regulation of the SMAD2/3 signalling antagonists, BMP4 and BMPR1 thus leading to the activation of the SMAD1/5/8 branch of the TGFß-signalling pathway and promoting the expression of somatic enriched genes [41]. To investigate miR-27 expression after RNAi-mediated knockdown of OCT4 inhESC, we isolated total RNA 72 h post transfection with siRNAs targeting either OCT4 (siOCT4) or EGFP (siEGFP) as a negative control [41]. To confirm the successful knockdown of OCT4, we quantified the expression of OCT4, and its downstream target NANOG, by RT-PCR in three biological replicates (Figure 3A). We observed a ∼80- to ∼95% repression of OCT4 and ∼75 to ∼95% reduced NANOG expression in all three biological replicates (siOCT#1-3). Successful knockdown of OCT4 was also confirmed by western blotting for the representative sample (siOCT4#1) (Figure 3B). Densitometry analysis revealed an ∼80% reduction in the level of OCT4 protein 48 h post transfection (Figure 3B). Further RT-PCR analyses revealed decreased expression of the pluripotency-associated genes, OCT4, SOX2, NANOG and LIN28 and an expected increased expression of BMP4, compared to the siEGFP control transfection (Figure 3C). Finally, we compared miR-27 expression in hESC 72 h after siRNA mediated knockdown of OCT4 with a TAQman miRNA assay. We observed for two samples with the most efficient OCT4 knockdown, a more than 16-fold increase in the levels of miR-27a and more than 6-fold increase in miR-27b expression (Figure 3D). The lowest, just 1-fold up-regulation of both, miR-27a and miR-27b, was observed with the less efficient OCT4 knockdown sample- siOCT4#3. These results confirm that OCT4 expression negatively correlates with miR-27a/b expression in hESC.


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

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

OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression.Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative OCT4 and NANOG expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.
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pone-0111637-g003: OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression.Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative OCT4 and NANOG expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.
Mentions: Ablating the function of OCT4 in hESC leads to reduced expression of pluripotency-associated genes such as NANOG, LEFTY1, LEFTY2 and NODAL. A consequence of this is the up-regulation of the SMAD2/3 signalling antagonists, BMP4 and BMPR1 thus leading to the activation of the SMAD1/5/8 branch of the TGFß-signalling pathway and promoting the expression of somatic enriched genes [41]. To investigate miR-27 expression after RNAi-mediated knockdown of OCT4 inhESC, we isolated total RNA 72 h post transfection with siRNAs targeting either OCT4 (siOCT4) or EGFP (siEGFP) as a negative control [41]. To confirm the successful knockdown of OCT4, we quantified the expression of OCT4, and its downstream target NANOG, by RT-PCR in three biological replicates (Figure 3A). We observed a ∼80- to ∼95% repression of OCT4 and ∼75 to ∼95% reduced NANOG expression in all three biological replicates (siOCT#1-3). Successful knockdown of OCT4 was also confirmed by western blotting for the representative sample (siOCT4#1) (Figure 3B). Densitometry analysis revealed an ∼80% reduction in the level of OCT4 protein 48 h post transfection (Figure 3B). Further RT-PCR analyses revealed decreased expression of the pluripotency-associated genes, OCT4, SOX2, NANOG and LIN28 and an expected increased expression of BMP4, compared to the siEGFP control transfection (Figure 3C). Finally, we compared miR-27 expression in hESC 72 h after siRNA mediated knockdown of OCT4 with a TAQman miRNA assay. We observed for two samples with the most efficient OCT4 knockdown, a more than 16-fold increase in the levels of miR-27a and more than 6-fold increase in miR-27b expression (Figure 3D). The lowest, just 1-fold up-regulation of both, miR-27a and miR-27b, was observed with the less efficient OCT4 knockdown sample- siOCT4#3. These results confirm that OCT4 expression negatively correlates with miR-27a/b expression in hESC.

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