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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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Related in: MedlinePlus

Schematic overview of our proposed regulatory network between miR-27 and pluripotency-associated genes.Genes highlighted in bold, black letters are those validated experimentally to be direct targets of miR-27.
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pone-0111637-g006: Schematic overview of our proposed regulatory network between miR-27 and pluripotency-associated genes.Genes highlighted in bold, black letters are those validated experimentally to be direct targets of miR-27.

Mentions: We have confirmed with our GFP-sensor approach that miR-27 directly inhibits the ACTIVIN/NODAL branch of TGFß-signalling by targeting ACVR2A, TGFßR1 and SMAD2 (Figure 1C). These results reveal that miR-27 negatively regulates SMAD2/3 and therefore inhibits self-renewal in hESC. Interestingly, our group recently demonstrated that activated SMAD2/3 promotes the expression of pluripotency-associated genes such as LEFTYA, LEFTYB, CER1 and NODAL. We also revealed that NANOG bears the SMAD2/3 binding motif within its promoter region [3]. Remarkably, we show here that miR-27 directly regulates NANOG by binding to its 3′-UTR and inhibiting its expression (Figure 2B). Additional evidence in support of miR-27 acting as an “off-switch” for self-renewal are as follows; (i) miR-27 moderately inhibits LIN28B by using the eGFP-sensor approach (Figure 2B). Additionally, over-expression of mir-27 in hEC cells represses LIN28 at the transcriptional and translational level (Figure 4C+D+E). LIN28B is a well-studied gatekeeper of pluripotency. LIN28B promotes OCT4 stability post-transcriptionally by interacting with polyribosomes in embryonic stem cells [14], [15]. Moreover, LIN28B binds the precursor form of miRNA let-7 and prevents its maturation [16], [17]. Let-7, one of the most potent differentiation-inducing miRNA, down-regulates a large number of pluripotency-associated genes such as, LIN28, MYC, CDK1 and HMGA2at the translational level [16], [26], [27]. To summarize, our results imply that (i) miR-27 indirectly promotes Let-7 maturation by modulating LIN28B. (ii) miR-27 over-expression leads to an elevated expression of the tumor suppressor oncogene, TP53 in hEC cells (Figure 4B). TP53 has been reported to activate miR-145, a repressor of pluripotency-associated genes such as OCT4, SOX2, LIN28 and KLF4[28]. TP53 also promotes the expression of miR-34, another miRNA that has been reported to suppress reprogramming efficiency by inhibiting expression of SOX2 and NANOG [29]. (iii) Another tumor suppressor oncogene, FOXO1, has been reported to be regulated by miR-27. While over-expression of miR-27 results in a reduction of FOXO1 expression in hEC-1B cells, inhibition of miR-27 with antagomiRs leads to a de-repression of FOXO1 in Ishiwaka cells [36]. FOXO1 is an essential regulator of pluripotency. Loss of FOXO1 induces loss of expression of OCT4, NANOG, KLF4, SOX2, TRA-1-31 and TRA-1-60 in the hESC line H1 [54]. (iv) We have confirmed two transcription factors, POLR3G and NR5A2, as direct targets of miR-27 (Figure 2B). Previous reports demonstrated that NR5A2 binds co-operatively with SOX2 within the promoter regions of OCT4 and NANOG and promotes their expression in embryonic stem cells [39]. In the case of the OCT4/NANOG downstream target POLR3G, it has been shown that down-regulation of POLR3G promotes differentiation of hESC [11]. (v) miR-27 has been reported to be linked to cancer progression where miR-27promotes cancer metastasis through epithelial–mesenchymal transition (EMT) in AGS cells [37], [55]. Moreover, miR-27 over-expression activates expression of ZEB1 and ZEB2, (two antagonist of E-CADHERIN), which then leads to activated ß-CATENIN expression and decreased E-CADHERIN levels [37]. E-CADHERIN is an important regulator promoting the undifferentiated state of hESC and is a key factor associated with the induction of pluripotency in somatic cells [56], [57]. Our transcriptome analysis revealed that over-expression of miR-27 in human embryonal carcinoma cells leads to down-regulation of pluripotency-associated genes, such as GDF3, LIN28, TRIM71, DNMT3A, DNMT3B and USP46 and an activated expression of developmental genes such as SMAD6, BMP2, FST and HAND1 (Figure 5C). We observed an increased expression of LEFTY2, a gene that has been previously reported to be abundantly expressed in hESC [58]. However, LEFTY2 is a key factor in various developmental processes and a previous knockdown study from our group reported an up-regulated expression of LEFTY2 after siRNA mediated knockdown of OCT4 or NANOG in the embryonal carcinoma cell line NCCIT [3]. Moreover, it has been recently reported that the NODAL inhibitor, LEFTY2, is down-regulated by miR-302, a microRNA that is highly enriched in hESC, thus revealing that modulating LEFTY2 at the translational level might be important to promote the undifferentiated stage [59]. More strikingly, we observed an up-regulation of genes that control developmental pathways such as p53-, WNT- and TGFß-signalling after miR-27 over-expression in NCCIT cells (Table 1). Finally, we have shown that over-expression of miR-27 in hEC leads to a dramatic reduction in expression of OCT4 mRNA and protein (Figure 4C+D+E) but, as shown with the eGFP-sensor approach, OCT4 is not a direct target gene of miR-27 (Figure 2B). The fact that loss of OCT4 induces activation of miR-27 expression in hES and that miR-27 over-expression results in reduced OCT4 expression in hEC, might imply that OCT4 and miR-27 form an indirect negative feedback loop but OCT4 rather than miR-27, is required for the maintenance of self-renewal in pluripotent stem cells [41] as depicted in Figure 6.


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

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

Schematic overview of our proposed regulatory network between miR-27 and pluripotency-associated genes.Genes highlighted in bold, black letters are those validated experimentally to be direct targets of miR-27.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219743&req=5

pone-0111637-g006: Schematic overview of our proposed regulatory network between miR-27 and pluripotency-associated genes.Genes highlighted in bold, black letters are those validated experimentally to be direct targets of miR-27.
Mentions: We have confirmed with our GFP-sensor approach that miR-27 directly inhibits the ACTIVIN/NODAL branch of TGFß-signalling by targeting ACVR2A, TGFßR1 and SMAD2 (Figure 1C). These results reveal that miR-27 negatively regulates SMAD2/3 and therefore inhibits self-renewal in hESC. Interestingly, our group recently demonstrated that activated SMAD2/3 promotes the expression of pluripotency-associated genes such as LEFTYA, LEFTYB, CER1 and NODAL. We also revealed that NANOG bears the SMAD2/3 binding motif within its promoter region [3]. Remarkably, we show here that miR-27 directly regulates NANOG by binding to its 3′-UTR and inhibiting its expression (Figure 2B). Additional evidence in support of miR-27 acting as an “off-switch” for self-renewal are as follows; (i) miR-27 moderately inhibits LIN28B by using the eGFP-sensor approach (Figure 2B). Additionally, over-expression of mir-27 in hEC cells represses LIN28 at the transcriptional and translational level (Figure 4C+D+E). LIN28B is a well-studied gatekeeper of pluripotency. LIN28B promotes OCT4 stability post-transcriptionally by interacting with polyribosomes in embryonic stem cells [14], [15]. Moreover, LIN28B binds the precursor form of miRNA let-7 and prevents its maturation [16], [17]. Let-7, one of the most potent differentiation-inducing miRNA, down-regulates a large number of pluripotency-associated genes such as, LIN28, MYC, CDK1 and HMGA2at the translational level [16], [26], [27]. To summarize, our results imply that (i) miR-27 indirectly promotes Let-7 maturation by modulating LIN28B. (ii) miR-27 over-expression leads to an elevated expression of the tumor suppressor oncogene, TP53 in hEC cells (Figure 4B). TP53 has been reported to activate miR-145, a repressor of pluripotency-associated genes such as OCT4, SOX2, LIN28 and KLF4[28]. TP53 also promotes the expression of miR-34, another miRNA that has been reported to suppress reprogramming efficiency by inhibiting expression of SOX2 and NANOG [29]. (iii) Another tumor suppressor oncogene, FOXO1, has been reported to be regulated by miR-27. While over-expression of miR-27 results in a reduction of FOXO1 expression in hEC-1B cells, inhibition of miR-27 with antagomiRs leads to a de-repression of FOXO1 in Ishiwaka cells [36]. FOXO1 is an essential regulator of pluripotency. Loss of FOXO1 induces loss of expression of OCT4, NANOG, KLF4, SOX2, TRA-1-31 and TRA-1-60 in the hESC line H1 [54]. (iv) We have confirmed two transcription factors, POLR3G and NR5A2, as direct targets of miR-27 (Figure 2B). Previous reports demonstrated that NR5A2 binds co-operatively with SOX2 within the promoter regions of OCT4 and NANOG and promotes their expression in embryonic stem cells [39]. In the case of the OCT4/NANOG downstream target POLR3G, it has been shown that down-regulation of POLR3G promotes differentiation of hESC [11]. (v) miR-27 has been reported to be linked to cancer progression where miR-27promotes cancer metastasis through epithelial–mesenchymal transition (EMT) in AGS cells [37], [55]. Moreover, miR-27 over-expression activates expression of ZEB1 and ZEB2, (two antagonist of E-CADHERIN), which then leads to activated ß-CATENIN expression and decreased E-CADHERIN levels [37]. E-CADHERIN is an important regulator promoting the undifferentiated state of hESC and is a key factor associated with the induction of pluripotency in somatic cells [56], [57]. Our transcriptome analysis revealed that over-expression of miR-27 in human embryonal carcinoma cells leads to down-regulation of pluripotency-associated genes, such as GDF3, LIN28, TRIM71, DNMT3A, DNMT3B and USP46 and an activated expression of developmental genes such as SMAD6, BMP2, FST and HAND1 (Figure 5C). We observed an increased expression of LEFTY2, a gene that has been previously reported to be abundantly expressed in hESC [58]. However, LEFTY2 is a key factor in various developmental processes and a previous knockdown study from our group reported an up-regulated expression of LEFTY2 after siRNA mediated knockdown of OCT4 or NANOG in the embryonal carcinoma cell line NCCIT [3]. Moreover, it has been recently reported that the NODAL inhibitor, LEFTY2, is down-regulated by miR-302, a microRNA that is highly enriched in hESC, thus revealing that modulating LEFTY2 at the translational level might be important to promote the undifferentiated stage [59]. More strikingly, we observed an up-regulation of genes that control developmental pathways such as p53-, WNT- and TGFß-signalling after miR-27 over-expression in NCCIT cells (Table 1). Finally, we have shown that over-expression of miR-27 in hEC leads to a dramatic reduction in expression of OCT4 mRNA and protein (Figure 4C+D+E) but, as shown with the eGFP-sensor approach, OCT4 is not a direct target gene of miR-27 (Figure 2B). The fact that loss of OCT4 induces activation of miR-27 expression in hES and that miR-27 over-expression results in reduced OCT4 expression in hEC, might imply that OCT4 and miR-27 form an indirect negative feedback loop but OCT4 rather than miR-27, is required for the maintenance of self-renewal in pluripotent stem cells [41] as depicted in Figure 6.

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