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Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.

Palagani A, Op de Beeck K, Naulaerts S, Diddens J, Sekhar Chirumamilla C, Van Camp G, Laukens K, Heyninck K, Gerlo S, Mestdagh P, Vandesompele J, Berghe WV - PLoS ONE (2014)

Bottom Line: Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells.Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir.Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerp, Belgium; Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University, Ghent, Belgium.

ABSTRACT
Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.

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Experimental validation of GC inducible microRNAs, potentially involved in regulation of the NR3C1(GR) pathway in MM1S cells(A) Venn-diagram which summarizes the overlap between the top 5 predicted molecular and cellular functions generated by IPA from GC regulated miRNAs and GC regulated mRNAs (B) Venn-diagram which represents the crosscomparison of the list of GC responsive miRNAs identified in MM1S with the list of GR(NR3C1) pathway specific miRNAs according to MiRWalk prediction freeware. (C) Real-time QPCR validation of the transcription levels of 9 common miRNAs selected in Fig. 3B, in MM1S and MM1R cells treated for 24, 48 or 72 h with 1 µM Dex. (D) GC dose responsive regulation of mir-150-5p levels in MM1S cells treated for 72 h with GC. All the miRNA QPCR validation studies have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs according to manufacturer instructions and relative to MM1S untreated condition (S UT). Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments as compared to control setups. Means with ***, **, * are significantly different to control setups (p<0.001, <0.01 or <0.05) as determined by one-way ANOVA (Dunnetts Post test).
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pone-0113842-g003: Experimental validation of GC inducible microRNAs, potentially involved in regulation of the NR3C1(GR) pathway in MM1S cells(A) Venn-diagram which summarizes the overlap between the top 5 predicted molecular and cellular functions generated by IPA from GC regulated miRNAs and GC regulated mRNAs (B) Venn-diagram which represents the crosscomparison of the list of GC responsive miRNAs identified in MM1S with the list of GR(NR3C1) pathway specific miRNAs according to MiRWalk prediction freeware. (C) Real-time QPCR validation of the transcription levels of 9 common miRNAs selected in Fig. 3B, in MM1S and MM1R cells treated for 24, 48 or 72 h with 1 µM Dex. (D) GC dose responsive regulation of mir-150-5p levels in MM1S cells treated for 72 h with GC. All the miRNA QPCR validation studies have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs according to manufacturer instructions and relative to MM1S untreated condition (S UT). Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments as compared to control setups. Means with ***, **, * are significantly different to control setups (p<0.001, <0.01 or <0.05) as determined by one-way ANOVA (Dunnetts Post test).

Mentions: Total RNA was isolated from MM1S and MM1R cells treated with 1 µM Dex for 72 h, qPCR profiling of 760 miRNAs as previously described [32]. This allowed to identify 30 GC inducible and 14 GC repressed miRNAs (>2-fold) in MM1S, which do not change transcription in MM1R cells (summarized in Table 3). Next, IPA analysis was applied to cross compare microRNA and mRNA profiles in MM1S to predict target genes and top pathways which are affected by microRNA expression. Venn diagram analysis of the top 5 ranked molecular and cellular pathways enriched for genes differentially expressed in MM1S upon Dex exposure and predicted microRNA target genes responsive to Dex treatment reveals that GC responsive microRNAs may target mRNAs involved in cell cycle and cell proliferation pathways (Fig. 3A). For example, Dex inducible microRNAs mir-150-5p, mir-152, mir-146b-5p, mir-1290, mir-125a-5p, mir-1206 are predicted to modulate multiple target genes involved in cell cycle, cellular growth and proliferation pathways (as yellow marked in Table S1).


Ectopic microRNA-150-5p transcription sensitizes glucocorticoid therapy response in MM1S multiple myeloma cells but fails to overcome hormone therapy resistance in MM1R cells.

Palagani A, Op de Beeck K, Naulaerts S, Diddens J, Sekhar Chirumamilla C, Van Camp G, Laukens K, Heyninck K, Gerlo S, Mestdagh P, Vandesompele J, Berghe WV - PLoS ONE (2014)

Experimental validation of GC inducible microRNAs, potentially involved in regulation of the NR3C1(GR) pathway in MM1S cells(A) Venn-diagram which summarizes the overlap between the top 5 predicted molecular and cellular functions generated by IPA from GC regulated miRNAs and GC regulated mRNAs (B) Venn-diagram which represents the crosscomparison of the list of GC responsive miRNAs identified in MM1S with the list of GR(NR3C1) pathway specific miRNAs according to MiRWalk prediction freeware. (C) Real-time QPCR validation of the transcription levels of 9 common miRNAs selected in Fig. 3B, in MM1S and MM1R cells treated for 24, 48 or 72 h with 1 µM Dex. (D) GC dose responsive regulation of mir-150-5p levels in MM1S cells treated for 72 h with GC. All the miRNA QPCR validation studies have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs according to manufacturer instructions and relative to MM1S untreated condition (S UT). Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments as compared to control setups. Means with ***, **, * are significantly different to control setups (p<0.001, <0.01 or <0.05) as determined by one-way ANOVA (Dunnetts Post test).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256227&req=5

pone-0113842-g003: Experimental validation of GC inducible microRNAs, potentially involved in regulation of the NR3C1(GR) pathway in MM1S cells(A) Venn-diagram which summarizes the overlap between the top 5 predicted molecular and cellular functions generated by IPA from GC regulated miRNAs and GC regulated mRNAs (B) Venn-diagram which represents the crosscomparison of the list of GC responsive miRNAs identified in MM1S with the list of GR(NR3C1) pathway specific miRNAs according to MiRWalk prediction freeware. (C) Real-time QPCR validation of the transcription levels of 9 common miRNAs selected in Fig. 3B, in MM1S and MM1R cells treated for 24, 48 or 72 h with 1 µM Dex. (D) GC dose responsive regulation of mir-150-5p levels in MM1S cells treated for 72 h with GC. All the miRNA QPCR validation studies have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs according to manufacturer instructions and relative to MM1S untreated condition (S UT). Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments as compared to control setups. Means with ***, **, * are significantly different to control setups (p<0.001, <0.01 or <0.05) as determined by one-way ANOVA (Dunnetts Post test).
Mentions: Total RNA was isolated from MM1S and MM1R cells treated with 1 µM Dex for 72 h, qPCR profiling of 760 miRNAs as previously described [32]. This allowed to identify 30 GC inducible and 14 GC repressed miRNAs (>2-fold) in MM1S, which do not change transcription in MM1R cells (summarized in Table 3). Next, IPA analysis was applied to cross compare microRNA and mRNA profiles in MM1S to predict target genes and top pathways which are affected by microRNA expression. Venn diagram analysis of the top 5 ranked molecular and cellular pathways enriched for genes differentially expressed in MM1S upon Dex exposure and predicted microRNA target genes responsive to Dex treatment reveals that GC responsive microRNAs may target mRNAs involved in cell cycle and cell proliferation pathways (Fig. 3A). For example, Dex inducible microRNAs mir-150-5p, mir-152, mir-146b-5p, mir-1290, mir-125a-5p, mir-1206 are predicted to modulate multiple target genes involved in cell cycle, cellular growth and proliferation pathways (as yellow marked in Table S1).

Bottom Line: Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells.Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir.Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerp, Belgium; Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Physiology, Ghent University, Ghent, Belgium.

ABSTRACT
Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.

Show MeSH
Related in: MedlinePlus