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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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Predicted role for mir-150-5p in regulation of cell death and survival, cell cycle, cellular growth and proliferation in MM1S cells.(A) Venn-diagram which represents the crosscomparison of 3626 predicted hsa-mir-150-5p targets according to miRWalk software and 1203 dexamethasone responsive target genes in MM1S cells, which remain unaffected in MM1R cells (B) IPA upstream analysis of the predicted microRNA target mRNAs in MM1S cells.
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pone-0113842-g004: Predicted role for mir-150-5p in regulation of cell death and survival, cell cycle, cellular growth and proliferation in MM1S cells.(A) Venn-diagram which represents the crosscomparison of 3626 predicted hsa-mir-150-5p targets according to miRWalk software and 1203 dexamethasone responsive target genes in MM1S cells, which remain unaffected in MM1R cells (B) IPA upstream analysis of the predicted microRNA target mRNAs in MM1S cells.

Mentions: To reduce the number of Dex responsive microRNA for further validation and functional studies, we next cross compared the list of GC responsive miRNA identified in MM1S with a list of miRNAs predicted to regulate the GR(NR3C1) pathway (Biocarta) according to the MiRWalk database [34] (Fig. 3B). This generated a shortlist of 11 miRNAs, which were selected for further QPCR validation. Along the same line, IPA correlation of Dex repressed mRNAs and 30 Dex inducible miRNAs identified a similar GR specific role for mir-150-5p, mir-146b-5p and mir-125a-5p in the cell cycle and cellular growth and proliferation pathway (Table S1). Time dependent GC responsive changes in transcription levels of 11 selected microRNAs were evaluated by QPCR in MM1S and MM1R cells exposed for different time points (6 h, 24 h, 48 h and 72 h) to 1 µM of Dex. QPCR analysis could confirm time and GC specific induction of 5 hsa-microRNAs i.e. of mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184 (Fig. 3C). Finally, hsa-mir-150-5p was selected for further functional studies because of its persistent GC responsive transcription between 6 and 72 h, and GC dose dependent regulation of mir-150-5p following 72 h treatment (Fig. 3D). Upon cross comparing 1203 Dex responsive target genes from MM1S cells with 1 µM Dex treatment for 72 h (gene selection was done based on p-value <0.05 and absolute fold change >1.5) and 3626 predicted hsa-mir-150-5p targets via the miRWalk database (prediction by at least 4 out of 10 available algorithms, i.e. DIANAmT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR4, PICTAR5, PITA, RNA22 and Targetscan), 196 common mRNA/gene targets (Fig. 4A) were identified in MM1S, which remain unaffected in MM1R cells (detailed list of genes can be found in Table S2). Upon employing IPA analysis, the 196 mRNA predicted GC responsive mir-150-5p target genes are enriched for cell death, cell morphology, cell cycle, cellular growth and proliferation pathways (Fig. 4A). Of special note, IPA upstream analysis predicts that mir-150-5p gene target genes are responsive for regulation by GC (Dex)/NR3C1, PPAR or STAT/IFNγ signaling (Fig. 4B).


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)

Predicted role for mir-150-5p in regulation of cell death and survival, cell cycle, cellular growth and proliferation in MM1S cells.(A) Venn-diagram which represents the crosscomparison of 3626 predicted hsa-mir-150-5p targets according to miRWalk software and 1203 dexamethasone responsive target genes in MM1S cells, which remain unaffected in MM1R cells (B) IPA upstream analysis of the predicted microRNA target mRNAs in MM1S cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113842-g004: Predicted role for mir-150-5p in regulation of cell death and survival, cell cycle, cellular growth and proliferation in MM1S cells.(A) Venn-diagram which represents the crosscomparison of 3626 predicted hsa-mir-150-5p targets according to miRWalk software and 1203 dexamethasone responsive target genes in MM1S cells, which remain unaffected in MM1R cells (B) IPA upstream analysis of the predicted microRNA target mRNAs in MM1S cells.
Mentions: To reduce the number of Dex responsive microRNA for further validation and functional studies, we next cross compared the list of GC responsive miRNA identified in MM1S with a list of miRNAs predicted to regulate the GR(NR3C1) pathway (Biocarta) according to the MiRWalk database [34] (Fig. 3B). This generated a shortlist of 11 miRNAs, which were selected for further QPCR validation. Along the same line, IPA correlation of Dex repressed mRNAs and 30 Dex inducible miRNAs identified a similar GR specific role for mir-150-5p, mir-146b-5p and mir-125a-5p in the cell cycle and cellular growth and proliferation pathway (Table S1). Time dependent GC responsive changes in transcription levels of 11 selected microRNAs were evaluated by QPCR in MM1S and MM1R cells exposed for different time points (6 h, 24 h, 48 h and 72 h) to 1 µM of Dex. QPCR analysis could confirm time and GC specific induction of 5 hsa-microRNAs i.e. of mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184 (Fig. 3C). Finally, hsa-mir-150-5p was selected for further functional studies because of its persistent GC responsive transcription between 6 and 72 h, and GC dose dependent regulation of mir-150-5p following 72 h treatment (Fig. 3D). Upon cross comparing 1203 Dex responsive target genes from MM1S cells with 1 µM Dex treatment for 72 h (gene selection was done based on p-value <0.05 and absolute fold change >1.5) and 3626 predicted hsa-mir-150-5p targets via the miRWalk database (prediction by at least 4 out of 10 available algorithms, i.e. DIANAmT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR4, PICTAR5, PITA, RNA22 and Targetscan), 196 common mRNA/gene targets (Fig. 4A) were identified in MM1S, which remain unaffected in MM1R cells (detailed list of genes can be found in Table S2). Upon employing IPA analysis, the 196 mRNA predicted GC responsive mir-150-5p target genes are enriched for cell death, cell morphology, cell cycle, cellular growth and proliferation pathways (Fig. 4A). Of special note, IPA upstream analysis predicts that mir-150-5p gene target genes are responsive for regulation by GC (Dex)/NR3C1, PPAR or STAT/IFNγ signaling (Fig. 4B).

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