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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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Related in: MedlinePlus

Weak sensitisation of low dose GC therapy response by mir-150-5p mimetic transfection can be reversed by transfection of a mir-150-5p-specific antagomir in MM1S cells.(A) MTT based quantification of MM1S cell survival, following 24, 48, or 72 h transfection with increasing doses of synthetic mir-150-5p mimetic as compared to mock transfection control (B) MTT based quantification of MM1S cell survival, following 72 h treatment with increasing doses of Dex, in combination with either mock transfection, else transfection with a synthetic mir-150-5p mimetic or mir-150-5p specific antagomir (as indicated in figure). Data represented are relative cell survival levels (mean ± SEM) of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttests). (C) Realtime quantitative PCR (QPCR) quantification of mir-150-5p transcription levels following 72 h treatment with 1 nM Dex, either or not in combination with transfection of a mir-150-5p specific antagomir. (D–E) Illumina BeadChip Gene Expression results of a subset of genes are presented as bargraphs, reflecting the mean fold change from three independent experiments of MM1S cells treated for 72 h with 1 nM Dex vs. control setup, either or not in combination with transfection of a mir-150-5p specific antagomir.
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pone-0113842-g010: Weak sensitisation of low dose GC therapy response by mir-150-5p mimetic transfection can be reversed by transfection of a mir-150-5p-specific antagomir in MM1S cells.(A) MTT based quantification of MM1S cell survival, following 24, 48, or 72 h transfection with increasing doses of synthetic mir-150-5p mimetic as compared to mock transfection control (B) MTT based quantification of MM1S cell survival, following 72 h treatment with increasing doses of Dex, in combination with either mock transfection, else transfection with a synthetic mir-150-5p mimetic or mir-150-5p specific antagomir (as indicated in figure). Data represented are relative cell survival levels (mean ± SEM) of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttests). (C) Realtime quantitative PCR (QPCR) quantification of mir-150-5p transcription levels following 72 h treatment with 1 nM Dex, either or not in combination with transfection of a mir-150-5p specific antagomir. (D–E) Illumina BeadChip Gene Expression results of a subset of genes are presented as bargraphs, reflecting the mean fold change from three independent experiments of MM1S cells treated for 72 h with 1 nM Dex vs. control setup, either or not in combination with transfection of a mir-150-5p specific antagomir.

Mentions: Since mir-150-5p modulates various genes related to cell cycle, cell proliferation and cell death pathways, we next wanted to measure cell death induced by ectopic mir-150-5p transfection. Surprisingly, no substantial induction of cell death could be observed by MTT-assay at 24, 48 and 72 h following transfection of MM1S cells by increasing amounts of mir150-5p (Fig. 10A). The latter suggests that mir-150-5p induced gene expression changes are not sufficient to trigger GC induced cell death and additional GC actions are required to kill the cells [15]. Next, we evaluated whether ectopic mir-150-5p transfection may sensitize for GC induced cell death. In this respect, mock and mir-150-5p transfected MM1S cells were exposed to various doses of Dex for 72 h and cell survival was again evaluated by a MTT-assay. From Fig. 10B, it can be observed that presence of mir-150-5p weakly increases GC induced cell death at nanomolar (nM) concentrations, although no significant effects could be discriminated at micromolar (µM) concentrations of Dex treatment. Moreover, upon GC treatment of MM1S cells transfected with a mir-150-5p specific antagomir, to reverse mir-150-5p effects, cell death could be reversed at low GC doses. This prompted us to evaluate corresponding changes in gene expression following GC treatment of MM1S cells transfected with mir-150-5p antagomir. Although we could confirm weak GC specific upregulation of mir150-5p and mild reduction of mir 150-5p levels upon GC treatment of MM1S cells transfected with mir-150-5p specific antagomir (Fig. 10C), array approaches seemed not sensitive enough to identify weak changes in gene repression or activation in GC treated cells in presence or absence of the antagomir (Fig. 10D–E and data not shown).


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)

Weak sensitisation of low dose GC therapy response by mir-150-5p mimetic transfection can be reversed by transfection of a mir-150-5p-specific antagomir in MM1S cells.(A) MTT based quantification of MM1S cell survival, following 24, 48, or 72 h transfection with increasing doses of synthetic mir-150-5p mimetic as compared to mock transfection control (B) MTT based quantification of MM1S cell survival, following 72 h treatment with increasing doses of Dex, in combination with either mock transfection, else transfection with a synthetic mir-150-5p mimetic or mir-150-5p specific antagomir (as indicated in figure). Data represented are relative cell survival levels (mean ± SEM) of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttests). (C) Realtime quantitative PCR (QPCR) quantification of mir-150-5p transcription levels following 72 h treatment with 1 nM Dex, either or not in combination with transfection of a mir-150-5p specific antagomir. (D–E) Illumina BeadChip Gene Expression results of a subset of genes are presented as bargraphs, reflecting the mean fold change from three independent experiments of MM1S cells treated for 72 h with 1 nM Dex vs. control setup, either or not in combination with transfection of a mir-150-5p specific antagomir.
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Related In: Results  -  Collection

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

pone-0113842-g010: Weak sensitisation of low dose GC therapy response by mir-150-5p mimetic transfection can be reversed by transfection of a mir-150-5p-specific antagomir in MM1S cells.(A) MTT based quantification of MM1S cell survival, following 24, 48, or 72 h transfection with increasing doses of synthetic mir-150-5p mimetic as compared to mock transfection control (B) MTT based quantification of MM1S cell survival, following 72 h treatment with increasing doses of Dex, in combination with either mock transfection, else transfection with a synthetic mir-150-5p mimetic or mir-150-5p specific antagomir (as indicated in figure). Data represented are relative cell survival levels (mean ± SEM) of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttests). (C) Realtime quantitative PCR (QPCR) quantification of mir-150-5p transcription levels following 72 h treatment with 1 nM Dex, either or not in combination with transfection of a mir-150-5p specific antagomir. (D–E) Illumina BeadChip Gene Expression results of a subset of genes are presented as bargraphs, reflecting the mean fold change from three independent experiments of MM1S cells treated for 72 h with 1 nM Dex vs. control setup, either or not in combination with transfection of a mir-150-5p specific antagomir.
Mentions: Since mir-150-5p modulates various genes related to cell cycle, cell proliferation and cell death pathways, we next wanted to measure cell death induced by ectopic mir-150-5p transfection. Surprisingly, no substantial induction of cell death could be observed by MTT-assay at 24, 48 and 72 h following transfection of MM1S cells by increasing amounts of mir150-5p (Fig. 10A). The latter suggests that mir-150-5p induced gene expression changes are not sufficient to trigger GC induced cell death and additional GC actions are required to kill the cells [15]. Next, we evaluated whether ectopic mir-150-5p transfection may sensitize for GC induced cell death. In this respect, mock and mir-150-5p transfected MM1S cells were exposed to various doses of Dex for 72 h and cell survival was again evaluated by a MTT-assay. From Fig. 10B, it can be observed that presence of mir-150-5p weakly increases GC induced cell death at nanomolar (nM) concentrations, although no significant effects could be discriminated at micromolar (µM) concentrations of Dex treatment. Moreover, upon GC treatment of MM1S cells transfected with a mir-150-5p specific antagomir, to reverse mir-150-5p effects, cell death could be reversed at low GC doses. This prompted us to evaluate corresponding changes in gene expression following GC treatment of MM1S cells transfected with mir-150-5p antagomir. Although we could confirm weak GC specific upregulation of mir150-5p and mild reduction of mir 150-5p levels upon GC treatment of MM1S cells transfected with mir-150-5p specific antagomir (Fig. 10C), array approaches seemed not sensitive enough to identify weak changes in gene repression or activation in GC treated cells in presence or absence of the antagomir (Fig. 10D–E and data not shown).

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