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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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Mir-150-5p mimetic transfection triggers GR specific gene expression in MM1S but not in MM1R cells, without modulating GR protein levels.(A) QPCR based quantification of synthetic mir-150-5p levels present 72 h after transfection of MM1S and MM1R cells. The miRNA expression levels have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs and are presented relative to MOCK control. Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setups according to two-way ANOVA (Bonferroni post-tests). (B) Comparison of top enriched pathways (IPA) of mRNA changes in MM1S cells treated for 72 h to 1 µM Dex versus changes in MM1S and MM1R cells after 72 h mock transfection of transfection with synthetic mir-150-5p. (C) NR3C1/GRα protein levels detected following 72 h transfection of a synthetic mir-150-5p mimetic in MM1S cells. (D) Venn-diagram which represents the overlap of experimentally validated GC inducible microRNAs in MM1S cells with the list of miRNAs predicted to target the 3′UTR of NR3C1. (E) QPCR analysis of NR3C1/GRα mRNA levels present in MM1S cells following 72 h transfection of a synthetic mir-150-5p mimetic (left panel) or following 72 h Dex (1 µM) treatment (right panel).
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pone-0113842-g005: Mir-150-5p mimetic transfection triggers GR specific gene expression in MM1S but not in MM1R cells, without modulating GR protein levels.(A) QPCR based quantification of synthetic mir-150-5p levels present 72 h after transfection of MM1S and MM1R cells. The miRNA expression levels have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs and are presented relative to MOCK control. Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setups according to two-way ANOVA (Bonferroni post-tests). (B) Comparison of top enriched pathways (IPA) of mRNA changes in MM1S cells treated for 72 h to 1 µM Dex versus changes in MM1S and MM1R cells after 72 h mock transfection of transfection with synthetic mir-150-5p. (C) NR3C1/GRα protein levels detected following 72 h transfection of a synthetic mir-150-5p mimetic in MM1S cells. (D) Venn-diagram which represents the overlap of experimentally validated GC inducible microRNAs in MM1S cells with the list of miRNAs predicted to target the 3′UTR of NR3C1. (E) QPCR analysis of NR3C1/GRα mRNA levels present in MM1S cells following 72 h transfection of a synthetic mir-150-5p mimetic (left panel) or following 72 h Dex (1 µM) treatment (right panel).

Mentions: To validate possible mir-150-5p effects on the predicted mRNA targets and/or cell cycle or cell proliferation pathways, we performed transfection experiments in MM1S and MM1R cells with a synthetic mir-150-5p mimetic in comparison to Dex treatment. Remarkably, although MM1S and MM1R show similar transcription levels of mir-150-5p upon mimetic transfection (Fig. 5A), we could only observe significant changes in mir-150-5p responsive mRNAs in MM1S but not in MM1R cells, upon analysing Illumina mRNA profiles. Interestingly, upon cross comparing Dex treated and mir-150-5p mimetic transfected MM1S/MM1R cells, IPA analysis revealed a remarkable overlap in mRNAs affected related to cell cycle-proliferation, DNA repair and cell death pathways in MM1S, as well as lack of effects for both setups in MM1R cells (Fig. 5B) (Table 4 and 5). To evaluate whether mir-150-5p may change GR/NR3C1 mRNA levels or protein stability, we performed QPCR and Western blot analysis following mir-150-5p transfection in MM1S cells. From Fig. 5C and D, it can be observed that mir-150-5p transfection does not change GR/NR3C1 mRNA or protein levels respectively. These results are in line with the in silico data, showing that none of the validated GC-inducible miRs are targeting GR-3′UTR mRNA (Fig. 5E and Table S3). Altogether, these results suggest that the mir-150-5p may mediate indirect GR like effects.


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)

Mir-150-5p mimetic transfection triggers GR specific gene expression in MM1S but not in MM1R cells, without modulating GR protein levels.(A) QPCR based quantification of synthetic mir-150-5p levels present 72 h after transfection of MM1S and MM1R cells. The miRNA expression levels have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs and are presented relative to MOCK control. Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setups according to two-way ANOVA (Bonferroni post-tests). (B) Comparison of top enriched pathways (IPA) of mRNA changes in MM1S cells treated for 72 h to 1 µM Dex versus changes in MM1S and MM1R cells after 72 h mock transfection of transfection with synthetic mir-150-5p. (C) NR3C1/GRα protein levels detected following 72 h transfection of a synthetic mir-150-5p mimetic in MM1S cells. (D) Venn-diagram which represents the overlap of experimentally validated GC inducible microRNAs in MM1S cells with the list of miRNAs predicted to target the 3′UTR of NR3C1. (E) QPCR analysis of NR3C1/GRα mRNA levels present in MM1S cells following 72 h transfection of a synthetic mir-150-5p mimetic (left panel) or following 72 h Dex (1 µM) treatment (right panel).
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pone-0113842-g005: Mir-150-5p mimetic transfection triggers GR specific gene expression in MM1S but not in MM1R cells, without modulating GR protein levels.(A) QPCR based quantification of synthetic mir-150-5p levels present 72 h after transfection of MM1S and MM1R cells. The miRNA expression levels have been normalized to SNORD 95 and SNORD 96A housekeeping miRNAs and are presented relative to MOCK control. Bar graphs represent relative miRNA (mean ± SEM) levels of three independent experiments. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setups according to two-way ANOVA (Bonferroni post-tests). (B) Comparison of top enriched pathways (IPA) of mRNA changes in MM1S cells treated for 72 h to 1 µM Dex versus changes in MM1S and MM1R cells after 72 h mock transfection of transfection with synthetic mir-150-5p. (C) NR3C1/GRα protein levels detected following 72 h transfection of a synthetic mir-150-5p mimetic in MM1S cells. (D) Venn-diagram which represents the overlap of experimentally validated GC inducible microRNAs in MM1S cells with the list of miRNAs predicted to target the 3′UTR of NR3C1. (E) QPCR analysis of NR3C1/GRα mRNA levels present in MM1S cells following 72 h transfection of a synthetic mir-150-5p mimetic (left panel) or following 72 h Dex (1 µM) treatment (right panel).
Mentions: To validate possible mir-150-5p effects on the predicted mRNA targets and/or cell cycle or cell proliferation pathways, we performed transfection experiments in MM1S and MM1R cells with a synthetic mir-150-5p mimetic in comparison to Dex treatment. Remarkably, although MM1S and MM1R show similar transcription levels of mir-150-5p upon mimetic transfection (Fig. 5A), we could only observe significant changes in mir-150-5p responsive mRNAs in MM1S but not in MM1R cells, upon analysing Illumina mRNA profiles. Interestingly, upon cross comparing Dex treated and mir-150-5p mimetic transfected MM1S/MM1R cells, IPA analysis revealed a remarkable overlap in mRNAs affected related to cell cycle-proliferation, DNA repair and cell death pathways in MM1S, as well as lack of effects for both setups in MM1R cells (Fig. 5B) (Table 4 and 5). To evaluate whether mir-150-5p may change GR/NR3C1 mRNA levels or protein stability, we performed QPCR and Western blot analysis following mir-150-5p transfection in MM1S cells. From Fig. 5C and D, it can be observed that mir-150-5p transfection does not change GR/NR3C1 mRNA or protein levels respectively. These results are in line with the in silico data, showing that none of the validated GC-inducible miRs are targeting GR-3′UTR mRNA (Fig. 5E and Table S3). Altogether, these results suggest that the mir-150-5p may mediate indirect GR like effects.

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