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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

Mir-150-5p transfection mimics GR like gene activation and repression response in MM1S and lack of response in MM1R cells.(A) Realtime quantitative PCR (QPCR) validation of GILZ (TSC22D3) and FKBP5 mRNA induction in MM1S and in MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex (left panel), or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof (right panel). Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttest). (B) Realtime quantitative PCR (QPCR) validation of MYB, IL23A, SKP2, BUB1, SREBF1 mRNA repression levels in MM1S or MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex, or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof. Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. 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) Illumina BeadChip Gene Expression Array results of selected genes are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S cells, treated for 72 h with 1 µM Dex, or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection, treated for 72 h with 1 nM Dex or a combination thereof versus control samples.
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pone-0113842-g007: Mir-150-5p transfection mimics GR like gene activation and repression response in MM1S and lack of response in MM1R cells.(A) Realtime quantitative PCR (QPCR) validation of GILZ (TSC22D3) and FKBP5 mRNA induction in MM1S and in MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex (left panel), or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof (right panel). Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttest). (B) Realtime quantitative PCR (QPCR) validation of MYB, IL23A, SKP2, BUB1, SREBF1 mRNA repression levels in MM1S or MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex, or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof. Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. 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) Illumina BeadChip Gene Expression Array results of selected genes are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S cells, treated for 72 h with 1 µM Dex, or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection, treated for 72 h with 1 nM Dex or a combination thereof versus control samples.

Mentions: The genes involved in cell cycle-proliferation, DNA repair and cell death pathways which are regulated in Dex treated and mir-150-5p mimetic transfected MM1S/MM1R cells are indicated in Figure 6A and 6B or summarized in Table S4. To further characterize crosstalk between GC treatment and mir-150-5p transfection, we also performed microarray analysis of combination setups of mir-150-5p with 1 nM Dex, since 1 µM Dex treatment combinations did not yield array quality RNA because of excessive cytotoxicity. As can be noted from Fig. 6A and 6B, 1 nM Dex treatment triggers weaker gene expression changes as compared to 1 µM Dex treatment. Furthermore, since mir-150-5p levels upon transfection increase 30-fold (Fig. 6A) as compared to 3-fold induction upon Dex treatment (Fig. 3D), mir-150-5p mimetic overexpression triggers stronger gene expression changes than 1 nM Dex treatment. As such, combining mir-150-5p transfection with 1 nM Dex treatment did not further increase gene expression changes elicited by mir-150-p transfection alone (Fig. 7A & B). A further detailed comparison of GC and mir-150-5p responsive activation (i.e. GILZ and FKBP5) (Fig. 7A) or repression (IL23A, SKP2, BUB, SREBF1) (Fig. 7B) of typical GR target genes revealed similar effects in MM1S but lack of effects in MM1R. For example, transactivation of the FKBP5 and GILZ genes can be observed following mir-150-5p transfection and GC treatment in MM1S cells, although mir-150-5p effects on FKBP5 expression are stronger. Along the same line, repression of GR target genes IL23A, SKP2, BUB or SREBF1 can be observed upon Dex treatment as well following mir-150-5p transfection. Finally, besides the common regulated genes, additional GR target genes could be identified which were more selectively affected by mir-150-5p transfection (SCNN1G) or else GC treatment (GILZ, DUSP1, SGK1) indicating sophisticated fine tuning of GR regulation (Fig. 7C).


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 transfection mimics GR like gene activation and repression response in MM1S and lack of response in MM1R cells.(A) Realtime quantitative PCR (QPCR) validation of GILZ (TSC22D3) and FKBP5 mRNA induction in MM1S and in MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex (left panel), or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof (right panel). Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttest). (B) Realtime quantitative PCR (QPCR) validation of MYB, IL23A, SKP2, BUB1, SREBF1 mRNA repression levels in MM1S or MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex, or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof. Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. 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) Illumina BeadChip Gene Expression Array results of selected genes are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S cells, treated for 72 h with 1 µM Dex, or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection, treated for 72 h with 1 nM Dex or a combination thereof versus control samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113842-g007: Mir-150-5p transfection mimics GR like gene activation and repression response in MM1S and lack of response in MM1R cells.(A) Realtime quantitative PCR (QPCR) validation of GILZ (TSC22D3) and FKBP5 mRNA induction in MM1S and in MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex (left panel), or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof (right panel). Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. Means with ***, **, * are significantly different (p<0.001, <0.01 or <0.05) from control setup as determined by two-way ANOVA (Bonferroni posttest). (B) Realtime quantitative PCR (QPCR) validation of MYB, IL23A, SKP2, BUB1, SREBF1 mRNA repression levels in MM1S or MM1R cells, following 72 h treatment with 1 nM or 1 µM Dex, or following 72 h transfection with synthetic mir-150-5p, 72 h treatment with 1 nM Dex, or a combination thereof. Data represent (mean ± SEM) values of three independent experiments normalized to 28S RNA housekeeping gene and relative to the respective untreated/solvent (UT) or MOCK control setups. 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) Illumina BeadChip Gene Expression Array results of selected genes are presented as bargraphs, reflecting mean of gene expression fold change from three independent experiments of MM1S cells, treated for 72 h with 1 µM Dex, or else of MM1S cells transfected for 72 h with synthetic mir-150-5p versus mock transfection, treated for 72 h with 1 nM Dex or a combination thereof versus control samples.
Mentions: The genes involved in cell cycle-proliferation, DNA repair and cell death pathways which are regulated in Dex treated and mir-150-5p mimetic transfected MM1S/MM1R cells are indicated in Figure 6A and 6B or summarized in Table S4. To further characterize crosstalk between GC treatment and mir-150-5p transfection, we also performed microarray analysis of combination setups of mir-150-5p with 1 nM Dex, since 1 µM Dex treatment combinations did not yield array quality RNA because of excessive cytotoxicity. As can be noted from Fig. 6A and 6B, 1 nM Dex treatment triggers weaker gene expression changes as compared to 1 µM Dex treatment. Furthermore, since mir-150-5p levels upon transfection increase 30-fold (Fig. 6A) as compared to 3-fold induction upon Dex treatment (Fig. 3D), mir-150-5p mimetic overexpression triggers stronger gene expression changes than 1 nM Dex treatment. As such, combining mir-150-5p transfection with 1 nM Dex treatment did not further increase gene expression changes elicited by mir-150-p transfection alone (Fig. 7A & B). A further detailed comparison of GC and mir-150-5p responsive activation (i.e. GILZ and FKBP5) (Fig. 7A) or repression (IL23A, SKP2, BUB, SREBF1) (Fig. 7B) of typical GR target genes revealed similar effects in MM1S but lack of effects in MM1R. For example, transactivation of the FKBP5 and GILZ genes can be observed following mir-150-5p transfection and GC treatment in MM1S cells, although mir-150-5p effects on FKBP5 expression are stronger. Along the same line, repression of GR target genes IL23A, SKP2, BUB or SREBF1 can be observed upon Dex treatment as well following mir-150-5p transfection. Finally, besides the common regulated genes, additional GR target genes could be identified which were more selectively affected by mir-150-5p transfection (SCNN1G) or else GC treatment (GILZ, DUSP1, SGK1) indicating sophisticated fine tuning of GR regulation (Fig. 7C).

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