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Regulation of RAB5C is important for the growth inhibitory effects of MiR-509 in human precursor-B acute lymphoblastic leukemia.

Tan YS, Kim M, Kingsbury TJ, Civin CI, Cheng WC - PLoS ONE (2014)

Bottom Line: Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509.Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509.Co-expression of the RAB5C open reading frame without its 3' untranslated region (3'UTR) blocked the growth-inhibitory effect mediated by miR-509.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
MicroRNAs (miRs) regulate essentially all cellular processes, but few miRs are known to inhibit growth of precursor-B acute lymphoblastic leukemias (B-ALLs). We identified miR-509 via a human genome-wide gain-of-function screen for miRs that inhibit growth of the NALM6 human B-ALL cell line. MiR-509-mediated inhibition of NALM6 growth was confirmed by 3 independent assays. Enforced miR-509 expression inhibited 2 of 2 additional B-ALL cell lines tested, but not 3 non-B-ALL leukemia cell lines. MiR-509-transduced NALM6 cells had reduced numbers of actively proliferating cells and increased numbers of cells undergoing apoptosis. Using miR target prediction algorithms and a filtering strategy, RAB5C was predicted as a potentially relevant target of miR-509. Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509. Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509. Co-expression of the RAB5C open reading frame without its 3' untranslated region (3'UTR) blocked the growth-inhibitory effect mediated by miR-509. These findings establish RAB5C as a target of miR-509 and an important regulator of B-ALL cell growth with potential as a therapeutic target.

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Enforced miR-509 resulted in inhibition of growth of 3 B-ALL cell lines, NALM6, REH and RCH-ACV.(A) Viable cell numbers measured via trypan blue dye exclusion counts of NALM6 cells transduced with either miR-509 lentivirus or empty vector (EV#1); 25,000 cells were plated for each sample starting at 3 days after transduction. (B) AlamarBlue cell growth assay on day 7 after transduction of NALM6 cells transduced with either miR-509 lentivirus or EV#1. Values for miR-509 were normalized to EV#1. (C) Viable cell counts of RCH-ACV cells based on trypan blue exclusion counts, initial plating of 25,000 cells for both samples on 3 days after transduction. Means ± SEMs are plotted, and SEMs for miR-509 were very small. (D) Cell growth of RCH-ACV transduced with either EV#1 or miR-509 overexpressing lentivirus using alamarBlue cell growth assay conducted on day 7 after transduction. Values for miR-509 were normalized to EV#1. (E) MiR-509-transduced REH cells reduced growth compared to EV#1 in an alamarBlue cell growth assay. Cells were transduced 7 days prior to addition of alamarBlue. (A to E) Means ± SEMs, 3 independent experiments done in triplicates. Statistical analysis was done by Student's t test. *p<0.05, **p<0.01.
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pone-0111777-g002: Enforced miR-509 resulted in inhibition of growth of 3 B-ALL cell lines, NALM6, REH and RCH-ACV.(A) Viable cell numbers measured via trypan blue dye exclusion counts of NALM6 cells transduced with either miR-509 lentivirus or empty vector (EV#1); 25,000 cells were plated for each sample starting at 3 days after transduction. (B) AlamarBlue cell growth assay on day 7 after transduction of NALM6 cells transduced with either miR-509 lentivirus or EV#1. Values for miR-509 were normalized to EV#1. (C) Viable cell counts of RCH-ACV cells based on trypan blue exclusion counts, initial plating of 25,000 cells for both samples on 3 days after transduction. Means ± SEMs are plotted, and SEMs for miR-509 were very small. (D) Cell growth of RCH-ACV transduced with either EV#1 or miR-509 overexpressing lentivirus using alamarBlue cell growth assay conducted on day 7 after transduction. Values for miR-509 were normalized to EV#1. (E) MiR-509-transduced REH cells reduced growth compared to EV#1 in an alamarBlue cell growth assay. Cells were transduced 7 days prior to addition of alamarBlue. (A to E) Means ± SEMs, 3 independent experiments done in triplicates. Statistical analysis was done by Student's t test. *p<0.05, **p<0.01.

Mentions: To further confirm the effect of miR-509 on NALM6 cell growth, we performed trypan blue dye exclusion cell counts and alamarBlue assays. At 8 days after transduction, cultures of miR-509-transduced NALM6 cells contained 43% fewer viable cells than empty vector-transduced cells by trypan blue counts (Figure 2A). Similarly, miR-509-transduced NALM6 cells had 48% reduced (p<0.05) cell growth, as compared to empty vector-transduced cells using the alamarBlue assay (Figure 2B). Since the alamarBlue dye-based assay measures the reducing environment within cells, which is linked to mitochondria metabolism [35], we examined whether miR-509 affected mitochondrial membrane potential. No difference in mitochondrial membrane potential was observed between miR-509-transduced and empty vector-transduced NALM6 cells (data not shown).


Regulation of RAB5C is important for the growth inhibitory effects of MiR-509 in human precursor-B acute lymphoblastic leukemia.

Tan YS, Kim M, Kingsbury TJ, Civin CI, Cheng WC - PLoS ONE (2014)

Enforced miR-509 resulted in inhibition of growth of 3 B-ALL cell lines, NALM6, REH and RCH-ACV.(A) Viable cell numbers measured via trypan blue dye exclusion counts of NALM6 cells transduced with either miR-509 lentivirus or empty vector (EV#1); 25,000 cells were plated for each sample starting at 3 days after transduction. (B) AlamarBlue cell growth assay on day 7 after transduction of NALM6 cells transduced with either miR-509 lentivirus or EV#1. Values for miR-509 were normalized to EV#1. (C) Viable cell counts of RCH-ACV cells based on trypan blue exclusion counts, initial plating of 25,000 cells for both samples on 3 days after transduction. Means ± SEMs are plotted, and SEMs for miR-509 were very small. (D) Cell growth of RCH-ACV transduced with either EV#1 or miR-509 overexpressing lentivirus using alamarBlue cell growth assay conducted on day 7 after transduction. Values for miR-509 were normalized to EV#1. (E) MiR-509-transduced REH cells reduced growth compared to EV#1 in an alamarBlue cell growth assay. Cells were transduced 7 days prior to addition of alamarBlue. (A to E) Means ± SEMs, 3 independent experiments done in triplicates. Statistical analysis was done by Student's t test. *p<0.05, **p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219775&req=5

pone-0111777-g002: Enforced miR-509 resulted in inhibition of growth of 3 B-ALL cell lines, NALM6, REH and RCH-ACV.(A) Viable cell numbers measured via trypan blue dye exclusion counts of NALM6 cells transduced with either miR-509 lentivirus or empty vector (EV#1); 25,000 cells were plated for each sample starting at 3 days after transduction. (B) AlamarBlue cell growth assay on day 7 after transduction of NALM6 cells transduced with either miR-509 lentivirus or EV#1. Values for miR-509 were normalized to EV#1. (C) Viable cell counts of RCH-ACV cells based on trypan blue exclusion counts, initial plating of 25,000 cells for both samples on 3 days after transduction. Means ± SEMs are plotted, and SEMs for miR-509 were very small. (D) Cell growth of RCH-ACV transduced with either EV#1 or miR-509 overexpressing lentivirus using alamarBlue cell growth assay conducted on day 7 after transduction. Values for miR-509 were normalized to EV#1. (E) MiR-509-transduced REH cells reduced growth compared to EV#1 in an alamarBlue cell growth assay. Cells were transduced 7 days prior to addition of alamarBlue. (A to E) Means ± SEMs, 3 independent experiments done in triplicates. Statistical analysis was done by Student's t test. *p<0.05, **p<0.01.
Mentions: To further confirm the effect of miR-509 on NALM6 cell growth, we performed trypan blue dye exclusion cell counts and alamarBlue assays. At 8 days after transduction, cultures of miR-509-transduced NALM6 cells contained 43% fewer viable cells than empty vector-transduced cells by trypan blue counts (Figure 2A). Similarly, miR-509-transduced NALM6 cells had 48% reduced (p<0.05) cell growth, as compared to empty vector-transduced cells using the alamarBlue assay (Figure 2B). Since the alamarBlue dye-based assay measures the reducing environment within cells, which is linked to mitochondria metabolism [35], we examined whether miR-509 affected mitochondrial membrane potential. No difference in mitochondrial membrane potential was observed between miR-509-transduced and empty vector-transduced NALM6 cells (data not shown).

Bottom Line: Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509.Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509.Co-expression of the RAB5C open reading frame without its 3' untranslated region (3'UTR) blocked the growth-inhibitory effect mediated by miR-509.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
MicroRNAs (miRs) regulate essentially all cellular processes, but few miRs are known to inhibit growth of precursor-B acute lymphoblastic leukemias (B-ALLs). We identified miR-509 via a human genome-wide gain-of-function screen for miRs that inhibit growth of the NALM6 human B-ALL cell line. MiR-509-mediated inhibition of NALM6 growth was confirmed by 3 independent assays. Enforced miR-509 expression inhibited 2 of 2 additional B-ALL cell lines tested, but not 3 non-B-ALL leukemia cell lines. MiR-509-transduced NALM6 cells had reduced numbers of actively proliferating cells and increased numbers of cells undergoing apoptosis. Using miR target prediction algorithms and a filtering strategy, RAB5C was predicted as a potentially relevant target of miR-509. Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509. Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509. Co-expression of the RAB5C open reading frame without its 3' untranslated region (3'UTR) blocked the growth-inhibitory effect mediated by miR-509. These findings establish RAB5C as a target of miR-509 and an important regulator of B-ALL cell growth with potential as a therapeutic target.

Show MeSH
Related in: MedlinePlus