Limits...
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.

Show MeSH

Related in: MedlinePlus

RAB5C mediates the growth-inhibitory effect of miR-509.(A) AlamarBlue assay of NALM6 cells transduced with either lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C on 7 days after transduction. Data represent means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (B) Representative western blot of NALM6 cells transduced with lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (C) Quantitation of western blots shown in (B) and 2 other independent experiments. Relative densitometry values were calculated relative to Scr Ctrl. Results show means ± SEMs with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (D) Enforced expression of RAB5C without its 3′UTR rescues miR-509-mediated growth inhibition. NALM6 cells were co-transduced with the indicated plasmids, and alamarBlue assay was read at 7 days after transduction. The empty lentiviral vector in this experiment is EV#3, which does not have RAB5C cloned in. Results show means ± SEMs of 3 independent experiments, with statistical analysis using Student's t test. *p<0.05 and ***p<0.001. (E) Representative western blot of NALM6 cells co-transduced with lentivirus of the indicated plasmids. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (F) Quantitation of western blots shown in (E) and 2 other independent experiments. Relative densitometry values were calculated relative to EV#1 and EV#3. Results show means ± SEMs with statistical analysis by Student's t test. *p<0.05 and ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4219775&req=5

pone-0111777-g006: RAB5C mediates the growth-inhibitory effect of miR-509.(A) AlamarBlue assay of NALM6 cells transduced with either lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C on 7 days after transduction. Data represent means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (B) Representative western blot of NALM6 cells transduced with lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (C) Quantitation of western blots shown in (B) and 2 other independent experiments. Relative densitometry values were calculated relative to Scr Ctrl. Results show means ± SEMs with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (D) Enforced expression of RAB5C without its 3′UTR rescues miR-509-mediated growth inhibition. NALM6 cells were co-transduced with the indicated plasmids, and alamarBlue assay was read at 7 days after transduction. The empty lentiviral vector in this experiment is EV#3, which does not have RAB5C cloned in. Results show means ± SEMs of 3 independent experiments, with statistical analysis using Student's t test. *p<0.05 and ***p<0.001. (E) Representative western blot of NALM6 cells co-transduced with lentivirus of the indicated plasmids. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (F) Quantitation of western blots shown in (E) and 2 other independent experiments. Relative densitometry values were calculated relative to EV#1 and EV#3. Results show means ± SEMs with statistical analysis by Student's t test. *p<0.05 and ***p<0.001.

Mentions: We then examined if reduced RAB5C is responsible for the functional effects of miR-509. To determine if repression of RAB5C would phenocopy the growth suppressive effect of miR-509, NALM6 cells were transduced with 3 different lentiviruses, each containing a distinct shRNA against RAB5C. In alamarBlue assays, all 3 shRNAs inhibited NALM6 cell growth by ≥42% (p<0.01) as compared to cells transduced with the scrambled control (Figure 6A). We verified that all 3 shRNAs resulted in ≥80% decreased RAB5C protein levels (p<0.01) in NALM6 cells (Figure 6B, 6C) using western blotting.


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)

RAB5C mediates the growth-inhibitory effect of miR-509.(A) AlamarBlue assay of NALM6 cells transduced with either lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C on 7 days after transduction. Data represent means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (B) Representative western blot of NALM6 cells transduced with lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (C) Quantitation of western blots shown in (B) and 2 other independent experiments. Relative densitometry values were calculated relative to Scr Ctrl. Results show means ± SEMs with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (D) Enforced expression of RAB5C without its 3′UTR rescues miR-509-mediated growth inhibition. NALM6 cells were co-transduced with the indicated plasmids, and alamarBlue assay was read at 7 days after transduction. The empty lentiviral vector in this experiment is EV#3, which does not have RAB5C cloned in. Results show means ± SEMs of 3 independent experiments, with statistical analysis using Student's t test. *p<0.05 and ***p<0.001. (E) Representative western blot of NALM6 cells co-transduced with lentivirus of the indicated plasmids. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (F) Quantitation of western blots shown in (E) and 2 other independent experiments. Relative densitometry values were calculated relative to EV#1 and EV#3. Results show means ± SEMs with statistical analysis by Student's t test. *p<0.05 and ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219775&req=5

pone-0111777-g006: RAB5C mediates the growth-inhibitory effect of miR-509.(A) AlamarBlue assay of NALM6 cells transduced with either lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C on 7 days after transduction. Data represent means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (B) Representative western blot of NALM6 cells transduced with lentivirus of scrambled control (Scr Ctrl), shRNA#1, shRNA#2 or shRNA#3 for RAB5C. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (C) Quantitation of western blots shown in (B) and 2 other independent experiments. Relative densitometry values were calculated relative to Scr Ctrl. Results show means ± SEMs with statistical analysis by Student's t test. **p<0.01 and ***p<0.001. (D) Enforced expression of RAB5C without its 3′UTR rescues miR-509-mediated growth inhibition. NALM6 cells were co-transduced with the indicated plasmids, and alamarBlue assay was read at 7 days after transduction. The empty lentiviral vector in this experiment is EV#3, which does not have RAB5C cloned in. Results show means ± SEMs of 3 independent experiments, with statistical analysis using Student's t test. *p<0.05 and ***p<0.001. (E) Representative western blot of NALM6 cells co-transduced with lentivirus of the indicated plasmids. Protein lysates were harvested 7 days after transduction and α-tubulin was used as a loading control. (F) Quantitation of western blots shown in (E) and 2 other independent experiments. Relative densitometry values were calculated relative to EV#1 and EV#3. Results show means ± SEMs with statistical analysis by Student's t test. *p<0.05 and ***p<0.001.
Mentions: We then examined if reduced RAB5C is responsible for the functional effects of miR-509. To determine if repression of RAB5C would phenocopy the growth suppressive effect of miR-509, NALM6 cells were transduced with 3 different lentiviruses, each containing a distinct shRNA against RAB5C. In alamarBlue assays, all 3 shRNAs inhibited NALM6 cell growth by ≥42% (p<0.01) as compared to cells transduced with the scrambled control (Figure 6A). We verified that all 3 shRNAs resulted in ≥80% decreased RAB5C protein levels (p<0.01) in NALM6 cells (Figure 6B, 6C) using western blotting.

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