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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 expression in decreased proportion of cells in S-phase, induced apoptosis and activated caspase-3/7.(A) Representative flow cytometric plots showing cell cycle distribution of NALM6 cells transduced with empty vector (EV#1) or miR-509 overexpressing lentivirus. On day 3 after transduction, cells were labeled with BrdU for 1 h. Cells were then fixed overnight and stained on the next day with both BrdU and 7-AAD before analysis by flow cytometry. Percent of cells at each phase of cell cycle are boxed as indicated. (B) Frequencies of cells at the different phases of cell cycle. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (C) Representative flow cytometric plots of cell death distribution of NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus. Cells were stained with both Annexin V and 7-AAD before analysis by flow cytometry on day 4 after transduction. Numbers represent the frequency in each quadrant. (D) Frequencies of apoptotic cells which are Annexin V-positive (Annexin V+) and 7-AAD negative (7-AAD–), as well as total Annexin V+. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (E) NALM6 cells were transduced with empty vector #2 (EV#2) or miR-509 overexpressing lentivirus, and cells were seeded in 384-well plate on day 3 after transduction. On day 7 after transduction, caspase-3/7 activity was measured and fold-change in caspase activity was normalized to EV#2. Means ± SEMs of 3 independent experiments done in triplicates, with statistical analysis by Student's t test. *p<0.05.
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pone-0111777-g003: Enforced miR-509 expression in decreased proportion of cells in S-phase, induced apoptosis and activated caspase-3/7.(A) Representative flow cytometric plots showing cell cycle distribution of NALM6 cells transduced with empty vector (EV#1) or miR-509 overexpressing lentivirus. On day 3 after transduction, cells were labeled with BrdU for 1 h. Cells were then fixed overnight and stained on the next day with both BrdU and 7-AAD before analysis by flow cytometry. Percent of cells at each phase of cell cycle are boxed as indicated. (B) Frequencies of cells at the different phases of cell cycle. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (C) Representative flow cytometric plots of cell death distribution of NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus. Cells were stained with both Annexin V and 7-AAD before analysis by flow cytometry on day 4 after transduction. Numbers represent the frequency in each quadrant. (D) Frequencies of apoptotic cells which are Annexin V-positive (Annexin V+) and 7-AAD negative (7-AAD–), as well as total Annexin V+. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (E) NALM6 cells were transduced with empty vector #2 (EV#2) or miR-509 overexpressing lentivirus, and cells were seeded in 384-well plate on day 3 after transduction. On day 7 after transduction, caspase-3/7 activity was measured and fold-change in caspase activity was normalized to EV#2. Means ± SEMs of 3 independent experiments done in triplicates, with statistical analysis by Student's t test. *p<0.05.

Mentions: To investigate the cellular mechanisms by which enforced miR-509 expression inhibits growth, we examined whether miR-509 regulates cell cycle progression by conducting BrdU/7-AAD staining [36]. 4 days after transduction, miR-509-transduced NALM6 had fewer cells in S-phase than empty vector-transduced cells (Figure 3A), and this was statistically significant (Figure 3B, p<0.05). In addition, there were slightly elevated numbers of cells in the subG1 and G2/M phases, but these differences were not statistically significant. To investigate if miR-509 promotes cell death via apoptosis, Annexin V/7-AAD staining was performed. 4 days after transduction, miR-509-transduced NALM6 cells had 1.5-fold (p<0.05) higher numbers of Annexin V+/7-AAD– apoptotic cells and 1.4-fold higher numbers of Annexin V+ dying/dead cells (p<0.05), as compared to empty vector-transduced cells (Figure 3C, 3D). Consistent with these findings, we detected a 1.5-fold increase (p<0.05) in activated capase-3/7 activity in miR-509-transduced NALM6 cells as compared to empty vector-transduced cells (Figure 3E).


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 expression in decreased proportion of cells in S-phase, induced apoptosis and activated caspase-3/7.(A) Representative flow cytometric plots showing cell cycle distribution of NALM6 cells transduced with empty vector (EV#1) or miR-509 overexpressing lentivirus. On day 3 after transduction, cells were labeled with BrdU for 1 h. Cells were then fixed overnight and stained on the next day with both BrdU and 7-AAD before analysis by flow cytometry. Percent of cells at each phase of cell cycle are boxed as indicated. (B) Frequencies of cells at the different phases of cell cycle. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (C) Representative flow cytometric plots of cell death distribution of NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus. Cells were stained with both Annexin V and 7-AAD before analysis by flow cytometry on day 4 after transduction. Numbers represent the frequency in each quadrant. (D) Frequencies of apoptotic cells which are Annexin V-positive (Annexin V+) and 7-AAD negative (7-AAD–), as well as total Annexin V+. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (E) NALM6 cells were transduced with empty vector #2 (EV#2) or miR-509 overexpressing lentivirus, and cells were seeded in 384-well plate on day 3 after transduction. On day 7 after transduction, caspase-3/7 activity was measured and fold-change in caspase activity was normalized to EV#2. Means ± SEMs of 3 independent experiments done in triplicates, with statistical analysis by Student's t test. *p<0.05.
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pone-0111777-g003: Enforced miR-509 expression in decreased proportion of cells in S-phase, induced apoptosis and activated caspase-3/7.(A) Representative flow cytometric plots showing cell cycle distribution of NALM6 cells transduced with empty vector (EV#1) or miR-509 overexpressing lentivirus. On day 3 after transduction, cells were labeled with BrdU for 1 h. Cells were then fixed overnight and stained on the next day with both BrdU and 7-AAD before analysis by flow cytometry. Percent of cells at each phase of cell cycle are boxed as indicated. (B) Frequencies of cells at the different phases of cell cycle. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (C) Representative flow cytometric plots of cell death distribution of NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus. Cells were stained with both Annexin V and 7-AAD before analysis by flow cytometry on day 4 after transduction. Numbers represent the frequency in each quadrant. (D) Frequencies of apoptotic cells which are Annexin V-positive (Annexin V+) and 7-AAD negative (7-AAD–), as well as total Annexin V+. Means ± SEMs of 3 independent experiments with statistical analysis by Student's t test. *p<0.05. (E) NALM6 cells were transduced with empty vector #2 (EV#2) or miR-509 overexpressing lentivirus, and cells were seeded in 384-well plate on day 3 after transduction. On day 7 after transduction, caspase-3/7 activity was measured and fold-change in caspase activity was normalized to EV#2. Means ± SEMs of 3 independent experiments done in triplicates, with statistical analysis by Student's t test. *p<0.05.
Mentions: To investigate the cellular mechanisms by which enforced miR-509 expression inhibits growth, we examined whether miR-509 regulates cell cycle progression by conducting BrdU/7-AAD staining [36]. 4 days after transduction, miR-509-transduced NALM6 had fewer cells in S-phase than empty vector-transduced cells (Figure 3A), and this was statistically significant (Figure 3B, p<0.05). In addition, there were slightly elevated numbers of cells in the subG1 and G2/M phases, but these differences were not statistically significant. To investigate if miR-509 promotes cell death via apoptosis, Annexin V/7-AAD staining was performed. 4 days after transduction, miR-509-transduced NALM6 cells had 1.5-fold (p<0.05) higher numbers of Annexin V+/7-AAD– apoptotic cells and 1.4-fold higher numbers of Annexin V+ dying/dead cells (p<0.05), as compared to empty vector-transduced cells (Figure 3C, 3D). Consistent with these findings, we detected a 1.5-fold increase (p<0.05) in activated capase-3/7 activity in miR-509-transduced NALM6 cells as compared to empty vector-transduced cells (Figure 3E).

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