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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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Identifying mRNA targets of miR-509.(A) Venn diagram showing the number of mRNAs that do not overlap, or are shared between each set in our in silico strategy to identify relevant targets of miR-509. Set 1 refers to the list of predicted targets of miR-509-5p or miR-509-3p from TargetScan6.2 or miRDB. Set 2 is the list of predicted targets of miRs tested to not inhibit NALM6 growth (i.e. miR-550a, miR-873, miR-381 and miR-432) from TargetScan6.2 or miRDB, while Set 3 is the list of mRNA that is expressed in NALM6, as determined by genome-wide microarray profiling downloaded from the Cancer Cell Line Encyclopedia and its expression levels are denoted in the microarray dataset as “marginal” or “present”. (B) Expression levels of 12 putative targets of miR-509 as determined by qRT-PCR. RNA was isolated from NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus at 7 days after transduction. All values were normalized to GAPDH and fold-change was calculated relative to EV#1 sample. Data represents means ± SEMs of 3 independent experiments, with statistical analysis by Student's t test. *p<0.05. (C) Representative western blots of RAB5C expression. NALM6 cells were transduced with either EV#1or miR-509 overexpressing lentivirus, and whole cell lysates were harvested at 7 days after transduction. α-tubulin was used for loading control. (D) Densitometry analysis of RAB5C expression of western blot in (C) and 2 other independent experiments. α-tubulin was used for normalization, and relative densitometry was then calculated compared to EV#1. Data shown represent means ± SEMs, with statistical analysis by Student's t test. ***p<0.001.
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pone-0111777-g004: Identifying mRNA targets of miR-509.(A) Venn diagram showing the number of mRNAs that do not overlap, or are shared between each set in our in silico strategy to identify relevant targets of miR-509. Set 1 refers to the list of predicted targets of miR-509-5p or miR-509-3p from TargetScan6.2 or miRDB. Set 2 is the list of predicted targets of miRs tested to not inhibit NALM6 growth (i.e. miR-550a, miR-873, miR-381 and miR-432) from TargetScan6.2 or miRDB, while Set 3 is the list of mRNA that is expressed in NALM6, as determined by genome-wide microarray profiling downloaded from the Cancer Cell Line Encyclopedia and its expression levels are denoted in the microarray dataset as “marginal” or “present”. (B) Expression levels of 12 putative targets of miR-509 as determined by qRT-PCR. RNA was isolated from NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus at 7 days after transduction. All values were normalized to GAPDH and fold-change was calculated relative to EV#1 sample. Data represents means ± SEMs of 3 independent experiments, with statistical analysis by Student's t test. *p<0.05. (C) Representative western blots of RAB5C expression. NALM6 cells were transduced with either EV#1or miR-509 overexpressing lentivirus, and whole cell lysates were harvested at 7 days after transduction. α-tubulin was used for loading control. (D) Densitometry analysis of RAB5C expression of western blot in (C) and 2 other independent experiments. α-tubulin was used for normalization, and relative densitometry was then calculated compared to EV#1. Data shown represent means ± SEMs, with statistical analysis by Student's t test. ***p<0.001.

Mentions: To identify targets of miR-509 that might mediate growth of B-ALL cells, we used a filtering strategy to prioritize the many predicted targets of miR-509 (Figure 4A). First, we downloaded the sets of predicted mRNA targets of miR-509-5p and miR-509-3p (Set 1), as well as those of the 4 miRs that we had shown not to inhibit NALM6 growth (i.e. miR-381, miR-432, miR-550a and miR-873; Set 2) from the TargetScan6.2 [37] and/or miRDB [38], [39] miR target prediction databases. Since NALM6 cells transduced with miR-432∼136 did not result in miR-136 overexpression, we did not include miR-136 targets in Set 2 (Figure 4A). Next, we downloaded the gene expression profile of NALM6, determined by genome-wide microarray profiling as listed in the Cancer Cell Line Encyclopedia (CCLE) [40] and focused on genes which have detectable expression in NALM6 (i.e. annotated as “marginal” or “present” in CCLE; Set 3). Then, we intersected these 3 sets of mRNAs [41] to identify the subset of genes expressed in NALM6 and predictively targeted by miR-509, but not predictively targeted by the 4 miRs that did not inhibit NALM6 growth. This resulted in a set of 395 genes (listed in Table S6). This list was subsequently reduced to 74 genes by selecting for genes known to participate in growth regulation based on annotations at NCBI's “Gene” database, DAVID bioinformatics resources [42], [43], as well as our own literature searches. Of these 74 predicted targets of miR-509, 12 genes previously demonstrated in the literature to be either involved in leukemia and oncogenesis (ERLIN2, FLI1, FOXP1, MAML1, RAC1, YWHAB and YWHAG), or predicted as miR-509 targets by both TargetScan6.2 and miRDB (PGRMC1, RAB5C, RAC1, TFDP2, UHMK1, USP9X) were selected for initial qRT-PCR analysis. We used this informatic filtering strategy, as compared to performing global differential gene expression analysis such as microarray analysis, to enable us to rapidly and at low cost identify target genes-of-interest. 3 of these 12 predicted targets (RAB5C, RAC1, and UHMK1) were down-regulated by miR-509 at the mRNA level (Figure 4B). RAB5C mRNA levels showed the greatest reduction, with a 40% lower level (p<0.05) in miR-509-transduced than in empty vector-transduced NALM6 cells (Figure 4B). Correspondingly, RAB5C protein was 85% (p<0.001) lower in miR-509-transduced cells by western blotting (Figure 4C, 4D). We also observed a ≥86% decrease in RAB5C protein levels in miR-509-transduced RCH-ACV and REH cells as compared to empty vector (Figure S4). Since RAB5 has been implicated in cell cycling [44], [45] and is one of the top 3 predicted targets of miR-509-3p by both TargetScan6.2 (Total context+ score  = −0.65) and miRDB (Target score  = 91), we focused our subsequent studies on RAB5C.


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)

Identifying mRNA targets of miR-509.(A) Venn diagram showing the number of mRNAs that do not overlap, or are shared between each set in our in silico strategy to identify relevant targets of miR-509. Set 1 refers to the list of predicted targets of miR-509-5p or miR-509-3p from TargetScan6.2 or miRDB. Set 2 is the list of predicted targets of miRs tested to not inhibit NALM6 growth (i.e. miR-550a, miR-873, miR-381 and miR-432) from TargetScan6.2 or miRDB, while Set 3 is the list of mRNA that is expressed in NALM6, as determined by genome-wide microarray profiling downloaded from the Cancer Cell Line Encyclopedia and its expression levels are denoted in the microarray dataset as “marginal” or “present”. (B) Expression levels of 12 putative targets of miR-509 as determined by qRT-PCR. RNA was isolated from NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus at 7 days after transduction. All values were normalized to GAPDH and fold-change was calculated relative to EV#1 sample. Data represents means ± SEMs of 3 independent experiments, with statistical analysis by Student's t test. *p<0.05. (C) Representative western blots of RAB5C expression. NALM6 cells were transduced with either EV#1or miR-509 overexpressing lentivirus, and whole cell lysates were harvested at 7 days after transduction. α-tubulin was used for loading control. (D) Densitometry analysis of RAB5C expression of western blot in (C) and 2 other independent experiments. α-tubulin was used for normalization, and relative densitometry was then calculated compared to EV#1. Data shown represent means ± SEMs, with statistical analysis by Student's t test. ***p<0.001.
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pone-0111777-g004: Identifying mRNA targets of miR-509.(A) Venn diagram showing the number of mRNAs that do not overlap, or are shared between each set in our in silico strategy to identify relevant targets of miR-509. Set 1 refers to the list of predicted targets of miR-509-5p or miR-509-3p from TargetScan6.2 or miRDB. Set 2 is the list of predicted targets of miRs tested to not inhibit NALM6 growth (i.e. miR-550a, miR-873, miR-381 and miR-432) from TargetScan6.2 or miRDB, while Set 3 is the list of mRNA that is expressed in NALM6, as determined by genome-wide microarray profiling downloaded from the Cancer Cell Line Encyclopedia and its expression levels are denoted in the microarray dataset as “marginal” or “present”. (B) Expression levels of 12 putative targets of miR-509 as determined by qRT-PCR. RNA was isolated from NALM6 cells transduced with EV#1 or miR-509 overexpressing lentivirus at 7 days after transduction. All values were normalized to GAPDH and fold-change was calculated relative to EV#1 sample. Data represents means ± SEMs of 3 independent experiments, with statistical analysis by Student's t test. *p<0.05. (C) Representative western blots of RAB5C expression. NALM6 cells were transduced with either EV#1or miR-509 overexpressing lentivirus, and whole cell lysates were harvested at 7 days after transduction. α-tubulin was used for loading control. (D) Densitometry analysis of RAB5C expression of western blot in (C) and 2 other independent experiments. α-tubulin was used for normalization, and relative densitometry was then calculated compared to EV#1. Data shown represent means ± SEMs, with statistical analysis by Student's t test. ***p<0.001.
Mentions: To identify targets of miR-509 that might mediate growth of B-ALL cells, we used a filtering strategy to prioritize the many predicted targets of miR-509 (Figure 4A). First, we downloaded the sets of predicted mRNA targets of miR-509-5p and miR-509-3p (Set 1), as well as those of the 4 miRs that we had shown not to inhibit NALM6 growth (i.e. miR-381, miR-432, miR-550a and miR-873; Set 2) from the TargetScan6.2 [37] and/or miRDB [38], [39] miR target prediction databases. Since NALM6 cells transduced with miR-432∼136 did not result in miR-136 overexpression, we did not include miR-136 targets in Set 2 (Figure 4A). Next, we downloaded the gene expression profile of NALM6, determined by genome-wide microarray profiling as listed in the Cancer Cell Line Encyclopedia (CCLE) [40] and focused on genes which have detectable expression in NALM6 (i.e. annotated as “marginal” or “present” in CCLE; Set 3). Then, we intersected these 3 sets of mRNAs [41] to identify the subset of genes expressed in NALM6 and predictively targeted by miR-509, but not predictively targeted by the 4 miRs that did not inhibit NALM6 growth. This resulted in a set of 395 genes (listed in Table S6). This list was subsequently reduced to 74 genes by selecting for genes known to participate in growth regulation based on annotations at NCBI's “Gene” database, DAVID bioinformatics resources [42], [43], as well as our own literature searches. Of these 74 predicted targets of miR-509, 12 genes previously demonstrated in the literature to be either involved in leukemia and oncogenesis (ERLIN2, FLI1, FOXP1, MAML1, RAC1, YWHAB and YWHAG), or predicted as miR-509 targets by both TargetScan6.2 and miRDB (PGRMC1, RAB5C, RAC1, TFDP2, UHMK1, USP9X) were selected for initial qRT-PCR analysis. We used this informatic filtering strategy, as compared to performing global differential gene expression analysis such as microarray analysis, to enable us to rapidly and at low cost identify target genes-of-interest. 3 of these 12 predicted targets (RAB5C, RAC1, and UHMK1) were down-regulated by miR-509 at the mRNA level (Figure 4B). RAB5C mRNA levels showed the greatest reduction, with a 40% lower level (p<0.05) in miR-509-transduced than in empty vector-transduced NALM6 cells (Figure 4B). Correspondingly, RAB5C protein was 85% (p<0.001) lower in miR-509-transduced cells by western blotting (Figure 4C, 4D). We also observed a ≥86% decrease in RAB5C protein levels in miR-509-transduced RCH-ACV and REH cells as compared to empty vector (Figure S4). Since RAB5 has been implicated in cell cycling [44], [45] and is one of the top 3 predicted targets of miR-509-3p by both TargetScan6.2 (Total context+ score  = −0.65) and miRDB (Target score  = 91), we focused our subsequent studies on RAB5C.

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