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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 inhibits growth of NALM6 cells.(A) Schematic of lentiviral vector used to express miRs. Arrow depicts the direction of human EF1α promoter. LTR: long terminal repeat; GFP: green fluorescent protein; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element. The parental plasmid without miR is denoted as empty vector #1 (EV#1). The miR sequence consists of the native miR hairpin with ∼200 bp of its flanking genomic sequences. (B) Assessment of %GFP+ cells by flow cytometry in the GFP competition assay. NALM6 cells were transduced with miR-509 lentivirus or empty vector (EV#1) at MOI  = 2, and transduced GFP+ cells were mixed with an equal number of mock-transduced cells (GFP–) 7 days later to achieve an initial culture of ∼50%GFP+ cells; this was designated Day 0 and the %GFP+ cells (pre-gated on viable cells) was assessed weekly by flow cytometry. Means ± SEMs are shown for 3 independent experiments. (C) Enforced expression of mature miR-509-5p and miR-509-3p NALM6 cells, as assayed by qRT-PCR. NALM6 cells were transduced with miR-509 lentivirus to MOI  = 2, and total RNA was collected at 7 days after transduction. U18 was used as the loading control, and normalized to EV#1-transduced NALM6 cells. Means ± SEMs of 3 independent experiments. (D) Expression of mature miR-509-5p was determined by miR microarray analysis in B-ALL, T-ALL and AML cell lines and primary samples, B cells, CD34+ HSPCs, granulocytes, monocytes and T cells. Dotted line represents normalized microarray intensity of 2 whereby any value <2 denotes undetectable expression. Data points shown are means ± SEMs. Expression data is accessible through GEO Series accession number GSE51908 [32]. (E) Expression of mature miR-509-3p and miR-18a as determined by miR microarray analysis similar to (D). (D, E) Data shown for miR-18a is only for the NALM6 cell line.
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pone-0111777-g001: Enforced miR-509 expression inhibits growth of NALM6 cells.(A) Schematic of lentiviral vector used to express miRs. Arrow depicts the direction of human EF1α promoter. LTR: long terminal repeat; GFP: green fluorescent protein; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element. The parental plasmid without miR is denoted as empty vector #1 (EV#1). The miR sequence consists of the native miR hairpin with ∼200 bp of its flanking genomic sequences. (B) Assessment of %GFP+ cells by flow cytometry in the GFP competition assay. NALM6 cells were transduced with miR-509 lentivirus or empty vector (EV#1) at MOI  = 2, and transduced GFP+ cells were mixed with an equal number of mock-transduced cells (GFP–) 7 days later to achieve an initial culture of ∼50%GFP+ cells; this was designated Day 0 and the %GFP+ cells (pre-gated on viable cells) was assessed weekly by flow cytometry. Means ± SEMs are shown for 3 independent experiments. (C) Enforced expression of mature miR-509-5p and miR-509-3p NALM6 cells, as assayed by qRT-PCR. NALM6 cells were transduced with miR-509 lentivirus to MOI  = 2, and total RNA was collected at 7 days after transduction. U18 was used as the loading control, and normalized to EV#1-transduced NALM6 cells. Means ± SEMs of 3 independent experiments. (D) Expression of mature miR-509-5p was determined by miR microarray analysis in B-ALL, T-ALL and AML cell lines and primary samples, B cells, CD34+ HSPCs, granulocytes, monocytes and T cells. Dotted line represents normalized microarray intensity of 2 whereby any value <2 denotes undetectable expression. Data points shown are means ± SEMs. Expression data is accessible through GEO Series accession number GSE51908 [32]. (E) Expression of mature miR-509-3p and miR-18a as determined by miR microarray analysis similar to (D). (D, E) Data shown for miR-18a is only for the NALM6 cell line.

Mentions: We applied our functional miR-HTS to screen a pooled lentivirus library of 578 human miRs or miR clusters for their growth-regulatory properties in human NALM6 B-ALL cells and identified candidate miRs as previously described [26]. 4 miRs (miR-381, miR-509, miR-550a, and miR-873) and 1 miR cluster (miR-432∼136) inhibited NALM6 growth in at least 2 of 3 replicate screens performed. In order to confirm the growth inhibitory effects of the candidate miRs identified from the functional screen, each of the 5 miR or miR cluster candidates was cloned into a lentiviral expression vector downstream of green fluorescent protein (GFP) (Figure 1A). We expressed the miR-432∼136 cluster as a single unit rather than as 2 individual miRs, to recapitulate the way they were screened and because the 2 miRs may cooperate. The growth inhibitory potential of each candidate miR or miR cluster was then tested, by performing multiple GFP competition assays [33], [34]. NALM6 cells were transduced with each of the 5 miR lentiviruses (>80% GFP+ cells), and each culture was then mixed with GFP– cells to obtain an initial culture with ∼50% GFP+ cells. If enforced expression of a given miR or miR cluster inhibited NALM6 growth, the %GFP+ cells in culture would decrease over time. For NALM6 cells transduced with the control empty vector, the %GFP+ cells remained stable at ∼50% over the 5-week GFP competition assay (Figure 1B). Similarly, no change in %GFP+ cells was observed over 35 days in the GFP competition assays for miR-381, miR-550a, miR-873 and miR-432∼136 (Figure S1A-S1D). In contrast, NALM6 cells transduced with miR-509 lentivirus were out-grown by the GFP– cells; the %GFP+ cells decreased from 46% at assay day 0 to 10% 35 days later (Figure 1B).


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 inhibits growth of NALM6 cells.(A) Schematic of lentiviral vector used to express miRs. Arrow depicts the direction of human EF1α promoter. LTR: long terminal repeat; GFP: green fluorescent protein; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element. The parental plasmid without miR is denoted as empty vector #1 (EV#1). The miR sequence consists of the native miR hairpin with ∼200 bp of its flanking genomic sequences. (B) Assessment of %GFP+ cells by flow cytometry in the GFP competition assay. NALM6 cells were transduced with miR-509 lentivirus or empty vector (EV#1) at MOI  = 2, and transduced GFP+ cells were mixed with an equal number of mock-transduced cells (GFP–) 7 days later to achieve an initial culture of ∼50%GFP+ cells; this was designated Day 0 and the %GFP+ cells (pre-gated on viable cells) was assessed weekly by flow cytometry. Means ± SEMs are shown for 3 independent experiments. (C) Enforced expression of mature miR-509-5p and miR-509-3p NALM6 cells, as assayed by qRT-PCR. NALM6 cells were transduced with miR-509 lentivirus to MOI  = 2, and total RNA was collected at 7 days after transduction. U18 was used as the loading control, and normalized to EV#1-transduced NALM6 cells. Means ± SEMs of 3 independent experiments. (D) Expression of mature miR-509-5p was determined by miR microarray analysis in B-ALL, T-ALL and AML cell lines and primary samples, B cells, CD34+ HSPCs, granulocytes, monocytes and T cells. Dotted line represents normalized microarray intensity of 2 whereby any value <2 denotes undetectable expression. Data points shown are means ± SEMs. Expression data is accessible through GEO Series accession number GSE51908 [32]. (E) Expression of mature miR-509-3p and miR-18a as determined by miR microarray analysis similar to (D). (D, E) Data shown for miR-18a is only for the NALM6 cell line.
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Related In: Results  -  Collection

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Show All Figures
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pone-0111777-g001: Enforced miR-509 expression inhibits growth of NALM6 cells.(A) Schematic of lentiviral vector used to express miRs. Arrow depicts the direction of human EF1α promoter. LTR: long terminal repeat; GFP: green fluorescent protein; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element. The parental plasmid without miR is denoted as empty vector #1 (EV#1). The miR sequence consists of the native miR hairpin with ∼200 bp of its flanking genomic sequences. (B) Assessment of %GFP+ cells by flow cytometry in the GFP competition assay. NALM6 cells were transduced with miR-509 lentivirus or empty vector (EV#1) at MOI  = 2, and transduced GFP+ cells were mixed with an equal number of mock-transduced cells (GFP–) 7 days later to achieve an initial culture of ∼50%GFP+ cells; this was designated Day 0 and the %GFP+ cells (pre-gated on viable cells) was assessed weekly by flow cytometry. Means ± SEMs are shown for 3 independent experiments. (C) Enforced expression of mature miR-509-5p and miR-509-3p NALM6 cells, as assayed by qRT-PCR. NALM6 cells were transduced with miR-509 lentivirus to MOI  = 2, and total RNA was collected at 7 days after transduction. U18 was used as the loading control, and normalized to EV#1-transduced NALM6 cells. Means ± SEMs of 3 independent experiments. (D) Expression of mature miR-509-5p was determined by miR microarray analysis in B-ALL, T-ALL and AML cell lines and primary samples, B cells, CD34+ HSPCs, granulocytes, monocytes and T cells. Dotted line represents normalized microarray intensity of 2 whereby any value <2 denotes undetectable expression. Data points shown are means ± SEMs. Expression data is accessible through GEO Series accession number GSE51908 [32]. (E) Expression of mature miR-509-3p and miR-18a as determined by miR microarray analysis similar to (D). (D, E) Data shown for miR-18a is only for the NALM6 cell line.
Mentions: We applied our functional miR-HTS to screen a pooled lentivirus library of 578 human miRs or miR clusters for their growth-regulatory properties in human NALM6 B-ALL cells and identified candidate miRs as previously described [26]. 4 miRs (miR-381, miR-509, miR-550a, and miR-873) and 1 miR cluster (miR-432∼136) inhibited NALM6 growth in at least 2 of 3 replicate screens performed. In order to confirm the growth inhibitory effects of the candidate miRs identified from the functional screen, each of the 5 miR or miR cluster candidates was cloned into a lentiviral expression vector downstream of green fluorescent protein (GFP) (Figure 1A). We expressed the miR-432∼136 cluster as a single unit rather than as 2 individual miRs, to recapitulate the way they were screened and because the 2 miRs may cooperate. The growth inhibitory potential of each candidate miR or miR cluster was then tested, by performing multiple GFP competition assays [33], [34]. NALM6 cells were transduced with each of the 5 miR lentiviruses (>80% GFP+ cells), and each culture was then mixed with GFP– cells to obtain an initial culture with ∼50% GFP+ cells. If enforced expression of a given miR or miR cluster inhibited NALM6 growth, the %GFP+ cells in culture would decrease over time. For NALM6 cells transduced with the control empty vector, the %GFP+ cells remained stable at ∼50% over the 5-week GFP competition assay (Figure 1B). Similarly, no change in %GFP+ cells was observed over 35 days in the GFP competition assays for miR-381, miR-550a, miR-873 and miR-432∼136 (Figure S1A-S1D). In contrast, NALM6 cells transduced with miR-509 lentivirus were out-grown by the GFP– cells; the %GFP+ cells decreased from 46% at assay day 0 to 10% 35 days later (Figure 1B).

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