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

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

Enforced miR-509 expression inhibits growth of NALM6 cells.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111777#pone.0111777-Candia1" target="_blank">[32]</a>. (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.</p

RAB5C is a direct target of miR-509.

<p>(A) Sequence alignment of RAB5C to miR-509-3p predicted by TargetScan6.2. The full length 3′UTR of RAB5C is 803 bases. Sequences shown in bold refer to position 66–72 and 759–766 of RAB5C 3′UTR where miR-509-3p is predicted to target. The underlined sequences were deleted in the RAB5C-3′UTR deletion constructs listed in (B) for the luciferase assay. (B) Schematic representation of luciferase vector constructs used in luciferase assay. Full length RAB5C 3′UTR was cloned downstream of the firefly luciferase gene (luc2) in the pmirGLO luciferase vector. Wild type RAB5C 3′UTR is listed as WT. Grey boxes indicate the 2 predicted miR-509-3p target sites (66–72 and 759–766), and the “X” indicates the deletion sites present in the deletion (Δ) constructs. (C) Luciferase assay demonstrates that RAB5C 3′UTR is targeted by miR-509-3p via 2 binding sites. 293T cells were transfected with the 300ng of the indicated luciferase plasmids and 50nM of miR mimics, and harvested for luciferase assay 48 h after transfection. All values were first normalized to Renilla luciferase. Relative luciferase activity was then calculated by normalizing co-transfection of miR mimics plus luciferase constructs to cells transfected with only the respective luciferase construct. MiR-551b was used as a non-targeting miR negative control. Data shown represent means ± SEMs of 3 independent experiments, with statistical analysis by Student's <i>t</i> test. **p<0.01, ***p<0.001.</p

Identifying mRNA targets of miR-509.

<p>(A) Venn diagram showing the number of mRNAs that do not overlap, or are shared between each set in our <i>in silico</i> 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 <i>t</i> test. *p<i><</i>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 <i>t</i> test. ***p<0.001.</p

Enforced miR-509 resulted in inhibition of growth of 3 B-ALL cell lines, NALM6, REH and RCH-ACV.

<p>(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 <i>t</i> test. *p<0.05, **p<0.01.</p

Absolute copy number of mature miR-509 and miR-18a RNA per NALM6 cell.

<p>RNA was isolated from NALM6 cells on day 7 after transduction with either control empty vector #1 or miR-509, and absolute qRT-PCR quantification was performed for miR-509-5p, miR-509-3p or miR-18a. Copy number per cell was estimated based on standard curves of miR-509-5p, miR-509-3p or miR-18a using DNA oligonucleotides. For reverse transcription, 10 ng RNA (equivalent to 800 cells, i.e. 12.5 pg of total RNA per cell) was used in each reaction. Means ± SEMs of 3 independent experiments.</p><p>Absolute copy number of mature miR-509 and miR-18a RNA per NALM6 cell.</p