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.