SUPPORTING INFORMATION ON THE INTERNET
Supplementary materials and methods
Colony formation. Colony formation was assessed using soft-agar assays in
quadruplicate cultures in 24-well plates in two independent experiments. Briefly, 48 h
after transfection, cells (5x105) were suspended (500µl in RPMI, 10% FBS) in 0.5% agar
layered over a 1.8 % agar in 24-well plates. Colonies were scored after different time
points for 10 days.
Cell cycle profile analysis. Following 2 or 3 days of transfection with the miR-29c mimic
and scrambled mimics, cells were trypsinised, resuspended in 70% cold ethanol (−20°C)
in PBS, and stored at −20°C for at least 30 min. Cells were centrifuged to remove
ethanol, washed with PBS, and resuspended in 2 μg/mL of propidium iodide (MP
Biomedicals) and 200 μg/mL of RNase A (Qiagen) in PBS. The cell cycle assay was
conducted on the CyAnADP high-performance Flow Cytometer (Beckman Coulter (UK)
Ltd, High Wycombe, UK) and analysed with the graphics software Summit v4.3.
Supplementary Figure S1. Gene expression levels of neurofibromin 1 in the 10
neurofibromas and 10 MPNST which were analysed for their miRNA profile, and
validation of the neurofibromin 1mutation in the sNF96.2 MPNST cell line. (A) Both
neurofibromas and MPNST cases showed a low level of mRNA expression for the
neurofibromin 1 gene as expected for tumours arising in the context of neurofibromatosis
type I. sNF96.2, a MPNST cell line known to harbour a mutation in neurofibromin 1
(3683delC) and to have loss of heterozygosity of the wild type allele were also found to
have very low expression of the neurofibromin 1 gene compared to control HEK293T
1
cells (wild type for neurofibromin 1). (B) Sanger sequencing of neurofibromin 1 exon 21
confirmed the homozygous deletion (3683delC) in sNF96.2 compared to HEK293T cells
(WT for neurofibromin 1).
Supplementary Figure S2 TaqMan validation for the most significantly deregulated
miRNAs in the 10 neurofibromas and 10 MPNST samples used in the microarray (A) and
in a larger cohort of 117 formal-fixed paraffin-embedded peripheral nerve sheath
tumours (B). (A) A strip chart representing the relative expression to RNU66 for miR340, miR-139-5p, miR-Let7g, miR-210, mi-R30e, and mi-R29c in the same tumour
samples as those analysed in the microarray, (B) Screening on 117 FFPE samples for
miR-210, miR-29c and miR-340. Statistical test used Wilcoxon. P value significant when
<0.05.
Supplementary Figure S3 miR-34a heat map. Expression values were log transformed
and values for each miRNA were median centred. The data were then average linkage
clustered using cluster software (Eisen lab) and the result for miR34a selected for display.
While black is median expression, red is higher and green lower.
Supplementary Figure S4. Functional studies involving miR-29c in the MPNST cell
line, sNF96.2, in vitro. (A) Viability assay assessed by MTS. sNF96.2 cells were
transfected in a 96-well plate by miR-29c, and scrambled mimics and analysed at 4 time
points, day 0 to day 5. The transfection was performed one day after plating, at time
point 1. The plates were read at 560 nm wavelength. The percentage of cell viability was
2
calculated by subtracting wavelengths from the test case to a blank control. (B) Rate of
cellular proliferation and kinetic growth curve assessed live with Incucyte. The sNF96.2
cells
transfected
with
miR-29c
mimics,
and
scrambled
mimics
were
monitored continuously for up to 120h after transfection (figure showing 50h) and
the result in the graph was calculated as the percentage of confluency. (C) Growth in 3-D
using soft agar showed no difference in growth and colony formation in sNF96.2 cells
expressing miR-29c mimics, or scrambled mimics. Briefly, 48h after transfection, cells
(5 105) were suspended (500ul in RPMI, 10% FBS) in 0.5 % agar over a layer of 1.8 %
agar in 24-well plates. The formation of colonies was monitored for 20 days. Bar sizes
indicate 200 µm. (D) Cell cycle profiling was analysed by propidium iodide staining (PI)
and performing FACS. Each population was measured as the percentage of the total cell
population.
Supplementary Table S1. Primer sequences used in the manuscript
Supplementary Table S2. Description of selected predicted target genes relevant to
migration and invasion. PITA, Target scan, PicTar and miRanda scores were reported
alongside the biological function of the predicted targets genes for miR-29c relevant to
migration and invasion.
Supplementary Table S3. List of predicted target genes for miR29c present in both
PITA and Targetscan predictions.
Supplementary Table S4. Genecodis 2.0 analysis for the predicted target genes.
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NGR = Number of annotated genes in the reference list (Total number of genes in
the reference list).
NG = Number of annotated genes in the input list (Total number of genes in the
input list).
Hyp = Hypergeometric pValue; Hyp* = Corrected hypergeometric p Value.
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