Page 146 - 2019_02-Haematologica-web

C. Hoareau-Aveilla et al.
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Figure 1. MiR-195 and miR-497 are down-regulated in human NPM-anaplastic lymphoma kinase (ALK)-positive(+) primary lymphoma samples and cell lines. (A) RNA sequencing from NPM-ALK+ KARPAS-299 cells following decitabine or DMSO treatment or transfection with a control siRNA (si-CTL) or siRNA targeting ALK mRNA (si-ALK). A set of 13 miRNAs was found to be up-deregulated >1.5 fold in treated cells in both conditions (si-ALK and decitabine) compared to either negative control siRNA (si-CTL) or drug vehicle alone (DMSO). (B) Quantitative real-time PCR (qRT-PCR) analysis of miR-195 and miR-497 in NPM-ALK+ anaplastic large cell lymphoma patients (n=52). Data were normalized against equivalent miRNA levels from reactive lymph node (RLN) tissue samples (n=19). Data represent means±Standard Error of Mean (bars), ***P<0.0001; unpaired two-tailed Student’s t-test with Welch’s correction.
Xenograft tumor assay
Mice were housed under pathogen-free conditions in an animal room at constant temperature (20-22°C), with a 12 h/12 h light:dark cycle and free access to food and water. All animal pro- cedures were performed following the principle guidelines of INSERM, and our protocol was approved by the Midi-Pyrénées Ethics Committee on Animal Experimentation. ALCL cell lines were transfected either with control (miR-CTL) or miR-497 miRNA mimics. Twenty-four hours later, a total of 3x106 COST or KARPAS-299 cells or 2x106 SU-DHL1 cells were injected subcuta- neously into both flanks of 7-week old female non-obese diabet- ic/severe combined immunodeficient (NOD-SCID) or NOD- SCID Gamma (NSG) mice (Janvier Labs, Le Genest, St Isle, France). Mouse body weight and tumor volumes were measured three times a week with calipers, using the formula “length x width2 x π/6”. At the end of the experiment, mice (6 per group) were humanely sacrificed. Subcutaneous tumors were then excised and sections were fixed in 10% neutral buffered formalin for histochemical analysis.
RNA sequencing
RNA sequencing was performed at the LISBP, Toulouse, France, on an Ion Proton (Thermofischer, Waltham, MA, USA) as single- end oriented 95pb-long reads. RNAs co-purified with biotinylated hsa-miR-497-5p or with a biotin-negative control mimic (biotin- miR-CTL) were used for library preparation using the Ion Total RNA-seq Kit V2 and Ion PI HiQ OT2 Kit (Thermofischer, Waltham, MA, USA). TopHat2 (v.2.1.0) was used for sequence alignment on the human genome GRCH38.83 and counting reads performed using HTSeq-Count (v.0.6.1P1).
Statistical analysis
Results are presented as mean values±Standard Error of Mean (SEM) from at least 3 independent experiments unless otherwise
indicated. Differences between groups were examined using the unpaired two-tailed Student’s t-test, unpaired two-tailed Student’s t-test with Welch’s correction or two-way ANOVA with Bonferroni post-test using GraphPad Prism software version 6.00 for Windows (GraphPad software) (La Jolla, CA, USA). For all tests, P<0.05 (*), P<0.01 (**) or P<0.001 (***) were considered sta- tistically significant. Event-free survival was analyzed using a Receiver Operating Characteristics (ROC) method. A cut-off value was then chosen which would assume a minimum error rate and a limited number of misclassifications within the cohort.
To determine the profile of methylated miRNA genes driven by NPM-ALK oncogene in ALCL cells, we per- formed a high throughput analysis of miRNA expression (small RNA sequencing) from NPM-ALK+ KARPAS-299 cells following decitabine treatment or transfection with a siRNA targeting ALK mRNA (si-ALK). A set of 13 miRNAs (miR-9-5p, miR-1287-5p, miR-15a-5p, miR-22-3p, miR- 26a-5p, miR-29c-3p, miR-194-5p, miR-195-5p, miR-199a- 3p, miR-199a-5p, miR-223-3p, miR-3613-5p and miR-497- 5p) was found to be up-regulated >1.5-fold in treated cells in both conditions (si-ALK and decitabine) compared either to negative control siRNA (si-CTL) or drug vehicle alone (DMSO) (Figure 1A). Amongst these 13 miRNAs, using microarray data previously published of miRNA- expression of NPM-ALK+ (n=52) and NPM-ALK– (n=5) ALCL patients,21 we observed that miR-195 and miR-497 were down-regulated only in NPM-ALK+ ALCL patients compared to normal reactive lymph node (RLNs, n=3) (Online Supplementary Table S2). To corroborate the
Results
The miR-497∼195 cluster is down-regulated in human NPM-ALK+ primary lymphoma samples and cell lines
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haematologica | 2019; 104(2)