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C. Hoareau-Aveilla et al.
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Figure 6. CDK6, CDC25A, CCNE1 and E2F3 are target genes of miR-497 in anaplastic lymphoma kinase (ALK)-positive(+) lymphoma cells. (A) Quantitative real-time (RT)-PCR analysis of CDK6, CDC25A, CCNE1 and E2F3 mRNA expression in NPM-ALK+ lymphoma COST, KARPAS-299 (KARPAS) and SU-DHL1 cells transfected with negative control miRNA (miR-CTL) or a mimic miR-497 (miR-497). GAPDH was used as internal control and the relative ratios of mRNA expression was expressed as 2–ΔΔCt relative to those in cells transfected with miR-CTL. Data represent means±Standard Deviation (bars), **P<0.001; ***P<0.0001; unpaired two-tailed Student’s t-test with Welch’s correction. (B) Western blotting analysis of CDK6, CDC25A, CCNE1 and E2F3 expression in NPM-ALK+ COST, KARPAS-299 (KARPAS) and SU-DHL1 cells transfected by mir-CTL or mir-497. The GAPDH protein served as an internal control to ensure equal loading. Results from one representative experiment are shown. (C) Densitometric analysis was performed using ImageJ software from Wayne Rasband (NIH). The relative levels of the proteins of interest were normalized to GAPDH levels. Data represent means±Standard Error of Mean (bars) from 3 independent experiments, **P<0.001; ***P<0.0001; ns: not significant; unpaired two-tailed Student’s t-test with Welch’s correction.
Effect of miR-497 on the expression of cell cycle regulatory genes in NPM-ALK+ lymphoma cells
To determine the mechanism(s) by which miR-497 reg- ulates tumor growth and progression of NPM-ALK+ can- cer cells, we searched for mRNA targets of miR-497. To capture miR-497 associated mRNAs, KARPAS-299 cells were transfected with biotinylated human miR-497. We isolated and sequenced RNAs that co-purified with biotin-miR-497 mimic to map miR-497-mRNA interac- tion. As a negative control, we performed the same exper- iment with a biotin-negative control mimic (biotin-miR- CTL). A large set of mRNAs is regulated by miR-497 (enrichment ratio >1.5). In this data set, 77 validated can- didate targets of miR-497 were collected from the miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/, Release 7.0) (Figure 5A). In addition, using the miRSystem database (http://mirsystem.cgm.ntu.edu.tw/), we identified 16 miR- 497 mRNA targets, experimentally validated and associ- ated with cell cycle regulation (KEGG pathway database) (Figure 5A). We identified 5 mRNA gene targets that were common to both databases: CCNE1, CDC25A, CDK6, CCND3 and E2F3 (Figure 5A). Next, we used gene expression array data (Daugrois et al., 2018, submitted publication) to identify the veracity of these 5 targets cap- tured by biotin-miR-497. Despite the presence of a mixed population of neoplastic and normal cells, CCNE1, CDC25A, CDK6 and E2F3 mRNAs were found to be sig-
nificantly over-expressed in NPM-ALK+ ALCL patients (n=55) compared to reactive lymph node controls (RLN) (Figure 5B). As previously reported by Thompson et al., CCND3 was not over-expressed in NPM-ALK+ ALCL patients.30 Finally, to validate the specificity of the biotin pulldown and applicability to other cell lines, biotinylated forms of miR-497 or negative miRNA-control were used to co-purify CCNE1, CCDN3, CDC25A, CDK6 and E2F3 mRNAs in three KARPAS-299, SU-DHL1 and COST cell lines. Enrichment of target mRNAs by streptavidin-medi- ated purification (enrichment ratio >2) was measured by qPCR and normalized to miR-CTL enrichment. In the biotin-miR-497 pulldown of COST, SU-DHL1 and KARPAS-299 cells, CDC25A, CCNE1 and E2F3 tran- scripts but not CCDN3 were captured 24 h after transfec- tion (Figure 5C). Moreover, biotin pulldown enriches CDK6 mRNA in COST and SU-DHL1 cells but not in KARPAS-299 cells (Figure 5C). This result was in accor- dance with data published by Nagen et al.,31 who reported very weak CDK6 expression in KARPAS-299 cells com- pared to SU-DHL1 cells (Online Supplementary Figure S5). Moreover, for the first time, we observed that CDK6 pro- tein is over-expressed in COST cells (similar to SU-DHL1 cells) in comparison to KARPAS-299 cells (Online Supplementary Figure S4). To corroborate the results of miR-497–bound mRNAs capture and transcriptomic analyses, the effect of miR-497 on the endogenous
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haematologica | 2019; 104(2)