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CDK4/CDK6 inhibitors in ALK-positive lymphomas
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Figure 7. Silencing of CDK6, CCNE1 and E2F3 genes reduces cell proliferation in anaplastic lymphoma kinase (ALK)-positive(+) lymphoma cells. NPM-ALK+ ALCL SU-DHL1 (A-C), COST (D-F) and KARPAS-299 (KARPAS) (G-I) cells were transfected with either an irrelevant siRNA as the negative control (si-CTL) or a siRNA targeting CDK6 mRNA (si-CDK6) or CDK6, CCNE1 and E2F3 mRNA together (si-Pool). Cell cycle distribution (A, D and G), cell count at three different time points (0, 1, 2 days) (B, E and H) and caspase 3/7 activity (C, F and I) were measured. Data represent means±Standard Error of Mean (bars) from 3 independent experiments, *P<0.05; **P<0.001; ***P<0.0001; ns: not significant; unpaired two-tailed Student’s t-test with Welch’s correction.
expression of CCNE1, CDC25A, CDK6 and E2F3 was subsequently examined using qPCR and Western blot- ting. Transfection of miR-497 mimic induced a significant decrease of all four mRNAs in all three cell lines com- pared to cells transfected with miR-CTL (Figure 6A). Overexpression of miR-497 significantly down-regulated the protein level of CDK6, CDC25A and E2F3 in all three cell lines (Figure 6B). In addition, CCNE1 expression was down-regulated in KARPAS-299 cells but not in COST or SU-DHL1 cells (Figure 6B). Densitometric analysis of the mean relative intensity for each target protein of miR-497 supports the Western blotting results (Figure 6C). Taken together, these results strongly suggest that miR-497 reg- ulates the expression of CCNE1, CDC25A, CDK6 and E2F3 in NPM-ALK+ cells.
Significance of identified miR-497 target genes in NPM-ALK+ lymphoma cells
To evaluate whether CCNE1, CDC25A, CDK6 and E2F3 may be functional downstream targets of miR-497 and involved in regulation of NPM-ALK+ cell proliferation, we individually knocked down the expression of these genes using specific siRNAs. In order to check that the knockdown of gene expression had been efficiently achieved, we performed qPCR and Western blotting to detect the mRNA (Online Supplementary Figure S5A) and
protein (Online Supplementary Figure S5B) levels, respec- tively. Densitometric analysis of the mean relative intensi- ty for each target protein of miR-497 supports the Western blot results (Online Supplementary Figure S5C). We observed that, in SU-DHL1 cells, silencing of CDK6 mRNA simultaneously induced a decrease of cell viability and an increase of both subG1 fraction and caspase 3/7 activity, thereby inhibiting cell growth (Figure 7A-C). Hence, CDK6 silencing alone is sufficient to recapitulate the phenotype observed upon miR-497 ectopic expression in SU-DHL1 cells. These results are similar to those pub- lished by Nagen et al.31 showing an addiction to CDK6 expression for proper cell cycle progression in this cell line. In contrast, even though siRNA directed against CDK6 led to a significant accumulation of COST and KARPAS-299 cells in G0/G1 phase (Online Supplementary Figure S6B and E), it is not sufficient to fully recapitulate miR-497-induced blockage of cell cycle. The same observation was made after E2F3 and CCNE1 mRNA downregulation (Online Supplementary Figure S6A and D, and C and F, respective- ly). Finally, we observed that CDC25A knockdown did not modify the cell cycle profiles of COST and KARPAS- 299 cells (data not shown). We also tested the effect of each siRNA on cellular apoptosis in COST and KARPAS-299 cells. Compared to the control condition (si-CTL), CDK6 and CCNE1 silencing induced apoptosis in COST cells
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