Figure 4.
miR-497/195 overexpression inhibits CDK4- and CCND3-mediated leukemia growth ex vivo. (A) Heatmap of 914 differentially expressed genes upon miR-497/195 overexpression in PDX-S2 (adjusted P < .05). (B) Downregulation of CCND3 and CDK4 messenger RNA (mRNA) levels in PDX-S2 and (C) in PDX-L8 cells upon miR-497/195 overexpression assessed by qRT-PCR (data are mean ± SD; Mann-Whitney U test). (D) CDK4 and CCND3 mRNA fold change in NALM-6 cells upon transduction with anti-miR-497 or anti-miR-195 miRZip constructs compared with scramble (scr) control vector, assessed by qRT-PCR (data are mean of triplicates ± SD; Student t test). (E-F) CCND3 and CDK4 mRNA levels in (E) xenograft samples (PXD) or (F) diagnostic samples from patients (PTS) with miR-497/195 expression above median (high) and below median (low) measured by qRT-PCR (data are mean ± SD; Mann-Whitney U test). (G) Downregulation at protein level of CDK4, CCND3, and RB1 phosphorylation on Ser708 upon miR-497/195 overexpression on PDX-S2 cells assessed by western blot. Loading control was β-actin. (H) Higher percentage of G1 cells (4′,6-diamidino-2-phenylindole [DAPI] staining), (I) lower cell count, (J) lower proliferation rate (lower decay in CellTrace Violet mean fluorescent intensity (MFI) relative to day 1 after staining and seeding) of miR-497/195 overexpressing PDX-S2 cells compared with empty vector (ev) control transduced cells cultured ex vivo on OP-9 feeder cells (data are mean ± SD of biological replicates: ev, n = 4-5; miR-497/195, n = 3; unpaired Student t-test. (I-J) Two-way analysis of variance (ANOVA). (K) Lower Ki-67 expression (MFI), (L) cell count, and (M) proliferation rate of miR-497/195 overexpressing PDX-L8 cells compared with ev control cultured ex vivo on OP-9 feeder cells (data are mean ± SD of biological replicates; ev, n = 6; miR-497/195, n = 6; two-way ANOVA). (N) Count of PDX-S2 green fluorescent protein-positive (GFP+) cells after transduction with anti-miR-497 or anti-miR-195 miRZip constructs or scr control cultured ex vivo on OP9 cells (data are mean of triplicates ± SD; two-way ANOVA). (O) Cell growth of PDX-L8 cells overexpressing control vector or miR-497/195 treated with palbociclib 0.1 or 1 µM cultured on OP9 cells. Growth was measured as count of palbociclib-treated cells relative to count of control cells at day 6 (data are mean ± SD; Student t test). (P) Cell growth of PDX-S6 cells transduced with miRZip or scr control construct and treated with palbociclib 0.1 or 1 µM cultured on OP9 cells. Treatment was started 4 days after transduction. Growth was measured as count of palbociclib-treated cells relative to count of control cells at day 7 of treatment (data are mean of triplicates ± SD; Student t test). ns, not significant.

miR-497/195 overexpression inhibits CDK4- and CCND3-mediated leukemia growth ex vivo. (A) Heatmap of 914 differentially expressed genes upon miR-497/195 overexpression in PDX-S2 (adjusted P < .05). (B) Downregulation of CCND3 and CDK4 messenger RNA (mRNA) levels in PDX-S2 and (C) in PDX-L8 cells upon miR-497/195 overexpression assessed by qRT-PCR (data are mean ± SD; Mann-Whitney U test). (D) CDK4 and CCND3 mRNA fold change in NALM-6 cells upon transduction with anti-miR-497 or anti-miR-195 miRZip constructs compared with scramble (scr) control vector, assessed by qRT-PCR (data are mean of triplicates ± SD; Student t test). (E-F) CCND3 and CDK4 mRNA levels in (E) xenograft samples (PXD) or (F) diagnostic samples from patients (PTS) with miR-497/195 expression above median (high) and below median (low) measured by qRT-PCR (data are mean ± SD; Mann-Whitney U test). (G) Downregulation at protein level of CDK4, CCND3, and RB1 phosphorylation on Ser708 upon miR-497/195 overexpression on PDX-S2 cells assessed by western blot. Loading control was β-actin. (H) Higher percentage of G1 cells (4′,6-diamidino-2-phenylindole [DAPI] staining), (I) lower cell count, (J) lower proliferation rate (lower decay in CellTrace Violet mean fluorescent intensity (MFI) relative to day 1 after staining and seeding) of miR-497/195 overexpressing PDX-S2 cells compared with empty vector (ev) control transduced cells cultured ex vivo on OP-9 feeder cells (data are mean ± SD of biological replicates: ev, n = 4-5; miR-497/195, n = 3; unpaired Student t-test. (I-J) Two-way analysis of variance (ANOVA). (K) Lower Ki-67 expression (MFI), (L) cell count, and (M) proliferation rate of miR-497/195 overexpressing PDX-L8 cells compared with ev control cultured ex vivo on OP-9 feeder cells (data are mean ± SD of biological replicates; ev, n = 6; miR-497/195, n = 6; two-way ANOVA). (N) Count of PDX-S2 green fluorescent protein-positive (GFP+) cells after transduction with anti-miR-497 or anti-miR-195 miRZip constructs or scr control cultured ex vivo on OP9 cells (data are mean of triplicates ± SD; two-way ANOVA). (O) Cell growth of PDX-L8 cells overexpressing control vector or miR-497/195 treated with palbociclib 0.1 or 1 µM cultured on OP9 cells. Growth was measured as count of palbociclib-treated cells relative to count of control cells at day 6 (data are mean ± SD; Student t test). (P) Cell growth of PDX-S6 cells transduced with miRZip or scr control construct and treated with palbociclib 0.1 or 1 µM cultured on OP9 cells. Treatment was started 4 days after transduction. Growth was measured as count of palbociclib-treated cells relative to count of control cells at day 7 of treatment (data are mean of triplicates ± SD; Student t test). ns, not significant.

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