Figure 6.
NR2F6 and SGMS1, two targets of miR-130b and miR-128a, have tumor-suppressive activity in MLL-AF4–driven leukemogenesis. (A) Overlap of deregulated genes in GSE79533 and GSE79450 data sets (black), predicted targets of miR-130b (green) or miR-128a (blue) using TargetScan and PicTar. (B) NR2F6 and (C) SGMS1 expression in t(4;11) MLL-AF4 pediatric leukemia blasts and nonblasts from our cohort. (D) Nr2f6 and (E) Sgms1 expression in FL Mll-AF4+ LSK (pre-leukemic) and BM (leukemic) of Mll-AF4+ pMIRH control mice and of Mll-AF4+ pMIRH-130b and Mll-AF4+ pMIRH-128a sick mice. (F) qRT-PCR of miR-130b, miR-128a, MLL-AF4, MEIS1, HOXA9, CDK6, BCL2, NR2F6, and SGMS1 in SEM cells transfected with a negative inhibitor control, miR-130b inhibitor, or miR-128a inhibitor. The log2 fold change (LOG2FC) is calculated using the negative inhibitor control as a reference. (G) Western blot against NR2F6 and SGMS1 in SEM cells transfected with a negative inhibitor control, miR-130b inhibitor, or miR-128a inhibitor. The brightness was adjusted manually in ImageJ to uniform the background, and each lane came from the same membrane. The WT lane was not directly next to the siRNA lane and is separated by a vertical line. Relative quantification for all 3 proteins was calculated by Image Laboratory using the sicontrol as a reference. The quantification presented for NR2F6 and SGMS1 is adjusted with the relative quantification of the β-actin signal intensity. The β-actin signal comes from the same membrane as the NR2F6/SGMS1 signal (n = 4). (H) Apoptosis and (I) proliferation of SEM leukemic cells upon NR2F6 and SGMS1 overexpression. (J) Expression of MLL-AF4 and its target genes (MEIS1, HOXA9, CDK6, BCL2, miR-130b, and miR-128a) and SGMS1 in SEM leukemic cells upon NR2F6 overexpression. The log2 fold change (LOG2FC) is calculated using SEM pCDH cells as a reference. (K) Survival curve of NSG mice transplanted with SEM control (pCDH-empty) and overexpressing NR2F6 (pCDH-NR2F6). NSG mice were culled 64 days after transplant due to facility concerns. This experiment could not be performed with SGMS1 due to the high amount of cell death in SEM cells. The overexpression of NR2F6 and SGMS1 in leukemic cells was achieved by lentiviral transduction, and transduced cells were monitored with GFP. A Gehan-Breslow-Wilcoxon test was used to compare survival curves. Unless stated otherwise, data were compared using a Mann-Whitney U test: *P < .05; **P < .01; ***P < .001; ****P < .0001. Graphs are presented as mean ± standard error of the mean. Blood Cancer UK CLCB, Childhoood Leukaemia Cell Bank.

NR2F6 and SGMS1, two targets of miR-130b and miR-128a, have tumor-suppressive activity in MLL-AF4–driven leukemogenesis. (A) Overlap of deregulated genes in GSE79533 and GSE79450 data sets (black), predicted targets of miR-130b (green) or miR-128a (blue) using TargetScan and PicTar. (B) NR2F6 and (C) SGMS1 expression in t(4;11) MLL-AF4 pediatric leukemia blasts and nonblasts from our cohort. (D) Nr2f6 and (E) Sgms1 expression in FL Mll-AF4+ LSK (pre-leukemic) and BM (leukemic) of Mll-AF4+ pMIRH control mice and of Mll-AF4+ pMIRH-130b and Mll-AF4+ pMIRH-128a sick mice. (F) qRT-PCR of miR-130b, miR-128a, MLL-AF4, MEIS1, HOXA9, CDK6, BCL2, NR2F6, and SGMS1 in SEM cells transfected with a negative inhibitor control, miR-130b inhibitor, or miR-128a inhibitor. The log2 fold change (LOG2FC) is calculated using the negative inhibitor control as a reference. (G) Western blot against NR2F6 and SGMS1 in SEM cells transfected with a negative inhibitor control, miR-130b inhibitor, or miR-128a inhibitor. The brightness was adjusted manually in ImageJ to uniform the background, and each lane came from the same membrane. The WT lane was not directly next to the siRNA lane and is separated by a vertical line. Relative quantification for all 3 proteins was calculated by Image Laboratory using the sicontrol as a reference. The quantification presented for NR2F6 and SGMS1 is adjusted with the relative quantification of the β-actin signal intensity. The β-actin signal comes from the same membrane as the NR2F6/SGMS1 signal (n = 4). (H) Apoptosis and (I) proliferation of SEM leukemic cells upon NR2F6 and SGMS1 overexpression. (J) Expression of MLL-AF4 and its target genes (MEIS1, HOXA9, CDK6, BCL2, miR-130b, and miR-128a) and SGMS1 in SEM leukemic cells upon NR2F6 overexpression. The log2 fold change (LOG2FC) is calculated using SEM pCDH cells as a reference. (K) Survival curve of NSG mice transplanted with SEM control (pCDH-empty) and overexpressing NR2F6 (pCDH-NR2F6). NSG mice were culled 64 days after transplant due to facility concerns. This experiment could not be performed with SGMS1 due to the high amount of cell death in SEM cells. The overexpression of NR2F6 and SGMS1 in leukemic cells was achieved by lentiviral transduction, and transduced cells were monitored with GFP. A Gehan-Breslow-Wilcoxon test was used to compare survival curves. Unless stated otherwise, data were compared using a Mann-Whitney U test: *P < .05; **P < .01; ***P < .001; ****P < .0001. Graphs are presented as mean ± standard error of the mean. Blood Cancer UK CLCB, Childhoood Leukaemia Cell Bank.

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