miR-582 Suppresses the Proliferation of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Cells and Protects Them From Natural Killer Cell-Mediated Cytotoxicity

Abstract

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a malignancy characterized by the aberrant accumulation of immature B-cell precursors in bone marrow and other lymphoid organs. Although several intrinsic regulatory signals participating in BCP-ALL have been clarified, detailed intrinsic and extrinsic mechanisms that regulate BCP-ALL progression have not been fully understood. In the current study, we report that miR-582 is downregulated in BCP-ALL cells compared with normal B cells. Forced overexpression of miR-582 attenuated BCP-ALL cell proliferation and survival. We found that miR-582 overexpression disturbed the mitochondrial metabolism of BCP-ALL cells, leading to less ATP but more ROS production. Mechanistically, we identified PPTC7 as a direct target of miR-582. MiR-582 overexpression inhibited the activity of CoQ10, which is downstream of PPTC7 and played an important positive regulatory role in mitochondrial electron transportation. Finally, we found that overexpression of miR-582 upregulated the expression of immune checkpoint molecule CD276 and reduced NK cell-mediated cytotoxicity against BCP-ALL cells. CD276 blockade significantly increased NK cell-mediated cytotoxicity against miR-582-overexpressing BCP-ALL cells. Together, our research demonstrates that miR-582 acts as a negative regulator of BCP-ALL cells by reducing proliferation and survival, but protects BCP-ALL cells from NK cell-mediated cytotoxicity, suggesting that miR-582 may be a new therapeutic biomarker for BCP-ALL with CD276 blocker.

Keywords: BCP-ALL; CD276; NK cells; PPTC7; immune checkpoint; metabolism; miR-582; mitochondria.

Figure 1

Overexpression of miR-582 inhibits the survival of BCP-ALL cells. (A, B) Relative expression of human miR-582-5p and miR-582-3p in normal B cells, BCP-ALL patients B cells, NALM-6, KOPN-8 and SUP-B15 cells were determined by qRT-PCR. (C) BCP-ALL patients B cells, NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus, and cultured for 72 h. Apoptosis was evaluated by using flow cytometry after staining of Annexin V and 7-AAD. The percentages of infected live (Annexin V^-/7-AAD^-) BCP-ALL cells, infected early and late apoptotic (Annexin V⁺/7-AAD^-/+) BCP-ALL cells were quantitatively compared (n = 3-4). (D) BCP-ALL cells were infected with pre-miR-582 lentivirus or their control lentivirus for 72 h, and Cleaved Caspase-3, Cleaved PARP proteins were determined by Western blotting, with β-actin as an internal control (n = 3). Bars represent means ± SD, *P < 0.05, **P < 0.01.

Figure 2

Overexpression of miR-582 inhibits the proliferation of BCP-ALL cells. (A) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus. Cell proliferation of infected cells was determined using the CFSE labeling assay (n = 3-6). (B) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus, and cultured for 72 h. Cell viability was detected by MTT method (n = 6). (C, D) Experimental timeline for in vivo study. luciferase (luci)-labeled NALM-6 were infected with pre-miR-582 lentivirus or their control lentivirus, then, an orthotopic xenograft BCP-ALL mouse model was established by i.v injecting 1 × 10⁶ luci-labeling pre-miR-582 NALM-6 cells or pre-miR-Ctrl NALM-6 cells into NCG mice on day 0. Tumor burden was determined on day 14 and 21. (E, F) On Day 21, single cell suspensions of BM and spleen from BCP-ALL mouse model were analyzed by flow cytometry (E), and the percentage of human CD19⁺ B cells were quantitatively compared (F) (n = 3). (G) Survival of NCG mice which injected with miR-582-overexpressing NALM-6 cells and control NALM-6 cells. Bars represent means ± SD, *P < 0.05, **P < 0.01.

Figure 3

Overexpression of miR-582 inhibits mitochondrial energy metabolism of BCP-ALL cells. (A) After infection with pre-miR-582 lentivirus or their control lentivirus for 72 h, the contents of ATP in NALM-6, KOPN-8 and SUP-B15 cells were measured by using a luciferase reporter assay system. The luminescence level was normalized to pre-miR-Ctrl group in each individual (n = 3). (B) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus for 72 h, the mitochondrial metabolic pressure was detected by using XF Extracellular Flux Analyzer (n = 3). (C, D) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus and their control lentivirus for 72 h, the total intracellular ROS and mitochondria ROS were determined by flow cytometry, total intracellular ROS was labeled by DCFH-DA, the mitochondria ROS was labeled by MitoTacker and DCFH-DA (n = 3-5). (E) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus for 72 h, 2-NBDG was added to glucose-free medium for 30 min and intracellular 2-NBDG was analyzed by flow cytometry and quantitatively by statistics the proportion of 2-NBDG⁺ cells (n = 3-5). Bars represent means ± SD, **P < 0.01.

Figure 4

miR-582 downregulates PPTC7 in BCP-ALL cells. (A) Significantly different expressed genes (differentially upregulated genes and differentially downregulated genes) in pre-miR-582 NALM-6 cells/pre-miR-Ctrl NALM-6 cells. (B) 94 genes which related to metabolism are significantly downregulated in pre-miR-582 NALM-6 cells. (C) 7 significantly downregulated metabolism-related genes were predicted target genes of human miR-582-5p by combined with RNA-seq data and TargetScan 6.2 database. (D, E) The mRNA expression of PPTC7 in pre-miR-Ctrl NALM-6 cells and pre-miR-582 NALM-6 cells were showed by volcano plot and heatmap. (F) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus for 72 h, and PPTC7 mRNA were determined by qRT-PCR, with β-actin as an internal control (n = 3). (G) Alignment of the seed sequence of human miR-582-5p with human PPTC7 3’UTR. Complementary bases are marked with blue color. (H) The sequences of PPTC7 3’UTR-wt and PPTC7 3’UTR-mut used for schematic of the reporter constructs. (I) HEK293T cells were transfected with miR-582-5p and different 3’UTR reporters of human PPTC7 for 72 h. Luciferase activity in cell lysates were determined by the dual luciferase reporter assay (n = 3). (J, K) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their controls for 72 h, and PPTC7 protein were determined by Western blotting, with β-actin as an internal control (n = 3). Bars represent means ± SD, *P < 0.05,**P < 0.01.

Figure 5

miR-582 attenuates BCP-ALL cells mitochondrial energy metabolism by inhibiting PPTC7/CoQ10 signaling. (A) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or their control lentivirus for 72 h, CoQ10 contents in cell lysates was evaluated and compared (n = 3). (B) NALM-6, KOPN-8 and SUP-B15 cells were infected with PPTC7 overexpression lentivirus (LV-PPTC7), with/without cultured with pre-miR-582 lentivirus for 72 h. CoQ10 contents in cell lysates was evaluated and compared (n = 3). (C) NALM-6, KOPN-8 and SUP-B15 cells were infected with LV-PPTC7, with/without cultured with pre-miR-582 lentivirus for 72 h, relative ATP level in cell lysates was evaluated and compared. (D, E) NALM-6, KOPN-8 and SUP-B15 cells were infected with LV-PPTC7, with/without cultured with pre-miR-582 lentivirus for 72 h, the total ROS and Mitochondria ROS were determined by flow cytometry, total intracellular ROS was labeled by DCFH-DA, the mitochondria ROS was labeled by MitoTacker and DCFH-DA. Bars represent means ± SD, *P < 0.05, **P < 0.01.

Figure 6

Overexpression of miR-582 protects BCP-ALL cells from NK cell-mediated cytotoxicity. (A) NALM-6 cells were infected with pre-miR-582 lentivirus or its control lentivirus for 72 h, and CD276 mRNA were determined by RNA-seq. (B, C) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus or its control lentivirus for 72 h, and CD276 mRNA were determined by qRT-PCR (B); the proportion of CD276⁺ cells were determined by flow cytometry (C). (D, E) NALM-6, KOPN-8 and SUP-B15 cells were infected with pre-miR-582 lentivirus and their control lentivirus, cultured with or without anti-CD276 antibody, and co-cultured with NK cells for 4 h, the proportion of CD107a⁺ NK and GZMB⁺ NK cells were determined by flow cytometry. (F) The signaling pathway of miR-582 regulate the progression of BCP-ALL and protect BCP-ALL cells from natural killer cell-mediated cytotoxicity, figure created with BioRender.com. Bars represent means ± SD, *P < 0.05, **P < 0.01.