Fig 1: RHOA activates IFGBP2 expression through serum response factor. (A) Luciferase expression analysis shows that in the presence of the IGFBP2 promoter there was an increase in activity that was proportional to the levels of serum response factor (SRF) transfected in the same cells. (B) When SRF was overexpressed in either NIH3T3 or BaF3 cells, there was a proportional increase in IGFBP2 expression. (C) When K562 cells were treated with RHOA activator I, levels of SRF declined in the cytoplasm but increased in the nucleus. The relative purity of the cytoplasmic and nuclear protein enrichment was demonstrated by the almost exclusive presence of proteins GAPDH (cytoplasm) and Lamin A (nucleus). (D) The same increased levels of SRF in the nucleus of adherent 3T3 cells following RHOA activation was seen using confocal microscopy. (E) Chromatin immunoprecipitation quantitative polymerase chain reaction analysis from K562 cells using primers P1, which target the IGFBP2 promoter region, showed occupancy of SRF on the IGFBP2 promoter (SRF-), which was increased when the cells were treated with the RHOA activator (SRF+). In the same experimental model, no significant changes were seen in the downstream intron region defined by the P2 primers. The scale bar in (D) represents 50 µm. Differences between the knockout and matched control cells were evaluated using the Student t test. Cells transduced with empty vector were used as the control for comparison in (B). *P<0.01, **P≤0.001, ***P≤0.0001, ****P≤0.00001, ns: not significant. MSCV: murine stem cell virus; EV: empty vector.
Fig 2: PGK1 and IGFBP2 are secreted miR-10b targets(A) The levels of PGK1 and IGFBP2 proteins are increased in CM of miR-10b-edited GSCs and glioma cells. Naive GBM8, GBM4, and LN229 cells were transduced with either EV (Cas9 only), G1, or G3 (miR-10b-editing) lentivirus, or UT, for 5 days, and the CM samples collected and analyzed by western blotting. Ponceau S staining was used as loading control. (B) miR-10b mimic regulates luciferase reporter containing a single miR-10b complementary site (mean ± SD, n = 5 samples/group, two-tailed unpaired t test). Scramble mimic (sc-mimic) was used as a control. (C) miR-10b mimic reduces the activity of luciferase reporters of PGK1 and IGFBP2 with several putative miR-10b-binding sites (mean ± SD, n = 5 samples/group, two-tailed unpaired t test). The detailed description of specific reporter constructs is in the materials and methods. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns-not significant.
Fig 3: PGK1 and IGFBP2 mediate bystander effects of miR-10b editing(A) CM samples derived from miR-10b-edited GBM8 cultures (G1 and G3) and the corresponding EV control cells were incubated with PGK1 inhibitor CBR-470-1 (10 μM) or DMSO and used for culturing naive GBM8 for 7 days. Representative microscopy images are shown. The effects on GSC growth were quantified (right panel, mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). (B) CM derived from miR-10b-edited GBM8 cultures (G1 and G3) and the corresponding EV control cells were incubated with 10 μg/mL anti-IGFBP2 neutralizing antibody or control goat IgG and used for culturing naive GBM8 for 7 days (mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). ∗∗∗∗ p < 0.0001.
Fig 4: Knockout of IFGBP2 partially recapitulates the phenotype of RHOA loss. (A) Analysis of the supernatant from K562 C8 and KU812 C7 knockout (KO) cells when compared with the respective mock control (MC) cells, showed a dramatic reduction in IGFBP2 protein levels in the KO cell culture medium. (B) In migration and adhesion analysis, K562 KO C8 cells showed a reduction in both phenotypes compared with MC cells. When exogenous, recombinant IGFBP2 (rIGFBP2) was added to the culture medium, there was a significant increase in both phenotypes in the KO C8 cells. (C, D) CRISPR knockout of IGFBP2 in K562 cells generated two clones, C5 and C11 (C), which when subjected to migration and adhesion assays (D) showed reduced levels compared with MC cells. (E) When these cells were xenografted into NSG hosts, mice receiving KO clones C5 and C11 showed an increased survival compared with MC-engrafted mice. (F) This result was consistent with luminescence intensity in the mice after 28 days, which showed that the tumor burden was significantly reduced in the mice grafted with the KO C5 cells compared to that of mice engrafted with MC cells. White blood cell count at the time of sacrifice was also reduced in the mice grafted with KO C5 cells. The scale bars in (B) and (D) represent 400 µm, with the magnification for the images in (B) and (D) being the same. Differences between the KO and MC cells were evaluated using the Student t test. Pairwise comparisons are indicated by the horizontal lines. **P≤0.001, ***P≤0.0001, ****P ≤0.00001. CM: culture medium; WBC: white blood cells.
Fig 5: Detection of the expression levels of potential RHOA downstream targets in chronic myeloid leukemia cells and primary patients’ samples. (A) Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis was used to verify differential expression of several of the most highly downregulated and upregulated genes in K562 RHOA knockout (KO) cells compared with mock control (MC) cells (N=3). Analysis of the same genes in the KU812 RHOA KO cells using RT-qPCR showed the same differential expression patterns. (B) The reduced expression levels for IGFBP2, IL20RB and CD24 were further confirmed using western blotting. (C) Analysis of the GSE47927 chronic myeloid leukemia (CML) dataset revealed a highly significant difference in IGFBP2 expression in CML samples (n=52) compared with normal controls (n=15). When cells from these CML cases were sub-fractionated into different stem/progenitor subpopulations, there was a highly significant increase in IGFBP2 expression in CML hematopoietic stem cells compared with normal counterparts. In similar comparisons, a significant increase in IGFBP2 expression was seen in common myeloid progenitors and megakaryocyte-erythroid progenitors but no difference was seen in granulocyte-monocytic progenitors. Statistical significance was established using the Student t test. **P≤0.001, ***P≤0.0001, ****P≤0.00001. HSC: hematopoietic stem cells; CMP: common myeloid progenitors; GMP: granulocyte-monocytic progenitors; MEP: megakaryocyte-erythroid progenitors.
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