Page 89 - Haematologica Vol. 109 - July 2024

ARTICLE - Targeting TNF/IL-17/MAPK in hE2A-PBX1 zebrafish H. Luo et al. E
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Figure 6. hE2A-PBX1 activate the TNF/IL-17/MAPK signaling pathway through upregulation of runx1 expression. (A) Whole mount in situ hybridization (WISH) of runx1 expressions in Tg(hsp70:E2A-PBX1-EGFP) (right panel) were higher than siblings (left panel) at 5 days post-fertilization (dpf). The caudal hematopoietic tissue (CHT) is enlarged in the red box (original magnification ×200). (A’) Statistical analysis of the runx1+ signals in panel (A). The black asterisks indicate statistical difference (Student t tests, mean ± standard error of the mean [SEM]; *P<0.05). N/N: number of zebrafish larvae showing representative phenotype/total number of zebrafish larvae examined. (B) Real time quantitative polymerase chain reaction (RT-qPCR) analysis showed increased runx1 mRNA expression in Tg(hsp70:E2A-PBX1-EGFP) compared to the sibling controls at 3 dpf. The black asterisks indicate statistical difference (Student t tests, mean ± SEM; ****P<0.0001) (C) Decreased number of lyz+ neutrophils in sibling and Tg(hsp70:E2A- PBX1-EGFP) larvae after injecting 0.2 mM runx1 morpholino (MO) at 3 dpf. The control groups were treated with 0.2 mM random sequence MO. The CHT is enlarged in the red box (original magnification ×200). (C’) Statistical analysis of the lyz+ signals shown in panel (C). The black asterisks indicate statistical difference (N≥15, one-way ANOVA, mean ± SEM; ***P<0.001, ****P<0.0001). (D) Top 20 enriched KEGG pathways identified in the analysis of differentially expressed genes (DEG) in KM cells from 6-month- old Tg(hsp70:E2A-PBX1-EGFP) compared to control groups after 3-month heat shock. The TNF/IL-17/MAPK signaling pathway is highlighted by the red arrow. (E) Heatmap of DEG involved in the TNF/IL-17/MAPK signaling pathway between WT and hE2A-PBX1 adult fish. Red and blue represent an increase and decrease gene expression levels, respectively; P<0.05. (F) RT-qPCR analysis showing mRNA expression of fosab, hsp27, junb, mmp9, socs3b, fosb and fosl1a in Tg(hsp70:E2A-PBX1-EGFP) compared to siblings after injecting 0.2 mM runx1 MO at 3 dpf. The control groups were treated with 0.2 mM random sequence MO (one-way ANOVA, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001). TNF: tumor necrosis factor; IL-17: interleukin-17; MAPK: mitogen-activated protein kinase.
IL-17/MAPK signaling pathway was enriched in hE2A-PBX1 zebrafish (Figure 6D), with ten genes (fosl1a, fosab, fosb, mmp13a, junb, hsp27, socs3a, socs3b, mmp9 and jund) were significantly upregulated in these pathways (Figure 6E). Additionally, we microinjected sibling and hE2A-PBX1 transgenic zebrafish with 0.2 mM runx1 MO, and detected the changes of significantly upregulated genes in the TNF/
IL-17/MAPK signaling pathway after knockdown of runx1. The results showed that knockdown of runx1 could significantly reverse the high expression of hsp27, junb, socs3b, fosb and fosl1a in hE2A-PBX1 transgenic line (Figure 6F). These data suggest that hE2A-PBX1 can induce myeloid expansion and activate the TNF/IL-17/MAPK signaling pathway through upregulation of runx1 expression.
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