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P. Lin et al.
C resistant AML patients (total 153 genes), we found a list of common up-regulated NRF2 target genes (n=37) (Figure 3B). Interestingly, dual-specificity protein phosphatase 1 (DUSP1) was one of the genes up-regulated in both high- risk MDS patients and Ara-C-resistant AML patients (Figure 3C). We then performed NRF2 ChIP-seq analysis based on a published dataset from human lymphoblastoid cell lines.24 In cells treated with NRF2 agonist, ChIP analy- sis vaildated the NRF2 binding site in the region of DUSP1 gene loci (Figure 3D). The NRF2 binding regions proximal to NQO1 and DUSP1 genes contained a conserved NRF2
binding TGAnnnnGG motif, as previously reported (Figure 3E).25 ChIP q-PCR analysis revealed that the NRF2 binding signals in the NQO1 and DUSP1 genes were sig- nificantly higher than the negative control loci. Lower NRF2 signals were detected in SKM-1 with 5 mM NRF2 inhibitor treatment (48 h, P<0.01) (Figure 3F).
Consistent with the mRNA expression of NRF2, mRNA expression of DUSP1 could also be inhibited by 2 mM NRF2 inhibitor Luteolin treatment in primary MDS cells (Figure 4A). Our q-PCR results confirmed that DUSP1 was an NRF2 direct target gene in SKM-1 and
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Figure 4. NRF2 and DUSP1 expressions were elevated in higher-risk myelodysplastic syndrome (MDS) or cytarabine (Ara-C)--resistant MDS patients. (A) NRF2 and DUSP1 mRNA levels were both repressed by Luteolin in primary MDS cells. (B) DUSP1 immunohistochemistry (IHC) staining of bone marrow (BM) biopsy samples (magnification ×400). (C) NRF2 and DUSP1 IHC scores in controls and MDS. (D) Immunoblotting analysis was conducted for NRF2 and DUSP1 protein levels in healthy controls, MDS cell lines, and primary MDS cells. (E) Elevations of NRF2 and DUSP1 were confirmed in the BM samples of Ara-C-resistant MDS by immunoblotting analysis. *P<0.05; **P<0.01.
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