miR-181d/RBP2/NF-κB p65 Feedback Regulation Promotes Chronic Myeloid Leukemia Blast Crisis

Abstract

Background: Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease. Once it progresses into the phase of blast crisis (CML-BP), the curative effect is poor, and the fatality rate is extremely high. Therefore, it is urgent to explore the molecular mechanisms of blast crisis and identify new therapeutic targets.

Methods: The expression levels of miR-181d, RBP2 and NF-κB p65 were assessed in 42 newly diagnosed CML-CP patients and 15 CML-BP patients. Quantitative real-time PCR, Western blots, and cell proliferation assay were used to characterize the changes induced by overexpression or inhibition of miR-181d, RBP2 or p65. Luciferase reporter assay and ChIP assay was conducted to establish functional association between miR-181d, RBP2 and p65. Inhibition of miR-181d expression and its consequences in tumor growth was demonstrated in vivo models.

Results: We found that miR-181d was overexpressed in CML-BP, which promoted leukemia cell proliferation. Histone demethylase RBP2 was identified as a direct target of miR-181d which downregulated RBP2 expression. Moreover, RBP2 inhibited transcriptional expression of NF-κB subunit, p65 by binding to its promoter and demethylating the tri/dimethylated H3K4 region in the p65 promoter locus. In turn, p65 directly bound to miR-181d promoter and upregulated its expression. Therefore, RBP2 inhibition resulting from miR-181d overexpression led to p65 upregulation which further forwarded miR-181d expression. This miR-181d/RBP2/p65 feedback regulation caused sustained NF-κB activation, which contributed to the development of CML-BP.

Conclusions: Taken together, the miR-181d/RBP2/p65 feedback regulation promoted CML-BP and miR-181d may serve as a potential therapeutic target of CML-BP.

Keywords: CML blast crisis; RBP2; cell proliferation; miR-181d; p65.

Figure 1

miR-181d is overexpressed in CML-BP and promotes leukemia cell proliferation. (A) qRT-PCR analysis of mature miR-181d in CML-CP and CML-BP patients. Human CML-CP and BP cells were obtained from patients' bone marrow. (B, C) EDU of K562 and HL60 cells following transfection with miR-181d mimics or inhibitor. The results were confirmed by 3 independent experiments. *p < 0.05, **p < 0.01.

Figure 2

miR-181d directly regulates RBP2 expression. (A, B) qRT-PCR and Western blot analysis of RBP2 mRNA and protein levels after transfection with miR-181d mimics or inhibitor. (C) Schematic illustration of the predicted miR-181d binding sites in RBP2 3′-UTR. (D–G) RBP2 wild type 3′UTR and its mutated activity following miR-181d mimics/inhibitor transfection in HL60 and HEK-293 cells. Luciferase activities were determined at 48 h and normalized by Renilla luciferase activity. The results are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not statistically significant.

Figure 3

p65 is directly and epigenetically downregulated by RBP2. (A, B) qRT-PCR of RBP2 and p65 mRNA level after transfection with RBP2 wild-type or RBP2-mutant (defective in demethylase activity, RBP2 H483A) plasmids in K562 and HL60 cells. (C) Western blot analysis of RBP2 and p65 protein expression levels after transfection with RBP2 wild-type or RBP2 H483A plasmids in K562 and HL60 cells. (D) Schematic illustration of the predicted RBP2 binding sites in the p65 promoter. (E, F) Regulation of p65 wild-type promoter by RBP2, including the first RBP2 binding site (p65 pro1) and the second RBP2 binding site (p65 pro2). HL60 and HEK-293 cells were transfected with p65 wild-type reporters, together with RBP2 expression plasmids, followed by luciferase activity assessment 48 h post-transfection. (G, H) p65 wild-type pro1 and its mutated activity following RBP2 wild-type plasmids transfection in HL60 and HEK-293 cells. (I, J) ChIP assay for RBP2 binding to the p65 promoter in K562 and HL60 cells. (K, L) The binding of RBP2 and H3K4me3/2 to p65 promoter after RBP2 plasmid transfection in K562 and HL60 cells. The results are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not statistically significant.

Figure 4

p65 is overexpressed in CML-BP and promotes leukemia cell proliferation. (A, B) qRT-PCR and Immunostaining analysis of p65 mRNA and protein levels in bone-marrow samples from patients with CML-CP and CML-BP. (C, D) EDU of K562 and HL60 cells following transfection with p65 plasmid or siRNA. The results were confirmed by 3 independent experiments. *p < 0.05, **p < 0.01.

Figure 5

miR-181d is directly upregulated by p65. (A, B) qRT-PCR of p65 mRNA and mature miR-181d levels after transfection with p65 plasmid or siRNA in K562 and HL60 cells. (C) Western blot analysis of p65 protein expression level after transfection with p65 plasmid or siRNA in K562 and HL60 cells. (D) Schematic illustration of the predicted p65 binding sites in the miR-181d promoter. (E–H) Regulation of miR-181d wild-type promoter by p65, including the first p65 binding site (miR-181d pro1) and the second p65 binding site (miR-181d pro2). HL60 and HEK-293 cells were transfected with miR-181d wild-type reporters together with p65 plasmid/siRNA and the luciferase activity was assessed 48 h or 72h post-transfection. (I–L) miR-181d wild-type pro1 and its mutated form activities following with p65 plasmid or siRNA transfection in HL60 and HEK-293 cells. (M, N) ChIP assay for p65 binding to the miR-181d promoter in K562 and HL60 cells. The results are from 3 independent experiments. *P < 0.05, **p < 0.01, ***p < 0.001, ns, not statistically significant.

Figure 6

Inhibition of miR-181d suppresses leukemia cell proliferation in vivo. (A) Xenograft model in NOD/SCID male mice. Timeline of the miR-181d antagomir therapeutic delivery experiment. Primary tumor gross appearance (B) and tumor growth curves (C) and the average tumor weight (D) of the miR-181d antagomir or control treated xenograft model. (E) RT–PCR analysis shows miR-181d antagomir efficacy in each group. (F–I) IHC staining and relative IHC score for RBP2, p65 and Ki67 were performed on the indicated tumors. Similar results were acquired in three independent experiments. (J) A summary of the findings. A new epigenetic mechanism involved in the pathogenesis of CML-BP: In CML progression, miR-181d overexpression directly inhibits RBP2 expression, low levels of RBP2 cannot repress the expression of p65, which increases miR-181d expression and stimulates cell proliferation. *p < 0.05, **p < 0.01, ***p < 0.001.