Novel regulation of nuclear factor-YB by miR-485-3p affects the expression of DNA topoisomerase IIα and drug responsiveness

Abstract

Nuclear factor (NF)-YB, a subunit of the transcription factor nuclear factor Y (NF-Y) complex, binds and activates CCAAT-containing promoters. Our previous work suggested that NF-YB may be a mediator of topoisomerase IIα (Top2α), working through the Top2α promoter. DNA topoisomerase II (Top2) is an essential nuclear enzyme and the primary target for several clinically important anticancer drugs. Our teniposide-resistant human lymphoblastic leukemia CEM cells (CEM/VM-1-5) express reduced Top2α protein compared with parental CEM cells. To study the regulation of Top2α during the development of drug resistance, we found that NF-YB protein expression is increased in CEM/VM-1-5 cells compared with parental CEM cells. This further suggests that increased NF-YB may be a negative regulator of Top2α in CEM/VM-1-5 cells. We asked what causes the up-regulation of NF-YB in CEM/VM-1-5 cells. We found by microRNA profiling that hsa-miR-485-3p is lower in CEM/VM-1-5 cells compared with CEM cells. MicroRNA target prediction programs revealed that the 3'-untranslated region (3'-UTR) of NF-YB harbors a putative hsa-miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p mediates drug responsiveness by decreasing NF-YB expression, which in turn negatively regulates Top2α expression. To test this, we overexpressed miR-485-3p in CEM/VM-1-5 cells and found that this led to reduced expression of NF-YB, a corresponding up-regulation of Top2α, and increased sensitivity to the Top2 inhibitors. Results in CEM cells were replicated in drug-sensitive and -resistant human rhabdomyosarcoma Rh30 cells, suggesting that our findings represent a general phenomenon. Ours is the first study to show that miR-485-3p mediates Top2α down-regulation in part by altered regulation of NF-YB.

Fig. 1.

Inverse relationship between the protein levels of Top2α and NF-YB in CEM and CEM/VM-1-5 cells and Rh30 and Rh30/v1 cells. Western blots of nuclear Top2α and NF-YB expression in CEM and CEM/VM-1-5 cells (A) and Rh30 and Rh30/v1 cells (D). PCNA served as loading control for nuclear proteins. Average of Top2α and NF-YB levels from three independent experiments ± S.D. are shown, determined by densitometric scanning on Western blots and normalized to PCNA signal; either CEM (B) or Rh30 (E) was assigned a value of 1 for comparison. **, p < 0.005; ***, p < 0.0001. C and F, NF-YB mRNA expression was analyzed by semiquantitative RT-PCR in CEM and CEM/VM-1-5 cells (C) and Rh30 and Rh30/v1 cells (F).

Fig. 2.

NF-YB is a putative target for hsa-miR-485-3p. Expression of hsa-miR-485-3p in CEM and CEM/VM-1-5 cells was determined by Applied Biosystems real-time PCR array (TaqMan Human MicroRNA Array version 1.0) according to the manufacturer's instructions. C_t values (A) or relative expression levels of miR-485-3p (B) are means of three pairs of microRNA profile results from CEM and CEM/VM-1-5 cells ± S.D. *, p < 0.05; **, p < 0.01. C, sequence alignment of putative has-miR-485-3p binding sites in 3′-UTR of NF-YB gene of four species. The base pairing nucleotides are in boldface type.

Fig. 3.

MiR-485-3p targets NF-YB 3′-UTR. A, luciferase reporter containing a putative miR-485-3p binding site, NF-YB-3′-UTR (pGL3-NF-YB-3′-UTR), was cotransfected with either miR-Crtl (pCDH-empty vector) or the miR-485-3p expression vector (pCDH-miR-485-3p) with schematic diagram on the left. B, MiR-485-3p expression vector (pCDH-miR-485-3p) was cotransfected with either luciferase reporter containing NF-YB-3′-UTR with or without putative miR-485-3p binding site, which was defined as pGL3-NF-YB-3′-UTR or pGL3-NF-YB-3′-UTR-d, respectively, with schematic diagram (left). Relative luciferase activities were measured and normalized against β-galactosidase activity. Values are average of three separate experiments done in triplicate ± S.E.; *, p < 0.05.

Fig. 4.

MiR-485-3p inhibition on NF-YB and mediation on drug responsiveness. Western blots of nuclear Top2α, NF-YB, and NF-YA expression in CEM/VM-1-5 (A) and Rh30/v1 cells (B). CEM/VM-1-5 and Rh30/v1 cells were transduced with either miR-485-3p expression virus or control virus (miR-Crtl). PCNA served as loading control for nuclear protein. IC₅₀ values of cells exposed to etoposide (C) or vinblastine (D) at various concentrations were calculated from the percentage of viable cells after exposure to treatment obtained from MTT assay. Values are average of three independent experiments done in triplicate ± S.E. *, p < 0.05.

Fig. 5.

NF-YB and Top2α mRNA expression levels in the NCI-60 panel of tumor cell lines. Shown is a graphical summary of NF-YB and Top2α mRNA levels in the form of a mean graph of the NCI-60 cells. Expression above the mean levels is drawn to the right of the centerline, and below the mean is drawn to the left.