HDAC1 promotes the migration of human myeloma cells via regulation of the lncRNA/Slug axis

Abstract

Understanding the mechanisms underlying malignancy in myeloma cells is important for targeted treatment and drug development. Histone deacetylases (HDACs) can regulate the progression of various cancer types; however, their roles in myeloma are not well known. In the present study, the expression of class I HDACs in myeloma cells and tissues was evaluated. Furthermore, the effects of HDAC1 on the migration of myeloma cells and the associated mechanisms were investigated. Among the class I HDACs evaluated, HDAC1 was upregulated in both myeloma cells and tissues. Targeted inhibition of HDAC1 suppressed the migration of myeloma cells. Of the assessed transcription factors, small interfering (si)‑HDAC1 decreased the expression of Slug. Overexpression of Slug reversed the si‑HDAC1‑mediated suppressed migration of myeloma cells. Mechanistically, the results revealed that HDAC1 regulated the mRNA stability of Slug, while it had no effect on its transcription or nuclear export. Furthermore, HDAC1 negatively regulated the expression of long non‑coding RNA (lncRNA) NONHSAT113026, which could bind with the 3'‑untranslated region of Slug mRNA to facilitate its degradation. The present study demonstrated that HDAC1 promoted the migration of human myeloma cells via regulation of lncRNA/Slug signaling.

Keywords: HDAC1; Slug; long non‑coding RNA; migration; myeloma.

Figure 1

HDAC1 is increased in MM cells and tissues. (A) The mRNA expression of class I HDACs in MM and nPCs cells were evaluated. (B) The protein expression of HDACs in MM and nPCs cells were determined. Expression of HDAC1 in human MM cancer tissues and normal mucosa tissues from (C) Talantov and (D) Haqq myeloma datasets from the Oncomine database. Data are presented as mean ± SD of three independent experiments. ^**P<0.01. HDAC1, histone deactylase 1; MM, multiple myeloma; nPCs, normal plasma cells.

Figure 2

HDAC8 regulates the migration of MM cells. (A) Cells were transfected with si-NC or si-HDAC1-1 or -2 for 24 h, the protein expression of HDAC1 was checked, si-HDAC1-1 was used for subsequent experiments. (B) Wound healing assay for cells transfected with si-NC or si-HDAC1 for 24 h. (C) U266 and (D) RPMI8226 cells were transfected with si-NC or si-HDAC1 for 24 h, the mRNA expression of MMP2, MMP9 and E-Cad was evaluated. (E) U266 cells were transfected with si-NC or si-HDAC1 for 24 h, the protein expression of MMP2, MMP9 and E-Cad was determined and quantitatively analyzed. Data are presented as the mean ± SD of three independent experiments. ^**P<0.01. HDAC1, histone deactylase 1; MM, multiple myeloma; si, small interfering; NC, negative control; E-Cad, E-Cadherin.

Figure 3

Slug is essential for HDAC1-regulated motility of MM cells. (A) U266 and (B) RPMI8226 cells were transfected with si-NC or si-HDAC1 for 24 h, the mRNA expression was determined via RT-qPCR. (C) Cells were transfected with si-NC or si-HDAC1 for 24 h, the protein expression of Slug was checked. U266 cells were co-transfected with si-NC, si-HDAC1, vector control or pcDNA/Slug for 24 h. (D) Dxpression of Slug was determined by western blot analysis. (E) Migration was assessed by wound healing assay. (F) the expression of E-Cad was checked by western blot analysis. Data are presented as the mean ± SD of three independent experiments. ^**P<0.01, NS, not significant; HDAC1, histone deactylase 1; MM, multiple myeloma; si, small interfering; NC, negative control; Vec, vector.

Figure 4

HDAC1 positively regulates the mRNA stability of Slug. (A) Cells were transfected with si-NC or si-HDAC1 for 24 h, the precursor mRNA of Slug was determined by RTq-PCR. (B) Cells were co-transfected with si-NC, si-HDAC1 or pGL3-Slug for 24 h, the promoter activity was evaluated via dual-luciferase assay. (C) Total RNA from the cytoplasm and nucleic fraction of U266 cells transfected with si-NC or si-HDAC1 for 24 h were isolated, the levels of Slug mRNA was measured. (D) U266 or (E) RPMI8226 cells were pre-transfected with si-NC or si-HDAC1 for 24 h and then further treated with Act-D for the indicated time periods before the mRNA expression of Slug was measured. Data are presented as the mean ± SD of three independent experiments. ^**P<0.01. HDAC1, histone deactylase 1; MM, multiple myeloma; si, small interfering; NC, negative control; Cyto, cytoplasm; Nucl, nucleus; Con, control.

Figure 5

HDAC1 negatively regulates the expression of lncRNA NONHSAT113026 in MM cells. (A) Cells were transfected with si-NC or si-HDAC1 for 24 h, the expression of lncRNA NONHSAT113026 was measured via RT-qPCR. (B) Cells were transfected with si-NC or si-lncRNA NONHSAT113026 for 24 h, the expression of lncRNA NONHSAT113026 was measured. (C) U266 cells were transfected with si-NC or si-lncRNA NONHSAT113026 for 24 h, cell migration was evaluated by wound healing assay. (D) U266 or (E) RPMI8226 cells were pre-transfected with si-NC or si-lncRNA NONHSAT113026 for 24 h and then expression of MMP2, MMP9 and E-Cad were measured via RT-qPCR. Data are presented as the mean ± SD of three independent experiments. ^**P<0.01. HDAC1, histone deactylase 1; MM, multiple myeloma; si, small interfering; NC, negative control; lnc, long non-coding; E-Cad, E-cadherin.

Figure 6

lncRNA NONHSAT113026 is involved in HDAC8-regulated expression of Slug. (A) U266 and (B) RPMI8226 cells were co-transfected with si-NC, si-HDAC1 and si-lncRNA NONHSAT113026 for 24 h before the mRNA expression of Slug was measured. (C) U266 and (D) RPMI8226 cells were co-transfected with si-NC, si-HDAC1 and si-lncRNA NONHSAT113026 for 24 h before the protein expression of Slug was measured. (E) U266 and (F) RPMI8226 cells were pre-transfected with si-NC or si-lncRNA NONHSAT113026 for 24 h and further treated with Act-D for indicated time periods before the mRNA of Slug was evaluated. (G) U266 cells were co-transfected with si-NC, si-HDAC1 and si-lncRNA NONHSAT113026 for 24 h, the expression of E-Cad was checked. Data are presented as the mean ± SD of three independent experiments. ^**P<0.01. NS, no significant; HDAC1, histone deactylase 1; MM, multiple myeloma; si, small interfering; NC, negative control; lnc, long non-coding.