miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

Abstract

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.

Keywords: epi-miRNA; methylation; miR-29b; microRNA; multiple myeloma.

Figure 1. SOCS-1 mRNA expression and its correlation with miR-29b levels in primary MM and PCL samples.(A). Differential expression of SOCS-1 mRNA in immunoselected CD138⁺ cells from 4 healthy donors (N), 55 MM classified according to the presence of the recurrent IGH chromosomal translocations and cyclin D expression (TC1: n = 12; TC2: n = 13; TC3: n = 12; TC4: n = 12; TC5: n = 6), 21 primary PCL (pPCL), and 8 secondary PCL (sPCL), as obtained by cDNA microarray; raw expression values are reported. Difference between normal and TC classes was assessed by t test using Welch correction or Wilcoxon rank sum test. SOCS-1 was significantly downregulated in all patient groups, except TC5, as compared with N samples (N vs TC1: P = 0.031; N vs TC2: P = 0.023; N vs TC3: P = 0.004; N vs TC4: P = 0.023; N vs TC5: P = 0.11; N vs pPCL: P = 0.03; N vs sPCL: P = 0.008).(B) Correlation analysis between endogenous miR-29b levels and SOCS-1 mRNA levels, determined by high density microarray of mRNA and miRNA expression in a panel of 34 MM (TC1:n = 6; TC2:n = 10; TC3:n = 9; TC4:n = 6; TC5:n = 3) and 18 pPCL samples (P = 0,09). Log values of raw data are reported in graph. Pearson test was used to evaluate the occurrence of correlation.

Figure 2. miR-29b demethylates SOCS-1 gene promoter.(A) Quantitative RT-PCR of miR-29b levels in NCI-H929 cells transfected with synthetic miR-29b mimics or scrambled oligonucleotides (NC). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. miR-29b levels in cells transfected with NC were set as internal reference. Data are the average of 2 independent transfection experiments performed in triplicate.(B) Sequenom MassARRAY platform was used for the quantitative methylation analysis. Mean methylation levels of CpG sites in SOCS-1 promoter are reported. Data are expressed as mean ± SD of 2 independent experiments performed in triplicate. CpGs whose methylation was detectable at basal level in NCI-H929 transfected cells, have been analyzed. °P ≤ 0.05; *P ≤ 0.01.(C) Graphic representation of all CpGs within amplicon 9 and 10 of SOCS-1 promoter. Red circles indicate all CpGs found demethylated after miR-29b transfection; blue circles represent CpGs unmethylated at basal level or unaffected by miR-29b.(D) Immunoblot of SOCS-1, 24 h after transfection of NCI-H929 cells with synthetic miR-29b or scrambled oligonucleotides (NC). GAPDH was used as loading control.

Figure 3. Bortezomib reduces global DNA methylation as well as SOCS-1 promoter methylation in MM cells.(A) Global DNA methylation levels were determined, as reported in the “Materials and Methods”, in NCI-H929 cells treated for 24 h with vehicle or bortezomib 0.5, 1.0 and 2.0 nM or(B) in NCI-H929 stably transduced with a control vector or miR-29b inhibitory sequences (anti-miR-29b) and then treated for 24 h with 1 nM bortezomib (BORT).(C) Mean methylation levels of CpG sites in SOCS-1 promoter, as determined by Sequenom MassARRAY analysis, in NCI-H929 cells treated for 24 h with vehicle or bortezomib 0.5, 1.0, or 2.0 nM. Data are expressed as mean ± SD of 2 independent experiments performed in triplicate.(D) Immunoblot of SOCS-1, 24 h after treatment of NCI-H929 cells with vehicle or bortezomib 0.5, 1.0 or 2.0 nM . GAPDH was used as loading control. *P ≤ 0.05; °P ≤ 0.01.(E) Quantitative RT-PCR of miR-29b levels in PCL patient-derived peripheral blood mononuclear cells, 24 and 72 h after systemic bortezomib treatment (1.3 mg/m²). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. *P ≤ 0.01.

Figure 4. miR-29b inhibits STAT3 and NF-κB signaling in MM cells.(A) Immunoblot of STAT3 phosphorylated at Y705 or unphosphorylated, 24 h after transfection of U266 cells with synthetic miR-29b or scrambled oligonucleotides (NC).(B) Quantitative RT-PCR of VEGF-A 24 h after transfection of U266 cells with synthetic miR-29b or scrambled oligonucleotides (NC). The results are shown as average mRNA expression, in 3 independent experiments, after normalization with GAPDH and ΔΔCt calculations *P < 0.05;(C) Dual luciferase assay was performed in U266 cells transfected with miR-29b or scrambled oligonucleotides (NC), together with a firefly luciferase constructs containing the NFκB-responsive elements and the pRL-TK vector for normalization. The firefly luciferase activity was normalized to renilla luciferase activity. The data are shown as relative luciferase activity of miR-29b-transfected cells as compared with the control (NC), in a total of six experiments from 3 independent transfections. *P < 0.05(D) Immunoblot of IκB-α phosphorylated at Ser32 or unphosphorylated, 24 h after transfection of U266 cells with synthetic miR-29b or scrambled oligonucleotides (NC). GAPDH was used as loading control.(E) Quantitative RT-PCR of IL-8, 24 h after transfection of U266 cells with synthetic miR-29b or scrambled oligonucleotides (NC). The results are shown as average mRNA expression, in 3 independent experiments, after normalization with GAPDH and ΔΔCt calculations. *P < 0.05.

Figure 5. miR-29b inhibits MM cell migration.(A) Quantitative RT-PCR of miR-29b levels in OPM2 cells, 24 h after transfection with synthetic miR-29b mimics or scrambled oligonucleotides (NC). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. miR-29b levels in cells transfected with NC were set as internal reference. Data are the average of 2 independent transfection experiments performed in triplicate.(B) Transwell migration assay of OPM2 cells, 24 h after transfection with miR-29b or scrambled oligonucleotides (NC), in the presence or absence of 10 ng/mL IL-8 added to the lower chamber of the transwell.(C). Quantitative RT-PCR of IL-8, MMP2 and VEGF-A, 24 h after transfection of OPM2 cells with synthetic miR-29b or scrambled oligonucleotides (NC). *P < 0.05, °P < 0.01.(D) Adhesion assay on HS-5 BM stromal cells performed with OPM2 cells, 24 h after transfection of MM cells with miR-29b or scrambled (NC) controls. Cells were stained, photographed under a light microscope equipped with a digital camera (original magnification, 20×) and then counted, as detailed in the “Materials and Methods”.(E) Quantitative RT-PCR of IL-8 and VEGF-A expression in HS-5 cells treated for 24 h with conditioned medium from OPM2 cells (0.5 × 10⁶), collected 24 h after transfection with synthetic miR-29b or scrambled oligonucleotides (NC). *P < 0.01.

Figure 6. miR-29b inhibits endothelial cell migration.(A) Transwell migration assay of HUVEC cells. HUVECs were incubated for 6 h using as chemo-attractant conditioned medium of OPM2 cells, collected 24 h after transfection with synthetic miR-29b mimics or scrambled oligonucleotides; migrating cells were quantified as indicated in materials and methods. A representative picture of migrating cells is shown (original magnification, 20×).(B) Quantitative RT-PCR of IL-8 and VEGF-A mRNA levels in HUVEC cells treated for 24 h with conditioned medium from OPM2 cells, collected 24 h after transfection with synthetic miR-29b or scrambled oligonucleotides (NC). *P < 0.05, °P < 0.01.