DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

Abstract

Aberrant DNA methylation plays a relevant role in multiple myeloma (MM) pathogenesis. MicroRNAs (miRNAs) are a class of small non-coding RNAs that recently emerged as master regulator of gene expression by targeting protein-coding mRNAs. However, miRNAs involvement in the regulation of the epigenetic machinery and their potential use as therapeutics in MM remain to be investigated. Here, we provide evidence that the expression of de novo DNA methyltransferases (DNMTs) is deregulated in MM cells. Moreover, we show that miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA methylation in MM cells. In vitro transfection of MM cells with synthetic miR-29b mimics significantly impairs cell cycle progression and also potentiates the growth-inhibitory effects induced by the demethylating agent 5-azacitidine. Most importantly, in vivo intratumor or systemic delivery of synthetic miR-29b mimics, in two clinically relevant murine models of human MM, including the SCID-synth-hu system, induces significant anti-tumor effects. All together, our findings demonstrate that aberrant DNMTs expression is efficiently modulated by tumor suppressive synthetic miR-29b mimics, indicating that methyloma modulation is a novel matter of investigation in miRNA-based therapy of MM.

Figure 1. Expression of DNMT3A and DNMT3B in MM and PCL patients or in MM cell lines

Differential expression of DNMT3A (A) and DNMT3B (B) in controls, MM and PCL patients. DNMT3A and DNMT3B mRNA levels were obtained by cDNA microarray and reported as raw expression values. The statistical significance of differences among the groups was assessed using Kruskal-Wallis test (P=0,0018 for DNMT3A; P=0,05 for DNMT3B). (C). Quantitative RT-PCR of DNMT3A or DNMT3B in KMS12 (left panel) and NCI-H929 (right panel) cells co-cultured with MM patient-derived BMSCs and then immunopurified by immunomagnetic sorting with anti-CD138 beads. Raw Ct values were normalized to GAPDH and expressed as ΔΔCt values calculated using the comparative cross threshold method. DNMT3A or DNMT3B levels in cells cultured in absence of BMSCs were set as internal reference. Data are the average of two independent co-culture experiments performed in triplicate. P values were obtained using two-tailed t test. * P<0,01.

Figure 2. MiR-29b targets DNMT3A and DNMT3B and reduces global DNA methylation levels in MM cells

(A). Quantitative RT-PCR of miR-29b levels in INA-6 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 two independent transfection experiments performed in triplicate.(B). Dual luciferase assay of INA-6 cells co-transfected with firefly luciferase constructs containing the 3'UTR of DNMT3A or DNMT3B and miR-29b or scrambled oligonucleotides (NC) as indicated. 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 to the control (NC) of a total of six experiments from three independent transfections. °P<0,01. (C). Quantitative RT-PCR of DNMT3A and DNMT3B 24 hours after transfection with synthetic miR-29b or scrambled oligonucleotides (NC) in INA-6 cells. The results are shown as average mRNA expression, in three independent experiments, after normalization with GAPDH and ΔΔCt calculations *P<0,05. (D). Immunoblot of DNMT3A and DNMT3B 24 hours after transfection of INA-6 with synthetic miR-29b or scrambled oligonucleotides (left panel) or in SKMM1 cells transduced with antagomiR-29b (anti-miR-29b) or the empty vector (right panel). GAPDH was used as loading control. (E). GDMi values of U266 and NCI-H929 cells transfected with synthetic miR-29b mimics or scrambled oligonucleotides (NC). The values represent the main of three independent triplicate experiments with standard error mean. °P<0,01.

Figure 3. Inverse correlation between miR-29b and DNMT3B levels in MM cell lines

Correlation of endogenous miR-29b levels with DNMT3A (A) and DNMT3B (B) mRNA levels determined by high density microarray of mRNA and miRNA expression in a panel of 17 MM cell lines. Log values of raw data are reported in graph. R= regression coefficient.

Figure 4. MiR-29b alters cell cycle progression and potentiates 5-azacitidine effects in MM cells

(A). Cell cycle analysis of NCI-H929 cells transduced with two different shRNAs against DNMT3A or DNMT3B or with a vector carrying a scrambled sequence (SCR). At least 20000 events for each point were analyzed in three independent experiments. Results are representative of one out of three experiments. (B). Cell growth curves of NCI-H929 cells transfected with synthetic miR-29b (miR-29b) or scrambled oligonucleotides (NC) with 5μM azacitidine (5-AZA) or vehicle (RPMI medium). Averaged values of three independent experiments are plotted including ±SD. P values 72hours after electroporation were obtained using two-tailed t test (P= 0,0039 for NC vs miR-29b; P=0,0028 for NC+AZA vs miR-29b+AZA). (C). Cell cycle analysis of NCI-H929 cells transfected with synthetic miR-29b mimics or scrambled oligonucleotides (NC) and then treated with 5μM azacitidine (5-AZA) or vehicle for 24, 48 or 72 hours. Results are representative of one out of three independent experiments.

Figure 5. In vivo anti-tumor activity of miR-29b mimics after intratumoral or systemic delivery in MM mouse-models

(A). In vivo tumor growth of SKMM1 xenografts intratumorally-treated with NLE (MaxSuppressor^TM In Vivo RNA-LANCER II)-miR-29b or controls. Palpable subcutaneous tumor xenografts were treated every 3 days (indicated by arrows) for a total of 4 injections, with 20 μg of formulated miR-29b or miR-NC (NC). As control 2 separate groups of tumor–bearing animals were injected with vehicle alone or NLE-formulated scrambled oligonucleotides (NC). Tumors were measured with an electronic caliper every 3 days, averaged tumor volume of each group ±SD are shown. P values were calculated for miR-29b versus miR-NC. *P<0.001. (B). Quantitative RT-PCR of miR-29b levels in retrieved xenografts after intratumor injection of miR-29b mimics or scrambled oligonucleotides. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. Data are the average of two independent triplicate experiments performed on two NC and two miR-29b injected animals °P<0,01. (C). In vivo tumor growth of OPM1 xenografts after systemic delivery of miR-29b or scrambled oligonucleotides (NC). Mice carrying palpable subcutaneous OPM1 tumor xenografts were treated with 20 μg of NLE-formulated miR-29b or scrambled oligonucleotides (NC) by intravenous tail vein injections (arrows indicate the day of injection). Caliper measurement of tumors were taken every day from the day of first treatment. Averaged tumor volumes of mice are reported ± SE. *P<0.05). (D). Quantitative RT-PCR of miR-29b levels in retrieved xenografts after system injection of miR-29b mimics or scrambled oligonucleotides (NC). Data are the average of two triplicate experiments performed on two NC and two miR-29b injected animals. °P<0,01. (E). Quantitative RT-PCR of DNMT3A or DNMT3B in retrieved xenografts after system injection of miR-29b mimics or scrambled oligonucleotides (NC). Raw Ct values were normalized to GAPDH and expressed as ΔΔCt values calculated using the comparative cross threshold method. DNMT3A or DNMT3B levels in NC-tumors were set as internal reference. Data are the average of two independent triplicate experiments performed on two NC and two miR-29b injected animals. °P<0,01.

Figure 6. In vivo analysis of miR-29b-effects in the SCID-synth-hu model

INA-6 cells were injected in synthetic recipients following engraftment of BMSCs. NLE-formulated miR-29b or NC were injected in groups of three animals, after detection of sIL6R in the mouse sera. Representative Histology (H&E), Caspase-3 and Ki-67 immunohistochemical staining of retrieved 3D biopolymeric scaffolds from treated animals is shown. Original magnification, x40.