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. 2014 May 30;5(10):3039-54.
doi: 10.18632/oncotarget.1747.

Targeting of multiple myeloma-related angiogenesis by miR-199a-5p mimics: in vitro and in vivo anti-tumor activity

Lavinia Raimondi  1 , Nicola Amodio, Maria Teresa Di Martino, Emanuela Altomare, Marzia Leotta, Daniele Caracciolo, Annamaria Gullà, Antonino Neri, Simona Taverna, Patrizia D'Aquila, Riccardo Alessandro, Antonio Giordano, Pierosandro Tagliaferri, Pierfrancesco Tassone
Affiliations

Targeting of multiple myeloma-related angiogenesis by miR-199a-5p mimics: in vitro and in vivo anti-tumor activity

Lavinia Raimondi et al. Oncotarget. .

Abstract

Multiple myeloma (MM) cells induce relevant angiogenic effects within the human bone marrow milieu (huBMM) by the aberrant expression of angiogenic factors. Hypoxia triggers angiogenic events within the huBMM and the transcription factor hypoxia-inducible factor-1α (HIF-1α) is over-expressed by MM cells. Since synthetic miR-199a-5p mimics negatively regulates HIF-1α, we here investigated a miRNA-based therapeutic strategy against hypoxic MM cells. We indeed found that enforced expression of miR-199a-5p led to down-modulated expression of HIF-1α as well as of other pro-angiogenic factors such as VEGF-A, IL-8, and FGFb in hypoxic MM cells in vitro. Moreover, miR-199a-5p negatively affected MM cells migration, while it increased the adhesion of MM cells to bone marrow stromal cells (BMSCs) in hypoxic conditions. Furthermore, transfection of MM cells with miR-199a-5p significantly impaired also endothelial cells migration and down-regulated the expression of endothelial adhesion molecules such as VCAM-1 and ICAM-1. Finally, we identified a hypoxia\AKT/miR-199a-5p loop as a potential molecular mechanism responsible of miR-199a-5p down-regulation in hypoxic MM cells. Taken together our results indicate that miR-199a-5p has an important role for the pathogenesis of MM and support the hypothesis that targeting angiogenesis via a miRNA/HIF-1α pathway may represent a novel potential therapeutical approach for this still lethal disease. VSports手机版.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. miR-199a-5p expression in myeloma cells and hypoxic-effect on miR-199a-5p expression in MM cells
(a) Quantitative RT-PCR analysis of miR-199a-5p using total RNA from 10 MM cell lines and 1 MM patient sample. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as fold increase over control CD138+ cells (black column, 1 arbitrary unit). Columns, means; Bars, S.D Values represent mean of three different experiments. (b) Western blotting analysis showing HIF-1α expression in nuclear (N) and cytoplasmic (C) -enriched cell fractions of MM cell lines treated for 4 hours with 100µM/L of the hypoxia-mimicking Cobalte Chloride. Histone H3 was used as loading control to discriminate the different cell fractions. (c) Quantitative RT-PCR of miR-199a-5p in OPM2, NCI-H929 and RPMI-8226 MM cell lines cultured in both normoxic and hypoxic conditions. Raw Ct were normalized to RNU44 housekeeping snoRNA and expressed as fold increase over normoxic MM cells (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. P values were obtained using two-tailed t test. *P<0,05; °P<0,01 (d) Quantitative RT–PCR of miR-199a-5p in OPM2 cells co-cultured for 24 hours with BMSCs and then immunopurified by immunomagnetic sorting with anti-CD138 beads. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as fold increase over normoxic OPM2 cells (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. °P<0,01.
Figure 2
Figure 2. miR-199a-5p targets HIF-1α and reduces HIF-1α and SIRT1 protein expression in hypoxic MM cells
(a) Western blotting analysis of HIF-1α 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in U266 and OPM2 cell lines treated for 4 hours with 100µM/L of the hypoxia-mimicking Cobalte Chloride. Tubulin was used as loading control. (b) Dual luciferase assay of OPM2 cells co-transfected with firefly luciferase constructs containing the 3'UTR of HIF-1α and miR-199a-5p 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-199a-5p-transfected cells as compared to the control (NC) of a total of six experiments from three independent transfections. P<0,05. (c) Western blotting analysis of SIRT1 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in U266 and OPM2 cell lines treated for 4 hours with 100µM/L of the hypoxia-mimicking Cobalte Chloride. Tubulin was used as loading control.
Figure 3
Figure 3. miR-199a-5p inhibits the hypoxia-induction of pro-angiogenic factors and reduces endothelial cells migration
(a) Quantitative RT-PCR of VEGF-A, IL-8 and b-FGF in both normoxic and hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Raw Ct were normalized to b-actin housekeeping and expressed as fold increase of negative control (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. °P<0,01; *P<0,05. (b) Vascular endothelial growth factor (VEGF-A) protein production in the conditioned medium derived from hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) was determined by enzyme-linked immunoadsorbent assay (ELISA). Data presented are the mean of three separate experiments. *P<0,05. (c) Chemotaxis assay of endothelial cells (HUVEC) exposed to conditioned medium from OPM2 cells transfected with either scrambled (miR-NC) or synthetic pre-miR-199a-5p (miR-199a-5p) (right panel); Huvec cells treated as in (c) and observed at contrast phase microscopy (left panel). *P<0,05. (d) Quantitative RT-PCR of VEGF-A, VCAM1, ICAM1 and IL-8 in Huvec cells exposed for 24 hours to conditioned medium from OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Raw Ct were normalized to b-actin housekeeping and expressed as fold increase of negative control (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. *P<0,05.
Figure 4
Figure 4. miR-199a-5p regulates DDR1 expression and decreases migration of MM cells
(a) Western blotting analysis of DDR1 and MMP2 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in OPM2 cells treated for 4 hours with 100µM/L of the hypoxia-mimicking Cobalte Chloride. Tubulin was used as loading control. (b) Quantitative RT-PCR of matrix metalloproteinase-2 (MMP2) in both normoxic and hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Raw Ct were normalized to b-actin housekeeping and expressed as fold increase of negative control (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. (c) Chemotaxis assay of both normoxic and hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). °P0,01.; *P<0,05.
Figure 5
Figure 5. miR-199a-5p overexpression increases adhesion of MM cells to a monolayer of BMSCs under hypoxic conditions
(a) Adhesion assay of hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) and seeded on to hypoxic BMSCs monolayer (right panel); OPM2 cells treated as in (a) and observed at contrast phase microscopy (left panel) *P<0,05. (b) Western blotting analysis of E-cadherin 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in OPM2 cells. Tubulin was used as loading control. (c) Quantitative RT-PCR of Snail in both normoxic and hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Raw Ct were normalized to b-actin housekeeping and expressed as fold increase of negative control (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. *P<0,05. (d) Quantitative RT-PCR of CXCR4 in both normoxic and hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Raw Ct were normalized to b-actin housekeeping and expressed as fold increase of negative control (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. *P<0,05. (e) Quantitative RT-PCR of IL6 and IL8 in normoxic HS5 cells exposed for 24 hours to conditioned medium from hypoxic OPM2 cells transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Raw Ct were normalized to b-actin housekeeping and expressed as fold increase of negative control (black column, 1 arbitrary unit). Columns, means; Bars, S.D. Values represent mean of three different experiments. *P<0,05.
Figure 6
Figure 6. miR-199a-5p overexpression reduces proliferation and promotes apoptosis in hypoxic MM cells
(a) Cell growth curves of OPM2 and U266 transfected with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC). Averaged values of three independent experiments are plotted including ±S.D. *P<0,05. (b) Western blotting analysis of p21Cip-1 and p27Kip-1 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in both normoxic and hypoxia-induced OPM2 cells. Tubulin was used as loading control. (c) Annexin V-staining of OPM2 cells 48 hours after transfection with synthetic miR-199a-5p or scrambled oligonucleotides (NC). The percentage of Annexin-V-positive cells is reported. Data are the average of three independent experiments ±S.D. °P<0,01. (d) Western blotting analysis of proteins caspase 3/7, cleaved poly ADP-ribose polymerase (PARP) and Bcl2, 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in both normoxic and hypoxia-induced OPM2 cells. Tubulin was used as loading control.
Figure 7
Figure 7. AKT control of miR-199a-5p expression in hypoxic MM cells
(a) NCI-H929 cells were electroporated with the pcDNA.3.1-HA-myr-AKT dominant active construct (AKT) or the empty vector pcDNA3.1 (vector) and 48 hours later analyzed for miR-199a-5p expression levels by quantitative RT–PCR. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as fold increase over vector (black column, 1 arbitrary unit). Columns, means; Bars, S.D Values represent mean of three different experiments. °P<0,01. (b) Quantitative RT–PCR of miR-199a-5p in NCI-H929 cells treated with 20 µM LY294002 or vehicle (DMSO) for 48 hours. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as fold increase over vehicle (black column, 1 arbitrary unit). Columns, means; Bars, S.D Values represent mean of three different experiments. P<0,05. (c) NCI-H929 cells were electroporated with an AKT short hairpin (sh)RNA- or control (SCR) shRNA plasmids and 48 hours later analyzed for miR-199a-5p expression levels by quantitative RT–PCR. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as fold increase over control (SCR) (black column, 1 arbitrary unit). Columns, means; Bars, S.D Values represent mean of three different experiments. P<0,05. (d) Western blotting analysis of total and phospho-AKT 24 hours after transfection with synthetic miR-199a-5p (miR-199a-5p) or scrambled oligonucleotides (NC) in OPM2 cells treated for 4 hours with 100µM/L of the hypoxia-mimicking Cobalte Chloride. Tubulin was used as loading control.
Figure 8
Figure 8. In vivo anti-tumor activity of miR-199a-5p after intratumoral delivery in MM mouse-models
(a) In vivo tumor growth of NCI-H929 xenografts intratumorally-treated with NLE (MaxSuppressor™ In Vivo RNA-Lancer II)-miR-199a-5p or miR-NC. Palpable subcutaneous tumor xenografts were treated every 3 days (indicated by arrows) for a total of six injections, with 20 µg of formulated miR-199a-5p or miR-NC. Tumors were measured with an electronic caliper every two days, averaged tumor volume of each group ±S.D. are shown. P<0,05. (b) Survival curves (Kaplan-Meier) of intratumorally miR-199a-5p treated mices show prolongation of survival compared to miR-NC (log-rank test, P=0.023). Survival was evaluated from the first day of treatment until death or sacrifice. Percent of mice alive is shown.
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