In vitro and in vivo activity of a novel locked nucleic acid (LNA)-inhibitor-miR-221 against multiple myeloma cells

Maria Teresa Di Martino, Annamaria Gullà, Maria Eugenia Gallo Cantafio, Emanuela Altomare, Nicola Amodio, Emanuela Leone, Eugenio Morelli, Santo Giovanni Lio, Daniele Caracciolo, Marco Rossi, Niels M Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone, Maria Teresa Di Martino, Annamaria Gullà, Maria Eugenia Gallo Cantafio, Emanuela Altomare, Nicola Amodio, Emanuela Leone, Eugenio Morelli, Santo Giovanni Lio, Daniele Caracciolo, Marco Rossi, Niels M Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone

Abstract

Background & aim: The miR-221/222 cluster is upregulated in malignant plasma cells from multiple myeloma (MM) patients harboring the t(4;14) translocation. We previously reported that silencing of miR-221/222 by an antisense oligonucleotide induces anti-MM activity and upregulates canonical miR-221/222 targets. The in vivo anti-tumor activity occurred when miR-221/222 inhibitors were delivered directly into MM xenografts. The aim of the present study was to evaluate the anti-MM activity of a novel phosphorothioate modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA-i-miR-221) specifically designed for systemic delivery.

Methods: In vitro anti-MM activity of LNA-i-miR-221 was evaluated by cell proliferation and BrdU uptake assays. In vivo studies were performed with non-obese diabetic/severe combined immunodeficient (NOD.SCID) mice bearing t(4;14) MM xenografts, which were intraperitoneally or intravenously treated with naked LNA-i-miR-221. RNA extracts from retrieved tumors were analyzed for miR-221 levels and modulation of canonical targets expression. H&E staining and immunohistochemistry were performed on retrieved tumors and mouse vital organs.

Results: In vitro, LNA-i-miR-221 exerted strong antagonistic activity against miR-221 and induced upregulation of the endogenous target p27Kip1. It had a marked anti-proliferative effect on t(4;14)-translocated MM cells but not on MM cells not carrying the translocation and not overexpressing miR-221. In vivo, systemic treatment with LNA-i-miR-221 triggered significant anti-tumor activity against t(4;14) MM xenografts; it also induced miR-221 downregulation, upregulated p27Kip1 and reduced Ki-67. No behavioral changes or organ-related toxicity were observed in mice as a consequence of treatments.

Conclusions: LNA-i-miR-221 is a highly stable, effective agent against t(4;14) MM cells, and is suitable for systemic use. These data provide the rationale for the clinical development of LNA-i-miR-221 for the treatment of MM.

Conflict of interest statement

Competing Interests: Dr. Niels Montano Frandsen is employed by Exiqon A/S, Vedbaek, Denmark. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1. LNA-i-miR-221 specifically recognizes the miR-221…
Figure 1. LNA-i-miR-221 specifically recognizes the miR-221 complementary sequence.
Luciferase reporter assay of NCI-H929 cells co-transfected with pLightSwitch_3′UTR Reporter Vector containing the miR-221-3p synthetic sequence cloned downstream of the Renilla luminescent reporter gene (RenSP) and miR-221/222 mimics (A) or LNA-i-miR-221 (B) or scrambled sequences as control. The firefly luciferase activity was normalized to Renilla luciferase activity. The data are shown as relative luciferase activity of miR-221/222-transfected cells versus the control (miR-NC) or LNA-i-miR-221-transfected cells versus the scrambled control (LNA-i-miR-NC). C) Dual-luciferase assay of NCI-H929 cells co-transfected with firefly luciferase constructs containing the 3′UTR of p27Kip1 and LNA-i-miR-221 or scrambled oligonucleotides (NC) as indicated. Firefly luciferase activity was normalized to r Renilla luciferase activity. The data are shown as relative luciferase activity of LNA-i-miR-221-transfected cells versus the control (NC).
Figure 2. Antiproliferative effects induced by transient…
Figure 2. Antiproliferative effects induced by transient expression of LNA-i-miR-221 in MM cell lines.
Effects on proliferation (A) and BrdU uptake (B) in NCI-H929 cells induced by transient LNA-miR-221 inhibitor (LNA-i-miR-221) transfection compared to scrambled control (LNA-i-miR-NC). C) OPM-2 cell growth inhibition. MM cells not bearing t(4;14): RPMI-8226 (D), KMS12-BM (E) and INA-6 (F) are not affected by transfection with LNA-i-miR-221 as compared to scrambled LNA sequences. Averaged values ±SD from 3 independent experiments.
Figure 3. Molecular effects induced by LNA-i-miR-221…
Figure 3. Molecular effects induced by LNA-i-miR-221 transfection in MM cells.
miR-221(A) q-RT-PCR 24, 48 and 72 hours after transfection with LNA-i-miR-221 and LNA-i-miR-NC in NCI-H929 cells. The results are shown as miRNA expression levels after normalization with RNU44 and ΔΔCt calculations. Data represent the average of 3 independent experiments ±SD. B) q-RT-PCR of p27Kip1 mRNA expression 24 and 48 hours after transfection with LNA-i-miR-221 or scrambled control in NCI-H929 cells. Data represent the average of 3 independent experiments ±SD after normalization with GAPDH mRNA and ΔΔCt calculations. (*) P

Figure 4. In vivo anti-tumor activity of…

Figure 4. In vivo anti-tumor activity of LNA-i-miR-221 in MM-xenografted SCID/NOD mice.

Effects of LNA-i-miR-221…

Figure 4. In vivo anti-tumor activity of LNA-i-miR-221 in MM-xenografted SCID/NOD mice.
Effects of LNA-i-miR-221 after 2 intraperitoneal injections of 25 mg/kg (A) and 4 intravenous injections of 25 mg/kg (B). MM-xenografted mice were treated with saline solution of LNA-i-miR-221 or LNA-i-miR-NC as control. The average tumor volume of each group ±SD is reported. P values were calculated for miRNA inhibitors versus scrambled oligonucleotides (NC) and significant P values (P<0.05) are indicated by asterisks. C) q-RT-PCR of miR-221 in retrieved tumors treated intravenously with LNA-i-miR-221 or LNA-i-miR-NC. The results are shown as averaged miRNA expression after normalization with RNU44 and ΔΔCt calculations as compared to control (miRNA expression in LNA-i-miR-NC treated animals) ±SD.*P<0.05. D) q-RT-PCR of miR-221 in liver, kidney and heart tissue biopsies of mice after 2 weeks of treatment. The results are shown for each organ as average miRNA expression after normalization with snoRNA-202 and ΔΔCt calculations with respect to miRNA expression in LNA-i-miR-NC-treated animals. The results represents the average value obtained in 3 different mice ±SD. **P<0.01.

Figure 5. LNA-i-miR-221 antiproliferative activity and target…

Figure 5. LNA-i-miR-221 antiproliferative activity and target silencing in retrieved MM xenografted tumors.

A) q-RT-PCR…

Figure 5. LNA-i-miR-221 antiproliferative activity and target silencing in retrieved MM xenografted tumors.
A) q-RT-PCR of p27Kip1 mRNA levels in treated tumors retrieved from mice after intravenous LNA-i-miR-221 treatment. Raw Ct values were normalized to GAPDH housekeeping mRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method (miRNA expression in LNA-i-miR-NC treated animals) ±SD. B) Western blot analysis of p27Kip1 protein in retrieved tumors from mice treated with LNA-i-miR-221 inhibitors or LNA-i-miR-NC. GAPDH was the protein loading control. C) H&E (200-fold magnification), p27Kip1 (200-fold magnification) and Ki-67 (10–fold magnification) immunohistochemistry staining of xenografted tumors retrieved from treated animals. Representative images are shown.
Figure 4. In vivo anti-tumor activity of…
Figure 4. In vivo anti-tumor activity of LNA-i-miR-221 in MM-xenografted SCID/NOD mice.
Effects of LNA-i-miR-221 after 2 intraperitoneal injections of 25 mg/kg (A) and 4 intravenous injections of 25 mg/kg (B). MM-xenografted mice were treated with saline solution of LNA-i-miR-221 or LNA-i-miR-NC as control. The average tumor volume of each group ±SD is reported. P values were calculated for miRNA inhibitors versus scrambled oligonucleotides (NC) and significant P values (P<0.05) are indicated by asterisks. C) q-RT-PCR of miR-221 in retrieved tumors treated intravenously with LNA-i-miR-221 or LNA-i-miR-NC. The results are shown as averaged miRNA expression after normalization with RNU44 and ΔΔCt calculations as compared to control (miRNA expression in LNA-i-miR-NC treated animals) ±SD.*P<0.05. D) q-RT-PCR of miR-221 in liver, kidney and heart tissue biopsies of mice after 2 weeks of treatment. The results are shown for each organ as average miRNA expression after normalization with snoRNA-202 and ΔΔCt calculations with respect to miRNA expression in LNA-i-miR-NC-treated animals. The results represents the average value obtained in 3 different mice ±SD. **P<0.01.
Figure 5. LNA-i-miR-221 antiproliferative activity and target…
Figure 5. LNA-i-miR-221 antiproliferative activity and target silencing in retrieved MM xenografted tumors.
A) q-RT-PCR of p27Kip1 mRNA levels in treated tumors retrieved from mice after intravenous LNA-i-miR-221 treatment. Raw Ct values were normalized to GAPDH housekeeping mRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method (miRNA expression in LNA-i-miR-NC treated animals) ±SD. B) Western blot analysis of p27Kip1 protein in retrieved tumors from mice treated with LNA-i-miR-221 inhibitors or LNA-i-miR-NC. GAPDH was the protein loading control. C) H&E (200-fold magnification), p27Kip1 (200-fold magnification) and Ki-67 (10–fold magnification) immunohistochemistry staining of xenografted tumors retrieved from treated animals. Representative images are shown.

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