Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth - PubMed (original) (raw)

. 2013 Apr 15;19(8):2096-106.

doi: 10.1158/1078-0432.CCR-12-3325. Epub 2013 Feb 27.

Eugenio Morelli, Maria T Di Martino, Nicola Amodio, Umberto Foresta, Annamaria Gullà, Marco Rossi, Antonino Neri, Antonio Giordano, Nikhil C Munshi, Kenneth C Anderson, Pierosandro Tagliaferri, Pierfrancesco Tassone

Affiliations

Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth

Emanuela Leone et al. Clin Cancer Res. 2013.

Erratum in

Abstract

Purpose: Deregulated expression of miRNAs plays a role in the pathogenesis and progression of multiple myeloma. Among upregulated miRNAs, miR-21 has oncogenic potential and therefore represents an attractive target for the treatment of multiple myeloma.

Experimental design: Here, we investigated the in vitro and in vivo anti-multiple myeloma activity of miR-21 inhibitors.

Results: Either transient-enforced expression or lentivirus-based constitutive expression of miR-21 inhibitors triggered significant growth inhibition of primary patient multiple myeloma cells or interleukin-6-dependent/independent multiple myeloma cell lines and overcame the protective activity of human bone marrow stromal cells. Conversely, transfection of miR-21 mimics significantly increased proliferation of multiple myeloma cells, showing its tumor-promoting potential in multiple myeloma. Importantly, upregulation of miR-21 canonical validated targets (PTEN, Rho-B, and BTG2), together with functional impairment of both AKT and extracellular signal-regulated kinase signaling, were achieved by transfection of miR-21 inhibitors into multiple myeloma cells. In vivo delivery of miR-21 inhibitors in severe combined immunodeficient mice bearing human multiple myeloma xenografts expressing miR-21 induced significant antitumor activity. Upregulation of PTEN and downregulation of p-AKT were observed in retrieved xenografts following treatment with miR-21 inhibitors.

Conclusion: Our findings show the first evidence that in vivo antagonism of miR-21 exerts anti-multiple myeloma activity, providing the rationale for clinical development of miR-21 inhibitors in this still incurable disease.

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Conflict of interest statement

Conflicts- of- interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. MiR-21 expression in MM patient cells and cell lines

(A) Quantitative RT-PCR analysis of miR-21 expression using total RNA from 7 MM cell lines and bone marrow-derived plasma cells (nPCs) from healthy donors. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values relative to miR-21 levels in nPCs. Values represent mean ±SE of three different experiments. (B) Quantitative RT-PCR of miR-21 expression in INA-6 cells cultured in the presence or absence of IL-6 (2.5 ng/mL) or co-cultured with hBMSCs, and then immunopurified by immunomagnetic sorting with anti-CD138 beads. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method. miR-21 expression levels in INA-6 cells cultured in the absence of IL-6 were set as an internal reference. Values represent mean ±SE of three different experiments. (C) Quantitative RT-PCR of miR-21 expression in nPCs (n=3) and ppMM (n=3) cells before and 6 hours after seeding on hBMSCs, and then immunopurified by immunomagnetic sorting with anti-CD138 beads. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method. MiR-21 expression levels in nPCs were used as an internal reference. Data are the average of three independent experiments performed in triplicate. P values were obtained using two-tailed t test.

Figure 2

Figure 2. Effects of ectopic expression of miR-21 inhibitors in MM cell lines

(A) Trypan blue exclusion growth curves were generated from KMS-26, U-266 and OPM-2 cells transfected with miR-21 inhibitors or scrambled controls (miR-NC inhibitors). (B) BrdU proliferation assay was performed in KMS-26, U-266 and OPM-2 cells 48h after transfection with miR-21 inhibitors or scrambled controls. (C) MTT survival assay was performed in KMS-26, U-266 and OPM-2 cells 48h after transfection with miR-21 inhibitors or scrambled controls. Averaged values ±SD of three independent experiments are plotted. P values were obtained using two-tailed t test.

Figure 3

Figure 3. Activity of miR-21 inhibitors in MM cell lines

BrdU proliferation assay and colony formation assay of (A) MM.1S and (B) U-266 cells transduced with a lentivirus carrying either the empty vector or miR-21 inhibitory sequences. Averaged values ±SD of three independent experiments are plotted. P values were obtained using two-tailed t test. (C) qRT-PCR of PTEN, BTG2 and Rho-B expression was performed in U-266 cells 48 hours after transfection with either miR-21 inhibitors or scrambled controls (miR-NC inhibitors). The results are shown as average mRNA expression after normalization with GAPDH and ΔΔCt calculations. Data represent the average ±SD of 3 independent experiments. (D) Western Blot of PTEN 24 – 48 hours after transfection of U-266 cells with miR-21 inhibitors or scrambled controls. (E) Levels of total AKT, total ERK, p-AKT and p-ERK 24 – 48 hours after transfection of U-266 cells with either miR-21 inhibitors or scrambled controls. Relative protein level values are derived from densitometric scan.

Figure 4

Figure 4. Effects of transient enforced expression of miR-21 mimics in MM cell lines

Growth curves and Brdu uptake (48 h time point) of (A) U-266 and (B) MM.1S cells transfected with either miR-21 mimics or scrambled controls (miR-NC). Enforced expression of miR-21 mimics was also triggered in MM.1S cells overexpressing a miRNA insensitive PTEN construct (mi-PTEN). (C) qRT-PCR of PTEN, BTG2 and Rho-B expression levels was done 48 hours after transfection of MM.1S cells with either miR-21 mimics or scrambled controls. The results shown are average mRNA expression after normalization with GAPDH and ΔΔCt calculations. (D) Western Blot of PTEN 48 hours after transfection of MM.1S cells with either miR-21 mimics or scrambled controls. Relative protein level values are derived from densitometric scan. (E) qRT-PCR of PTEN expression levels in MM.1S cells co-transfected with either mi-PTEN or an empty vector and miR-21 mimics or scrambled controls (48h time point). The results shown are average mRNA expression after normalization with GAPDH and ΔΔCt calculations. Data represent the average ±SD of 3 independent experiments.

Figure 5

Figure 5. miR-21 inhibition antagonizes pro-survival effect of BM milieu

(A) MTT assay of INA-6 cells cultured adherent to hBMSCs, in IL-6-enriched culture medium, or in IL-6-free culture medium. The assay was performed 48 hours after transfection of co-cultured cells with miR-21 inhibitors or scrambled controls (miR-NC inhibitors). (B) MTT assay of INA-6 cells cultured with hBMSCs was performed 48 hours after either INA-6 cells or hBMSCs were independently transfected with miR-21 inhibitors or scrambled controls. (C) MTT assay performed in ppMM cells cultured in the presence or absence of hBMSCs. The assay was performed 48 hours after transfection with miR-21 inhibitors or scrambled controls. Averaged values ±SD of three independent experiments are plotted including. P values were obtained using two-tailed t test.

Figure 6

Figure 6. MiR-21 inhibition antagonizes MM tumor growth in vivo

(A) In vivo tumor growth of OPM-2 xenografts intratumorally-treated with miR-21 inhibitors or scrambled controls (miR-NC inhibitors). Palpable subcutaneous tumor xenografts were treated every 2 days with 20 µg of miR-21 inhibitors for a total of 8 injections (indicated by arrows). A separate control group of tumor-bearing animals was injected with scrambled controls. Tumors were measured with an electronic caliper every 2 days, and averaged tumor volume of each group ±SD are shown. P values were obtained using two-tailed t test. (B) Quantitative RT-PCR of PTEN expression in lysates from retrieved OPM2 xenografts. The results shown are average mRNA expression levels after normalization with GAPDH and ΔΔCt calculations. Data represent the average ±SD of 3 independent experiments. (C) Western Blot of PTEN, total AKT and p-AKT levels in lysates from a representative retrieved OPM-2 xenograft. Relative protein level values are derived from densitometric scan.

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