Regulation of Selected MicroRNAs in Multiple Myeloma,

Multiple myeloma is clinically and biologically heterogeneous. Certain translocations and chromosomal losses (t{14;16}, t{14;20}, del 17p) and gene expression profiles define high-risk disease. Recently, several groups found microRNAs (miRs) dysregulated in multiple myeloma. A profile composed of 28 miRs was found to define high-risk disease. Among the dysregulated miRs, miR15a, miR16 were down-regulated, miR19b, miR20a, miR181b were increased according to most publications. MiR-21 was generally upregulated in high-risk disease and inducible by interleukin-6. We hypothesized that commonly administered treatments for multiple myeloma would alter the expression pattern of these miRs.

Materials and Methods: For these in-vitro experiments, 4 established cell lines were used: RPMI8226, OPM-2 (t4;14), Kas-6 (IL-6-dependent) and MM1-S (t14;16). The cells were treated with ionizing radiation (3- 6 Gy), lenalidomide (10 μM), doxorubicin (50 ng/ml), bortezomib (2- 50 nM), SAHA (1–3x 10⁻⁶ M), pegylated interferon α (3–300 ng/ml) and nutlin-3 (10 μM) between 2 and 48 hours. RNA was extracted and quantitative real-time RT-PCR was performed for miR-15a, miR-16, miR19b, miR-20a, miR21, miR-181b and a control gene (U6). The expression was calculated and compared by the ΔΔ CT method.

Ionizing radiation increased miR15a in 1/2 cell lines at early time points, increased miR-19b at early time points in 2/2 cell lines (decreased later) and increased MiR20a in 2/2 cell lines at early time points. Lenalidomide induced miR15a in 2/4 cell lines, miR19b in 3/ 4 cell lines and miR-20a in 3/ 4 cell lines. Doxorubicin increased miR-16 in 2/3 cases and miR-20a in 2/3 cases (in 1 cell line decreased). Bortezomib overall induced few changes in miR-expression. SAHA induced miR-15a in 2/3 cell lines and decreased miR-16 in 1/3. MiR-19a was decreased with SAHA in 2/4 and increased in 1/4 cell lines. MiR-20 decreased in 1/4 and increased in 1/4. MiR-21 decreased in 1 and increased in 1/4 SAHA-treated cell lines. MiR-181b increased in 2/4 cell lines. Pegylated interferon decreased MiR-15a in 3/4 cell lines, decreased miR-16a in 3/4 cell lines, increased miR19b in 2/4 cell lines. MiR-20a was increased in 2/4 and decreased in 1/4 cell lines. MiR-181b was decreased in 2/ 4 cell lines. Nutlin-3 increased miR16 in 1/3 cell lines, increased miR-20a in 2/4 cell lines, increased miR-181b in 2/4, decreased miR-181b in 1/4 cell lines. Most changes observed are in the range of −50 – + 200%.

Many miRs are induced at early time points under non-cytotoxic conditions. The variability observed in these experiments may be due to the genetic heterogeneity of the cell lines. Interferon mostly down-modulates the expression of the miRs studied. Previous experiments, for example using endothelial cells also showed an induction of certain miRs after cytotoxic or cytostatic treatments. This can be explained as a stress response or protective mechanism enhancing tumor cell survival. However, the functional relevance of our data was not investigated. The downregulation of miRs following interferon treatment is surprising and would argue for a combination of interferon with cytostatic treatments. If confirmed using CD138 selected samples from patients with multiple myeloma, our data may be used to develop a treatment profile which ultimately might prognosticate treatment response. Our results are also relevant for future miR-based treatments for multiple myeloma.

Orlowski:Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.