MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis

Abstract

Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n = 49) and CD138+ bone marrow PCs from subjects with MM (n = 16), monoclonal gammopathy of undetermined significance (MGUS) (n = 6), and normal donors (n = 6). We identified overexpression of miR-21, miR-106b approximately 25 cluster, miR-181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17 approximately 92 cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17 approximately 92 cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target of the miR106b approximately 25 cluster, miR-181a and b, and miR-32. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.

Fig. 1.

MM and MGUS express a distinct spectrum of miRNA in comparison to normal CD138+ PCs. (A) Schematic drawing showing the multistep molecular process of PC transformation. (B) Representative list of the miRNAs significantly deregulated in MGUS versus normal PCs analyzed in this study. The asterisks indicate the specific associated cluster. (C) Representative list of the common deregulated miRNAs in the comparison classes MM patient versus normal PCs and PCs MM versus normal PCs analyzed in this study. The asterisks indicate the specific associated cluster. (D) miR-17 microRNA clusters. Three paralog families of miRNA precursors can be identified: miR-17/18/18X/20/93/106a/106b/93 (yellow), mir-19a/19b-1/19b-2 (blue) and mir-92-1/92-2/25 (green).

Fig. 2.

miR-181s, 106b-25 cluster, 32 target PCAF. (A) miRNAs predicted to interact with PCAF gene in several consensus binding sites at its 3′-UTR, according to “in silico” target Target Scan prediction software. (B–D) Luciferase assay showing decreased luciferase activity in cells cotransfected with pGL3-PCAF-3′UTR and miR-181s (B), miR-32/miR-25/miR-92 (C), and miR-93/miR-106b (D) oligonucleotides. Deletion of six bases in three putative miR-181s, miR-106b/miR-93, and miR-32/miR-25/miR-92 binding sites, complementary to miRNAs seed regions, abrogates this effect (MUT). Bars indicate firefly luciferase activity normalized to Renilla luciferase activity ± SD. Each reporter plasmid was transfected at least twice (on different days) and each sample was assayed in triplicate. (E, F) Immunoblot analyses showing PCAF and GADPH expression after transfection with miR-181s and miR-106b/25 cluster ASOs in U266 cells (E) and with miR-181s, miR-32 and miR-92 ASOs in JJN3 cells (F), or mimics in K562 cells (G, H). (I) Real-time RT-PCR analyses for p53 and PCAF expression in MM1s cells transfected with miR-181a and b or with miR-181a and b and miR-106b/25 together ASOs (pool) or with scrambled oligonucleotide at 48 h after transfection and after 4 h of UV treatment. The PCR products for both genes were normalized to GADPH and ACTIN expression. The bar-graph represents the mean values observed in four separate studies ± SE. (L) Immununoblot analysis showing p53 protein expression after 48 h of miR-181a, miR-181b, miR-92 and scrambled ASOs transfection in MM1 cells after 9 h and overnight incubation with 10 μM nutlin-3a; GADPH was internal loading control. Densitometry based on GADPH levels shows increased level of p53 in presence of miR-181a and miR-181b ASOs in MM1s cells.

Fig. 3.

miR-19s target SOCS-1 in MM cell lines. (A) Immunoblot analysis with antisera against SOCS-1 and GADPH in 15 MM cell lines and two CD138+PCs from healthy donors (control). (B) Predicted highly conserved consensus binding site in human, mouse, rat, and dog for miR-19s on the 3′UTR of SOCS-1. (C) Relative luciferase activity in MEG01 cells transiently cotransfected with luciferase reporter vector containing the 3′ UTR of SOCS-1 and miR-19s or scrambled oligonucleotides. Deletion of the six bases in the putative miR-19s binding site, complementary to miRNA seed region, abrogates this effect (MUT). Bars indicate firefly luciferase activity normalized to Renilla luciferase activity ± SD. Each reporter plasmid was transfected at least twice (on different days) and each sample was assayed in triplicate. (D) Western blot showing SOCS-1 protein in whole cell lysates from U266 and JJN3 cells at 48 h after transfection with scrambled oligonucleotides or miR-19a, miR-19b, or both ASOs. GADPH was used as loading control. Densitometry based on GADPH levels shows increased level of SOCS-1 in presence of miR-19a or miR-19b or together ASOs in U266 and JJN3 cells. (E) Western blot showing that miR-19s modulate expression of activated STAT-3 in U266 cells in vitro. Cells were transfected with anti-miR-19s or negative control (Scr) miRNA inhibitors in vitro, and cell lysates were obtained after 72 h. Densitometry based on STAT-3 levels shows decreased level of P-STAT-3 in presence of miR-19s. (F) Stem-loop q-RT-PCR to validate the expression of endogenous miR-19a and miR-19b, at 72 h after transfection of U266 cells with antagonizing oligonucleotides, after normalization with RNU6B.

Fig. 4.

Antagonizing miR-19s and miR-181s expression in MM cell lines resulted in significant tumor suppression in nude mice. U266 and JJN3 cells (30 × 10⁶ cells) were injected subcutaneously into the flanks of nude mice with 100 μl of matrigel solution 24 h after transfection with miR-19s and miR-181s ASOs or scrambled oligonucleotide. Mice were killed on day 35 and tumor volumes were calculated. (A) Time course of tumor growth of U266 cell line. (B) Time course of tumor growth of JJN3 cell line; tumors treated with miR-19s and miR-181s ASOs were significantly smaller than tumors of scrambled groups for both cell lines. (Scale bar, 10 mm.)