MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma

Moshe E. Gatt, Jian-Jun Zhao, Margaret S. Ebert, Yunyu Zhang, Zhangbo Chu, Mala Mani, Roi Gazit, Daniel E. Carrasco, Jui Dutta-Simmons, Sophia Adamia, Stéphane Minvielle, Yu-Tzu Tai, Nikhil C. Munshi, Hervé Avet-Loiseau, Kenneth C. Anderson and Daniel R. Carrasco
This article has been retracted 117(26):7188

Data supplements

  • Supplemental materials for: Gatt et al

    Files in this Data Supplement:

    • Table S1. Most significant GO categories of the top 100 upregulated genes in MMS1 and RPMI 8226 miR16 inhibited cells (PDF, 51.8 KB)
    • Table S2. Top 100 upregulated genes in MMS1 and RPMI 8226 miR16 inhibited cells with their possible miR16 binding sites (PDF, 111 KB)
    • Table S3. Top upregulated gene probes clustered to MAPK signaling pathway (PDF, 65.1 KB)
    • Table S4. Primers used in the article (PDF, 60.3 KB)
    • Figure S1. miR-15a/16 downregulation in other cell lines and controls (JPG, 513 KB) -
      (A) Northern blot analysis showing reduced expression of miR-16 in RPMI 8226, OPM1 and KMS34 MM cell lines, as well as in SKW6.4 transformed B-cell line after lentiviral transduction with vector expressing the miR-16 GFP sponge (sp16). (B) Northern blot analysis showing higher expression of miR-16 in MMS1 and RPMI 8226 MM cell lines, as well as in 293T cell line, stably expressing the miR-15a/16 overexpression plasmid (pRS16), but not the control miR-15a/16 mutant (pRSmut) plasmid. miR suppression is specific for both miR-16 and -15a, but not for a dissimilar miR, miR-20, in cell lines transduced with sp16 as compared to control spCX, evidenced by Northern blot analysis in MM cell lines (C) or by luciferase activity in 293T cells transfected with a reporter plasmid driving luciferase expression under the control of a CMV promoter and UTR binding sites for miR-16, -15a or -20 (D). (E) No change in relative luciferase activity of the miR-16 target reporter and cell proliferation (F) as evaluated by H3 thymidine incorporation, comparing GFP only expressing (GFP) and GFP-control sponge (spCX) MMS1 cells. (G) Flow cytometric analysis by PI staining showing enhanced S and G2/M cell cycle phases in MMS1 miR-15a/16 knockdown cells. The experiment was repeated three separate times. (H) IB analysis showing marked upregulation of known miR-16 targets BCL2 and Cyclin D1, and partial upregulation of CDC25a, in miR-15a/16 knockdown cells.

    • Figure S2. Principle Component analysis of miR-15a/16 inhibition by gene expression profiling (JPG, 38.7 KB) -
      MMS1 and RPMI 8226 cells were grown in triplicate under identical conditions and subjected to RNA extraction for subsequent GEP. The PCA showed a clustering within each treated cell line group, with a wide number of genes that were changed after the treatment among the two cell lines. Shown are the MMS1 samples (black) and RPMI 8226 (red), stably expressing the control spCX (○) or the miR-16 sponge sp16 (△).

    • Figure S3. Validation of target genes upregulated upon miR-15a/16 inhibition (JPG, 995 KB) -
      (A) Using qRT-PCR with β-actin as the reference gene, upregulated target gene mRNA levels were measured in both MMS1 and RPMI 8226 cell lines, comparing stably transduced sponge 16 cells (sp16) with their control sponge cells (spCX). Also compared were stably selected miR-16�overexpressing MMS1 and RPMI 8226 cell lines (pRS16) with their control miR-16 mutants (pRSmut). Experiments were done in triplicate, and a representative of at least two independent assays is shown. (B) IB analysis of miR-15a/16 target genes (previously validated by qRT-PCR) and their downstream affected proteins in OPM-1, and KMS34 MM cell lines. Note in the middle lane the number of MM lines (MMS1, RPMI8226, OPM1 and KMS34) in which there was an upregulation of the designated target at the protein level. Also shown for control purposes are the target protein levels in a B-cell line skw6.4, as well as in 293T cells. In each cell line the stably transduced sponge 16 cells (sp16) are compared with their control sponge cells (spCX). Actin is used as a loading control. (C) Target genes (previously validated by qRT-PCR), and their downstream affected proteins, as shown by IB of both MMS1 and RPMI 8226 MM cell lines. In each line, the stably transfected miR-16�overexpressing cells (pRS16) are compared with their control miR-16 mutated cells (pRSmut). Actin is used a loading control.

    • Figure S4. Treatment of miR-15a/16-inhibited cells with chemotherapeutic agents (JPG, 135 KB) -
      Viability assays showing that miR-15a/16-inhibited cells are not more susceptible to treatment with conventional chemotherapeutic agents including Adriamycin (A) and melphalan (B), but are highly sensitive to Bortezomib (C). In each cell line, the stably transduced sponge 16 cells (sp16) are compared with their control sponge cells (spCX). MM cells were treated at the designated concentrations, and viability was measured after 36�48 hours compared with untreated controls. Experiments were done in triplicate and repeated three times.