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Blood, Vol. 110, Issue 4, 1330-1333, August 15, 2007
Interleukin-6–dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer
Blood Löffler et al.
110: 1330
Supplemental materials for Löffler et al, Vol 110, Issue 4, 1330-1333
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 77.3 KB)
- Table S1. List of all footprint clusters detected in the upstream region of miR-21 (PDF, 55.3 KB) -
The table contains the complete list of phylogenetic footprints (PF) in the region from 4 kb upstream to 2 kb downstream of the miR-21 gene as detected by the program tracker. The list has been modified from the raw data by removing duplicate hits and multiple entries of the same cluster, and by arranging clusters in a linear order. Footprints are listed with their start position and length. The cluster containing the two Stat3 binding sites is marked with STAT. Two exons of TMEM49 that fall into the analyzed sequence region are covered by clusters 31 and 62 (marked TMEM49 in the table). The duplicated teleost sequences are: DR-021-1 and DR-021-2 Danio rerio, TN-021-1 and TN-021-2 Tetraodon nigroviridis, TR-021-1 and TR-021-2 Fugu rupribes. For two teleost species only one cluster was available: GA-021-1 Gasterostreus aculeatus, and OL-021-1 Oryzias latipes. For Fugu rupribes, genome assembly was imperfect. However, when region TR-021-2, corresponding to Scaffold_99, position 697298-697390 (see Table S2), was assembled with the reverse complement of Trace ACAX160258.b1 from the Ensembl trace repository, the Stat3 enhancer region emerged for this species as well. We observe phylogenetic footprints resembling the last two TMEM49 exons (as annotated in human) in the upstream region of both miR-21 copies of teleosts (Table S1). The annotation in Ensembl 40 (September 21, 2006) consistently shows a TMEM49 homologue upstream of the miR-21 copy that is not associated with the Stat3 binding motifs. Teleosts have inherited a second copy of this gene (Ensembl gene family ENSF00000004033). This second copy of TMEM49, however, is located upstream of the Stat3-related miR-21 paralog only in Fugu. In zebrafish, both TMEM49 paralogs map to different locations on chromosome 15, in Tetraodon, the second copy is located on a different scaffold than miR-21 and the Stat3 binding site, and in Gasterosteus only a single homologue is reported. Unless we see dramatic assembly and/or annotation errors in multiple genomes, these genomic data suggest that miR-21 and its associated Stat3 binding sites are functionally independent of the TMEM49 gene. Abbreviations of the sequences used: BT-021-1 Bos taurus, CF-021-1 Canis familaris, GG-021-1 Gallus gallus, HS-021-1 Homo sapiens, MM-021-1 Mus musculus, PT-021-1 Pan troglodytes.
- Table S2. Genomic sequence data of the miR-21 upstream region (PDF, 19.6 KB) -
Chromosome positions or trace numbers miR-21 upstream region used for phylogenetic footprinting are listed in the table. Genomic sequence data of the miR-21 upstream region for Homo sapiens, Mus musculus, Bos taurus, Canis familaris, Gallus gallus, Danio rerio, Tetraodon nigroviridis, Gasterosteus aculeatus, and Oryzias latipes were obtained from Ensembl (v36), and for Monodelphis domestica from Ensembl (v39). For the following organisms, traces that contain the conserved region around the Stat3 binding sites were retrieved from the Ensembl trace repository: Sorex araneus, Spermophilus tridecemlineatus, Myotis lucifugus, Procavia capensis, Echinops telfairi, Ornithorhynchus anatinus, Xenopus tropicalis, Taeniopygia guttata, and Trichosurus vulpecula.
- Figure S1. Structure of the miR-21 gene (JPG, 37.7 KB)
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(A) Conserved clusters in the miR-21 gene region. Teleost species harbor two copies of miR-21 (red arrowheads), but only one of them contains the conserved upstream regulatory region (STAT). Two of the detected footprints are conserved in vertebrates and both gene copies of teleosts. These conserved regions match the exons of TMEM49 (Ex). The transcription start site of the human miR-21 gene resides between these two TMEM49 exons. Other conserved clusters (small circles) are either tetrapod-specific or shared by the copy of teleost miR-21 that contains the Stat3 binding sites. (B) The distance (in bp) between the miR-21 sequence and the Stat3 binding sites is given for all organisms where genomic data were available. The analysis shows a stable distance of 3 kb to 3.8 kb between miR-21 and the Stat3 enhancer throughout all vertebrates. This is remarkable as the intervening coding sequences have lost their integrity and the length of such sequences normally correlates with the genome size of organisms.11 Teleost genomes are significantly smaller than the genomes of tetrapod species, and the size of a coding gene immediately upstream of miR-21 ranges from 140 kb in tetrapods to 5.5 kb in teleosts. Therefore, this observation strongly suggests that the stable distance between miR-21 and the Stat3 sites is of functional relevance.
- Figure S2. Verification of siRNA specificity towards Stat3 (JPG, 42.1 KB)
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(A) Reporter gene assays were performed in HepG2 cells transfected with a luciferase vector driven by the Stat3-responsive promoter of the α1-antichymotrypsin gene and a  -galactosidase expression vector. Empty pSUPER (control) or pSUPER-siStat3 was contransfected. Stat3 expression was reconstituted by cotransfection of a vector encoding a Stat3 variant made resistant to siRNA attack by introducing silent mutations into the target sequence (Stat3res). Cells were either left untreated or stimulated with IL-6 for 4 h, and luciferase and -galactosidase levels determined. Luciferase values were normalized to -galactosidase activities to account for transfection efficiency. Data represent mean values plus or minus standard deviation derived from three independent experiments. (B,C) INA-6 cells cultured in the presence of IL-6 were transiently transfected with pSUPER-siStat3 or pSUPER-scrambled encoding a scrambled-sequence shRNA (control) together with a vector-encoding enhanced green fluorescence protein. At the posttransfection time points indicated, cells were subjected to a flow-cytometric Annexin V apoptosis assay. Transfected cells were gated on the basis of green fluorescence and viable, Annexin V-negative cells counted. Values were calculated relative to the numbers of viable cells in the control experiment. Data represent mean values of two independent experiments (panel B). 48 hours after transfection, green fluorescent cells were sorted using a FACS sorter and cell lysates subjected to immunoblot analysis with antibodies to Stat3 or Stat1 as indicated (panel C).
- Figure S3. IL-6 induces miR-21 in LnCAP human prostate carcinoma and HepG2 human hepatocellular carcinoma cells (JPG, 31.2 KB)
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LnCAP and HepG2 cells were treated with IL-6 for the times indicated. Levels of (A) pri-miR-21 and (B) mature miR-21 were determined by real-time PCR. Values obtained for cells cultivated in the absence of IL-6 were set to 1.
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