Article Figures & Data
Figures
- Figure 1.
Identification of LOGs. A, introduction of let-7b for 72 h caused down-regulation of 103 genes in both HepG2 and A549 cells ( 13). Purple circle: Of these 103 genes, 62 were found in a newly annotated data set of 22,000 genes found in mouse embryos (Supplementary Table S2). Green circle: 8,224 mouse genes found to be down-regulated between E9.5 and E15 of embryonic development. Of the 62 genes down-regulated in the two cancer cell lines, 16 were among the predicted let-7 targets (yellow circle) according to TargetRank. Of these 16 genes, 11 were down-regulated >1.4-fold in mouse embryos between E9.5 and E15. *, the number of LOGs containing one more gene (LIN28B) that was annotated through sequence match. B, the 12 LOGs ranked according to their average ranking position in the three analyses. Genes with known function as either oncogenes or tumor markers are in boldface. For footnotes, see Supplementary data.
- Figure 2.
Identification of IMP-1 as a direct target of let-7. A, schematic of the 3′-UTR at the human IGF2BP1 genomic locus located on chromosome 12. Gray box, 3′-end of the open reading frame; horizontal line, 3′-UTR spanning ∼6.7 kb. Solid rectangles, locations of the six putative let-7 binding sites and the nucleotide positions within the 3′-UTR. Sequences of the seed match to the let-7 family of miRNAs are shown below the nucleotide positions. Sites conserved among mammalian species are in boldface. Location of fragments IMP-1/2 containing LCS2 and LCS3 and fragment IMP-1/5 containing LCS5 are shown as stippled lines. B, A549 cells were transiently transfected with let-7g or a scrambled (scr) precursor miRNA. HeLa cells were transfected with an LNA-anti-let-7 or a scrambled LNA oligonucleotide. β-Actin was detected to show equal loading. C, IMP-1 is posttranscriptionally regulated through the 3′-UTR by let-7. Luciferase reporter assays were done in 293TN cells with reporter plasmid psiCHECK-HMGA2 3′-UTR (HMGA2), psiCHECK-IMP-1 3′-UTR (IMP-1), psiCHECK-IMP-1 fragment 2,3 3′-UTR (IMP-1/2), psiCHECK-IMP-1 fragment 2,3-mut 3′-UTR (IMP-1/2 mut), psiCHECK-IMP-1 fragment 5 3′-UTR (IMP-1/5), or psiCHECK-IMP-1 fragment 5-mut 3′-UTR (IMP-1/5 mut) together with 1 pmol of either pre-miR scrambled (S) or pre-miR let-7g (7g). Renilla luciferase activity was normalized to the internally controlled firefly luciferase activity. D, luciferase reporter assay with cell extracts of HeLa cells transfected with empty vector (/) or psiCHECK-IMP-1 3′-UTR and transfected with 50 nmol/L of a scrambled LNA oligonucleotide, or transfected with the LNA-let-7 inhibitor.
- Figure 3.
Let-7 regulates properties of tumor cells through down-regulation of IMP-1. A549 cells were transfected with scrambled oligo, let-7g, or a siRNA specific for IMP-1 for 72 h and subjected to cell count (A), cell cycle analysis (B), or motility assay (C). Insets in A show the levels of reduction of IMP-1 expression achieved by transfecting cells with let-7g or si-IMP-1. D, A549 cells were transfected with either a scrambled si-control oligo or si-IMP-1. Forty-eight hours after transfection, expression of cell cycle regulators and control genes was quantified by real-time PCR relative to the expression of GAPDH mRNA. E, A549 cells were infected with a shIMP-1 lentivirus or control virus. Forty-eight hours after infection, cells were counted (day 2), either control-transfected or transfected with let-7g, and replated at 20,000 per well. Cells were again counted 4 and 6 d after infection with viruses. Inset, Western blot analysis of all cells for HMGA2, IMP-1, and actin. A similar result was obtained with another independent IMP-1–specific shRNA virus (data not shown).
Additional Files
Supplementary Data, Boyerinas, et al.
Files in this Data Supplement:
Volume 68, Issue 8
