miR-21 and miR-31 are up-regulated during TGF-β/TNF-α-induced EMT in LIM 1863 organoids. A, phase contrast images of LIM 1863 organoids with or without TGF-β/TNF-α (T+T) treatment (24 h). A schematic depiction of the morphological and adhesive changes is shown (lower). B, LNA-based microarray was used to measure the relative ratio of each miRNA in LIM 1863 organoids with or without TGF-β/TNF-α (T+T) treatment. A total of 455 miRNAs were profiled. miR-21 and miR-31 were most significantly up-regulated by TGF-β/TNF-α. C and D, Northern blot analyses detecting miR-21 (C) or miR-31 (D) in LIM 1863 organoids with indicated cytokine treatment for 24 h. The ∼21-nt mature miRNAs (miR-21 or miR-31) are shown. 5 S rRNA expression was used as an internal control. E, extracts from LIM 1863 cells after indicated cytokine treatments were analyzed by Western blotting using indicated antibodies. F, real-time quantitative RT-PCR analysis of SMAD7 mRNA levels in LIM 1863 cells after vehicle or TGF-β/TNF-α (T+T) stimulation (mean ± S.D., **, p < 0.01). U6 snRNA was used as the internal standard.

TGF-β/TNF-α regulates miR-21 and miR-31 abundance at the transcription and processing steps. A, TGF-β/TNF-α (T+T) induction of mature miR-21 was abrogated by cycloheximide (CHX). LIM 1863 organoids were incubated with DMSO or 15 μg/ml cycloheximide for 30 min followed by treatment with both TGF-β and TNF-α (T+T) for the indicated times. Shown are Northern blotting analysis of miR-21 precursor (pre-miR-21, ∼70 nt) and mature miR-21 (∼21 nt), with 5 S rRNA as the loading control. B, TGF-β/TNF-α (T+T) induced a rapid increase in the miR-21 primary transcript (pri-miR-21), which was not affected by cycloheximide. Northern blotting of total RNA shows three prominent miR-21 primary transcripts with ∼1, ∼2, and ∼4 kb in size (arrows). GAPDH served as the loading control. C, similar experiment as in A showing the increase in mature miR-31 in response to TGF-β/TNF-α (T+T) was affected by cycloheximide. D, LIM 1863 cells expressing a control shRNA (con) or a Drosha shRNA vector were treated with TGF-β/TNF-α (T+T) for 36 h, and the levels of mature miR-21 and miR-31 were measured by the Taqman miRNA assay (mean ± S.D., **, p < 0.01; ***, p < 0.001). U6 snRNA was used as the internal standard.

TGF-β-induced LIM 1863 morphological change is potentiated by miR-21 and miR-31. A, representative LIM 1863 organoids exhibiting “spreading” or “not spreading” morphologies after 24 h TGF-β treatment (upper). A schematic drawing of “spreading” and “not spreading” morphologies is shown (lower). B, LIM 1863 organoids transfected with indicated miRNA precursors were stimulated with TGF-β, and the morphology was scored as “spreading” or “not spreading” at 24 h or 48 h post TGF-β addition. The results are plotted (mean ± S.D., > 200 organoids were counted in each experiment, data represent >3 experiments). *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, same experiment as in B (only the 24 h time point), and the mRNA levels of indicated markers were measured by quantitative real-time RT-PCR (mean ± S.D., n>3), using U6 snRNA as an internal reference. FN1: fibronectin 1; IL8: interleukin 8; LAMC2: laminin γ2; MMP7: matrix metalloproteinase 7. D, inhibition of miR-21 or miR-31 resulted in markedly diminished induction of EMT markers by TGF-β/TNF-α. LIM 1863 cells were transfected with 250 nm 2′O-methyl RNA inhibitors of miR-21, miR-31, or control (Con). Eighteen hours later, cells were stimulated with TGF-β/TNF-α (T+T), and the mRNA abundance of LAMC2 and MMP7 was measured by quantitative real-time RT-PCR at indicated time points (mean ± S.D.).

miR-21 and miR-31 regulate LIM 1863 migration and invasion. A and B, LIM 1863 organoids were transfected with 100 nm miR-21 or miR-31 precursors or a negative precursor control (C). Forty-eight hours later, the organoids were dissociated by trypsin and 1 × 10⁵ cells were seeded into the upper wells of transwell chambers coated without (A) or with Matrigel (B). After 72 h, cells that migrated to the lower chambers were counted (mean ± S.D., 8 fields per filter were examined per experiment). LIM 1863 cells transfected with miR-21 and miR-31 precursors spontaneously attached and spread on Matrigel-coated filters (phase contrast images in B, right). C and D, Inhibition of miR-21 and miR-31 activities affects TGF-β//TNF-α-induced LIM 1863 cell migration and invasion. LIM 1863 cells were transfected with 250 nm 2′O-methyl RNA inhibitors of miR-21, miR-31, or control. Eight hours post-transfection, cells (5 × 10⁵ for migration and 1 × 10⁶ for invasion assay) were seeded into the upper wells of transwell chambers coated without (C) or with Matrigel (D) and treated with TGF-β//TNF-α (T+T) or without. After 72 h, the number of cells that migrated to the lower chamber was counted (mean ± S.D., 8 fields per filter were examined). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Context-dependent pro-migration and pro-invasion activities of miR-21 and miR-31 in colon and breast cancer cells. A–C, control, miR-21, or miR-31 precursors (100 nm) were transfected into DLD1 (A), SW480 (B), or MDA-MB-231 (C) cells. The motility and invasiveness of these cells were analyzed by transwell migration and invasion assays as in Fig. 4, A and B. Plotted is the number of cells that migrated to the lower chamber was counted (mean ± S.D., >3 fields per filter were examined, **, p < 0.01; ***, p < 0.001).

TIAM1 is an endogenous target of both miR-21 and miR-31. A, LIM 1863 organoids were treated with TGF-β/TNF-α (T+T) for 24 h and Western blotting was performed to determine TIAM1 levels (C16, Santa Cruz Biotechnology), with β-tubulin as a loading control. B and C, miR-21 and miR-31 down-regulate TIAM1 protein abundance in multiple colon cancer cell lines. LIM 1863 (B), DLD1 (C), or SW480 (C) cells were transfected with 100 nm miR-21 or miR-31 precursors, or a negative control precursor. After 72 h, TIAM1 protein level was measured by Western blotting. D, LIM 1863 cells were transfected with 100 nm indicated miRNA precursors and 48 h later, the levels of mature miR-21 and miR-31 were determined by the TaqMan miRNA assay (mean ± S.D., n>3). E, (left) Alignment of TIAM1 3′-UTR with the miR-21 sequence. The numbering starts from the first residue after the stop codon. Asterisks indicate the nucleotides that were mutated (CT->TG for both site 1 and site 2 mutations). (right), miR-21 directly targets the 3′-UTR of TIAM1. LIM 1863 cells were transfected first with 100 nm of miR-21 precursor or a control. Eighteen hours later the same cells were transfected with a Renilla luciferase reporter containing the wild type or mutant TIAM1 3′-UTR (e.g. S1: site 1 mutation; S2: site 2 mutation; S1/2: double mutation). A constitutively active firefly luciferase reporter was used as the internal control. The luciferase activities were measured and plotted (mean ± S.D., n>3). *, p < 0.05. F, Similar experiments as in E, analyzing the inhibitory impact of miR-31 on the 3′-UTR of TIAM1. The mutated TIAM1 3′-UTR residues are marked by * (CC->TT), the numbering is the same as in E. **, p < 0.05; ***, p < 0.001.

Elevated TIAM1 level antagonizes promotion of LIM 1863 morphological changes, motility, and invasiveness by TGF-β/TNF-α and miR-21/miR-31. A, phase contrast images showing no obvious morphological changes in LIM 1863 organoids with or without TIAM1 overexpression. B, TIAM1 overexpression greatly reduced the ability of LIM 1863 cells to undergo EMT. Cells transduced with TIAM1 or empty vector (control) were stimulated with TGF-β/TNF-α (T+T). The morphology of the organoids (n >75 per group) was scored as “spreading” or “not spreading” as in Fig. 3B at 6 h and 24 h time points (mean ± S.D., >75 organoids were counted in each experiment, data represent >3 experiments). **, p < 0.01. C and D, Transwell migration (C) and Matrigel invasion (D) assays measuring LIM 1863 cell motility and invasiveness, respectively. LIM 1863 cells were transduced with lentiviral vectors encoding TIAM1 or control. Cells (5 × 10⁵ for the migration assay and 1 × 10⁶ for the invasion assay) were then seeded into the upper chambers and treated with vehicle or TGF-β/TNF-α (T+T) as indicated. After 72 h, the number of cells that migrated to the lower chambers was counted (mean ± S.D., 8 fields per filter were examined). ***, p < 0.001. E and F, LIM 1863 cells transduced with TIAM1-expressing or control vectors were further transfected with miR-21, miR-31 or control (C) precursors as indicated. Cells were then analyzed for motility (E) and invasion (F) (mean ± S.D., 8 fields per filter were examined). ***, p < 0.001.