miRNA Microarray

Total RNA was extracted using the mirVana^TM RNA Isolation kit (Ambion) according to the manufacturer's instructions from LIM 1863 cells that were treated with both TGF-β and TNF-α or media only for 24 h. Total RNA was submitted to Exiqon (Vebaek, Denmark) for miRNA microarray profiling services, in which RNA was labeled with Hy3 and Hy5 fluorophores and hybridized to a miRCURY LNA microRNA Array (version 8.1). All subsequent data analysis was performed by Exiqon.

Northern Blotting

Total RNA was extracted using the mirVana^TM RNA Isolation Kit according to manufacturer's instructions (Ambion). Twenty micrograms of total RNA was resolved on denaturing agarose gels and transferred to nylon membranes. Following UV-cross-linking, membranes were incubated with random-primed miR-21 or GAPDH cDNA probes in Church's hybridization buffer (0.5 m NaHPO₄, pH 7.2; 1 mm EDTA; 7% SDS) at 42 °C for 18 h. For detecting the miR-21 primary transcript, the probe encompassed 250 bp on both sides of the mature miR-21 sequence. Membranes were washed three times in 2× SSC, 0.1% SDS at room temperature. To detect precursor and mature miRNA species, 20–40 μg of total RNA was resolved on 20% denaturing polyacrylamide gels, transferred to nylon membranes, UV-cross-linked and probed with 5′-end-labeled DNA oligonucleotide probes to detect miR-21 (5′-TCAACATCAGTCTGA TAAGCTA-3′), miR-31 (5′-CAGCTATGC CAGCATCTTGCC-3′), or 5 S rRNA (5′-TTAGCTTCCGAGATCA-3′) in Church's hybridization buffer at 37 °C for 18 h. Membranes were washed as above, except at 37 °C. All membranes were exposed to phosphor imaging screens, and scanned with a Storm 860 PhosphorImager (Molecular Dynamics).

Overexpression and Inhibition of miRNA Function

Human miR-21 and miR-31 precursors (AM17100) and a Cy3-labeled pre-miR negative control (AM17120) were purchased from Ambion. MiRNA activity was inhibited using chemically synthesized 2′O-methyl-modified RNA oligonucleotides (Dharmacon) that were antisense to miR-21 (5′-mUmCmAmAmCmAmUmCmAmG mUmCmUmGm-AmGmCmUmA-3′) or miR-31 (5′-mCmAmGmCmUmAmUmGmCmC mAmGmCmAmUmCmUmUmGmCmC-3′). A 5′-Cy3-labeled RNA oligonucleotide (Integrated DNA Technologies) targeting eGFP (5′-Cy3mA mAmGmGmCmAm-AmGmCmUmGmAmC mCmCmUmGmAmAmGmU-3′) was used as a negative control.

All transfections were performed utilizing Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. LIM 1863 organoids were resuspended in RPMI 1640 supplemented with 5% FBS and transfected with 100 nm of miRNA precursors in 24-well tissue culture plates. For migration and invasion assays, LIM 1863 organoids were trypsinized and dissociated before transfection. In 24-well tissue culture plates, 3 × 10⁵ cells were transfected with 100 nm miRNA precursors or 250 nm miRNA inhibitors.

Real-time RT-PCR

Total RNA was isolated using the mirVana^TM RNA Isolation kit following manufacturer's instructions (Ambion). One microgram of RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). SYBR-green real-time quantitative PCR was performed using a Bio-Rad MyiQ PCR detection system with the following gene-specific primers: fibronectin-1 (FN1), forward 5′-GAGCCATGTGTCT-TACCATT-3′ and reverse 5′-AGTATTTCTGGTCCTGCTCA-3′; interleukin-8 (IL8), forward 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ and reverse 5′-TCTCAGCCCTCTTCAAAAAACTTCTC-3′; laminin-γ-2 (LAMC2), forward 5′-CTGCAGGTGGACAACA-GAAA-3′ and reverse 5′-TCTGCTGTCACATTGGCTTC-3′; matrix metalloproteinase-7 (MMP7), forward 5′-CATGAGTGAGCTACAGTGGG-A-3′ and reverse 5′-CTATGACGCGGGAGTTTAACAT-3′; T-lymphoma invasion and metastasis-1 (TIAM1), forward 5′-AAGACGTACTCAGGCCATGTCC-3′ and reverse 5′-GACCCAAATGTCGCAGTCAG-3′; and U6 snRNA, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′.

To measure mature miR-21 and miR-31 levels by quantitative real-time PCR, 10 ng of total RNA was reverse-transcribed using the TaqMan miRNA reverse transcription kit and RT primers for miR-21, miR-31, and U6 snRNA (Applied Biosystems). The cDNAs were then analyzed by real-time PCR using TaqMan probes for miR-21, miR-31, and U6 snRNA (Applied Biosystems).

Cell Migration and Invasion Assay

Uncoated or Matrigel-coated transwells containing 8 μm pores were used for the assays (BD Biosciences). Cells were seeded into the upper chamber in serum-free RPMI 1640 medium. Conditioned DME media from NIH 3T3 cells containing 10% FBS was added to the lower chamber. Cells were fixed in 100% methanol 72 h later and stained with a 1:5 dilution of Giemsa (Sigma) for 40 min at room temperature. Cells remaining on the upper side of the filter were removed with a cotton swab. The filters were then mounted onto slides and images were taken at 10X magnification. From these images, the number of migratory or invasive cells was counted.

Dual Luciferase Reporter Assay

The full-length TIAM1 3′-UTR (1970 nt) was cloned into the XbaI and NotI sites of a modified pRL-TK vector (Promega) immediately downstream of the Renilla luciferase stop codon and designated pRL-TIAM1. The putative miR-21 and miR-31 recognition elements (MRE) in the TIAM1 3′UTR were mutated by site-directed mutagenesis (Stratagene). For the dual luciferase assay, LIM 1863 cells were transfected with 100 nm of pre-miR-21, pre-miR-31, or a negative precursor control using Lipofectamine 2000 (Invitrogen). After 24 h, cells were co-transfected with 200 ng of pRL-TIAM and 100 ng of pGL3-luc as the internal control. Cell extracts were prepared 48 h later and the Dual-Glo luciferase reporter assay (Promega) was performed according to the manufacturer's protocol.

TGF-β/TNF-α Up-regulate miR-21/miR-31 at the Transcription and Processing Levels

In a time course analysis, we found that while the miR-21 precursor (a ~70-nt stem-loop processing intermediate) in LIM 1863 was rapidly increased and reached a plateau 2 h after TGF-β/TNF-α stimulation, the increase in the mature miR-21 was delayed and did not reach its peak until 24 h later (Fig. 2A). Whereas this observation suggests activation of miR-21 gene transcription by TGF-β/TNF-α, it is also clear that the processing from precursor to mature miR-21 is the more rate-limiting step in TGF-β/TNF-α induction of this miRNA. Interestingly, pretreating the LIM 1863 organoids with cycloheximide effectively abrogated the increase in mature miR-21 by TGF-β/TNF-α, and yet had no effect on the increase in miR-21 precursor (Fig. 2A). This suggests that TGF-β/TNF-α enhances the Argonaute-mediated processing of miR-21 precursor through an indirect mechanism, requiring the synthesis of unknown factors. Northern blotting of total RNA revealed three primary transcripts of the miR-21 gene, ranging in size from 1 to 4 kb (Fig. 2B). All three transcripts rapidly increased 2 h after TGF-β/TNF-α treatment, which was not affected by cycloheximide (Fig. 2B). Therefore, the level of miR-21 is regulated by TGF-β/TNF-α at the initial transcription and the processing steps, and only miR-21 gene transcription is a direct downstream target of TGF-β/TNF-α signaling.

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FIGURE 2.

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.

The increase in mature miR-31 by TGF-β/TNF-α became noticeable 2 h after the cytokine treatment, and continued to accumulate until 24 h later (Fig. 2C). Cycloheximide also effectively inhibited such up-regulation of mature miR-31 by TGF-β/TNF-α (Fig. 2C). However, neither the primary nor the precursor forms of miR-31 were detectable by Northern blotting, so we could not determine whether miR-31 is also regulated at the primary transcription level like in the case of miR-21. Therefore, for both miR-21 and miR-31, the up-regulation by TGF-β/TNF-α appears to be indirect and requires new synthesis of unknown factors.

Furthermore, when we knocked down the essential miRNA-processing factor Drosha using a previously validated shRNA, TGF-β/TNF-α failed to elevate the level of mature miR-21 and miR-31 (Fig. 1D and supplemental Fig. S1) (36). This observation suggested that the increase in miR-21/miR-31 upon TGF-β/TNF-α stimulation is due to new biosynthesis and/or processing of miRNAs.

miR-21 and miR-31 Are Important Regulators in TGF-β-induced EMT of LIM 1863 Cells

TGF-β-induced EMT in LIM 1863 is a slow process. By the first 24 h, most organoids attached to the tissue culture plate, but only a small percentage of them began to spread out into a monolayer (Fig. 3A). By counting the organoids exhibiting either a “spreading” or “not spreading” morphology, we quantified TGF-β-induced EMT and evaluated the impact of miR-21 and miR-31 overexpression. Indeed, when examined 24 h after TGF-β addition, LIM 1863 organoids transfected with the precursors of either miR-21 or miR-31 had a significantly higher percentage adopting a “spreading” morphology, compared with organoids transfected with a non-targeting control miRNA precursor (Fig. 3B). The same was observed 48 h after TGF-β stimulation (Fig. 3B). These data suggest that miR-21 and miR-31 are able to accelerate the EMT process initiated by TGF-β.

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FIGURE 3.

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.).

To better quantify the EMT process, we measured the mRNA levels of a number of EMT markers. In our LIM 1863 culture, TGF-β/TNF-α induced re-distribution of E-cadherin from cell surface to cytoplasm without much change in its protein level (data not shown), so we measured the expression of other known markers including fibronectin 1 (FN1), interleukin 8 (IL-8), laminin-γ-2 (LAMC2), and matrix metalloproteinase 7 (MMP7) (37, 38). The levels of these markers were all increased 24 h after TGF-β treatment (Fig. 3C). Transfection of either miR-21 or miR-31 precursors further potentiated TGF-β in increasing the mRNA levels of FN1, IL8, and LAMC2 (Fig. 3C). In the case of MMP7, only miR-31 had a significant effect (Fig. 3C). Even in cells without any exposure to TGF-β, either miR-21 or miR-31 elevated the expression of these EMT markers as well (Fig. 3C). These more quantitative analyses further substantiated our conclusion that a high level of miR-21 or miR-31 facilitates TGF-β-induced EMT of LIM 1863. Importantly, TGF-β-induced up-regulation of SMAD7 was not enhanced by either miR-21 or miR-31 overexpression, suggesting that these two miRNAs only facilitate a subset of the TGF-β responses, and do not enhance TGF-β signaling in general (supplemental Fig. S2).

We further carried out loss-of-function studies to evaluate the importance of miR-21 and miR-31 in TGF-β/TNF-α-induced EMT of LIM 1863 cells. Indeed, when miR-21 or miR-31 activity was neutralized by antisense 2′O-methyl RNA oligonucleotides, TGF-β/TNF-α induction of the mesenchymal markers LAMC2 and MMP7 was substantially decreased (Fig. 3D). This further supported the requirement of miR-21 and miR-31 in LIM 1863 cell EMT induced by TGF-β/TNF-α.

miR-21 and miR-31 Regulate the Migration and Invasion of LIM 1863 Cells

EMT is often linked to a gain in the migratory and invasive properties of the cell. Even though the LIM 1863 organoids hardly migrated in the standard transwell assay, we found that if cells were immediately plated after dissociation of the organoids by trypsin, there was migration in the transwell assay with 10% FBS and media from NIH 3T3 culture as the chemoattractant (Fig. 4A). The same was observed in the Matrigel invasion assay (Fig. 4B). When LIM 1863 cells transfected with miR-21 or miR-31 precursors were tested in these assays, they exhibited significantly enhanced migratory and invasive properties compared with cells transfected with a control miRNA precursor (Fig. 4, A and B). Interestingly, corroborating the results in Matrigel invasion assays, we noticed that when plated onto the Matrigel filter, LIM 1863 cells transfected with miR-21 or miR-31 readily adhered and spread out, whereas cells transfected with a control miRNA precursor did not show such characteristics (Fig. 4B, phase contrast images). These observations support the notion that an increase in miR-21 or miR-31 enhances the migration and invasion properties of LIM 1863 cells.

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FIGURE 4.

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.

If the dissociated LIM 1863 cells were treated with TGF-β/TNF-α upon plating into the transwells, the motility was markedly increased (Fig. 4C). Importantly, when miR-21 function was inhibited by the 2′O-methyl anti-miR-21 oligonucleotide, TGF-β/TNF-α-induction of LIM 1863 motility was significantly reduced (Fig. 4C). This suggests that miR-21 is required for TGF-β/TNF-α-regulation of LIM 1863 cell migration. In contrast, we observed little effect with anti-miR-31, suggesting miR-31 is not as rate-limiting as miR-21 is in regulating LIM 1863 motility. In Matrigel assays, TGF-β/TNF-α also stimulated invasion of the LIM 1863 cells (Fig. 4D). Interestingly, inhibition of either miR-21 or miR-31 significantly decreased LIM 1863 invasion in response to TGF-β/TNF-α, so both of these two miRNAs have non-overlapping targets that are important for LIM 1863 to invade through the extracellular matrix (Fig. 4D). From these gain- and loss- of function studies, we conclude that both miR-21 and miR-31 positively regulate migration and invasion properties of the LIM 1863 cancer cells.

Pro-migration and Pro-invasion Functions of miR-21 and miR-31 in Other Colon Cancer Cell Lines

To determine if the activities of miR-21/miR-31 we observed so far are unique to LIM 1863 cells, we overexpressed these two miRNAs in SW480 and DLD1 colon cancer cell lines. Because in our hands these two cell lines do not undergo EMT in response to TGF-β, we only evaluated their motility and invasion. Indeed in both cell lines, transfection of miR-21 and miR-31 precursors resulted in a marked increase in cell migration and invasion (Fig. 5, A and B). Given that SW480 cells do not express SMAD4 and experiments in Fig. 5 were all done without TGF-β stimulation, our observations also suggest that the pro-metastasis activities of miR-21 and miR-31 do not depend on a functional TGF-β/SMAD pathway.

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FIGURE 5.

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).

Interestingly, under exactly the same conditions, miR-31 overexpression substantially suppressed motility and invasion of a breast cancer cell line MDA-MB-231, consistent with previous reports of an anti-metastasis function of miR-31 in breast cancer (29, 30). Therefore, the impact of miR-31 on cancer cell migratory properties is dependent on cell contexts.

All of the above evidence points to pro-cancer invasion and metastasis functions of miR-21 and miR-31 in multiple colon cancer cell lines. These results also agree with previous studies of colon cancer tissues in which higher levels of miR-21 and miR-31 have been linked to colon cancer progression to a late stage and metastasis (39, 40).

TIAM1 Is a Target for Both miR-21 and miR-31

One critical question is the downstream targets of miR-21 and/or miR-31 that contribute to the cellular impact of these two miRNAs. Using TargetScan to search for 3′-UTR sequences with 7 nucleotide matches to the seed region of miR-21 or miR-31, TIAM1 (T lymphoma invasion and metastasis 1) emerged as a possible target for miR-21 (41). Upon visual inspection we found a weak recognition site for miR-31 (e.g. a 6 nucleotide match) in the TIAM1 3′-UTR as well. TIAM1 is a guanine nucleotide exchange factor (GEF) of Rac, and has been implicated in regulating cell migration, invasion and tumor progression (42,–44). Indeed, treating the LIM 1863 organoids with TGF-β/TNF-α substantially reduced the protein level of TIAM1, further prompting us to investigate TIAM1 as a possible target of miR-21 and/or miR-31 (Fig. 6A). Transfecting the LIM 1863 cells with precursors for either miR-21 or miR-31 resulted in markedly decreased abundance of TIAM1 protein when compared with the non-targeting miRNA precursor control (Fig. 6B). Quantitative real-time PCR revealed no change in the mRNA level of TIAM1 by either miR-21 or miR-31 (supplemental Fig. S3). Thus miR-21 and miR-31 appear to control TIAM1 expression mainly through repression of the protein translation, not degradation of the mRNA. Consistent with the functional data in Fig. 5, miR-21 and miR-31 also down-regulated TIAM1 in DLD1 and SW480 cells (Fig. 6C). Importantly, we found that transfection of miR-21 did not cause a significant change in mature miR-31 level, and vice versa (Fig. 6D). Therefore, miR-21 and miR-31 do not influence the expression levels of each other and likely impact the expression of TIAM1 independently.

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FIGURE 6.

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.

To further validate whether TIAM1 is a direct target of miR-21 or miR-31, we engineered a luciferase reporter construct containing the 1970 bp 3′-UTR of the TIAM1 gene. MiR-21 precursor indeed significantly reduced the reporter expression (Fig. 6E). We identified two potential miR-21 target sites (matching 7 nt of the miR-21 seed sequence) in the TIAM1 3′-UTR (Fig. 6E, left), and mutations in either one of them reduced, but did not fully abolish the suppression by miR-21 (Fig. 6E, left). Only when both sites were mutated did the miR-21 precursor completely fail to repress the reporter expression (Fig. 6F, right). These data strongly support the hypothesis that TIAM1 is a direct target of miR-21 in LIM 1863 cells. Transfection of miR-31 precursor also significantly reduced the TIAM1 3′-UTR reporter (Fig. 6F). However, we could only find one stretch of 6 nucleotides complementing the miR-31 seed sequence (Fig. 6F). Nevertheless, mutation of this sequence significantly alleviated the repression by miR-31 (Fig. 6F). This strongly suggests that miR-31 also directly represses TIAM1 translation. The remaining inhibitory effect of miR-31 may be due to additional cryptic sites within the TIAM1 3′-UTR or other indirect mechanisms.