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Keywords:

  • Caco-2;
  • colon;
  • differentiation;
  • miR-146a;
  • MMP16

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Cellular differentiation in the gut is vital in maintaining the cellular and functional specialization of the epithelial layer. MicroRNAs (miRNAs) have recently emerged as one of the key players in orchestrating the differentiation process in the gut. Using the spontaneously differentiating Caco-2 cell line, we observed an increased expression of miR-146a but not miR-146b in the course of differentiation. Bioinformatic analyses revealed that the membrane type matrix metalloprotease 16 (MMP16, MT3-MMP) was a predicted target of miR-146a and a decrease in the mRNA and protein expression of MMP16 was observed in the course of differentiation. Transfection of a luciferase reporter vector containing the 3′UTR of MMP16 showed decreased luciferase activity due to miR-146a expression. With forced expression of miR-146a in undifferentiated Caco-2 cells, a decrease in the mRNA and protein levels of MMP16 and a lower gelatinase activity in a gelatin zymogram were observed. Additionally, forced expression of miR-146a in HT-29 colon cancer cells also resulted in decreased expression of MMP16, along with a decrease in the invasion through Matrigel. Taken together, we have shown here that MMP16 is regulated by miR-146a in spontaneously differentiated Caco-2 cells. As MMP16 activates the zymogen of MMP2, which is known to degrade extracellular matrix proteins, the regulation of MMP16 by miR-146a may account, at least in part, for lower motility of well-differentiated cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

The intestinal epithelium, formed of a single layer of columnar cells, forms projections (crypts) into the underlying connective tissue. These crypts house multipotent stem cell niches, which differentiate into absorptive cells, mucus producing goblet cells or endocrine cells as they move upwards towards the villi to be finally expelled into the lumen (Humphries & Wright 2008). As the cells differentiate, they not only lose their ability to proliferate, but also form tight junctions that prevent the paracellular movement of molecules, thereby providing the intestinal barrier function (Beaurepaire et al. 2009). Formation of polarized cells during epithelial differentiation involves contact with fibroblast and extracellular matrix proteins, the activity of transcription factors and microRNA (miRNA) mediated regulation (Simon-Assmann et al. 2007; Dalmasso et al. 2010).

The importance of miRNAs in intestinal differentiation and function was recently demonstrated by knocking out Dicer1, a key enzyme in miRNA biogenesis, in the intestinal epithelium (Mckenna et al. 2010). These authors reported that the Dicer1 knockout mice had disorganized crypts with a loss of goblet cells and an increased inflammatory phenotype with greater neutrophil infiltration into the lamina propria and dramatic increases in paracellular permeability, indicating that miRNAs and/or an as yet unknown function of Dicer1 potentially regulate each of these functions in the intestine (Mckenna et al. 2010).

An emerging key player in intestinal physiology appears to be miR-146. Two closely related mature miR-146 sequences (miR-146a and miR-146b) are transcribed from genes on chromosomes 5 and 10, respectively, and differ by only two bases in the 3′ region of their seed sequences (Taganov et al. 2006). They may thus target similar mRNAs for translational repression or destabilization. The importance of miR-146a in the intestine was highlighted in a recent study showing that tolerance to intestinal microbes in neonates was dependent on the loss of IRAK1, an nuclear factor-κB (NF-κB) target protein, by degradation in the proteosome and lysosome as well as its translational repression by miR-146a (Chassin et al. 2010).

miR-146a/b have also been shown to inhibit the invasive potential in pancreatic cancer, brain glioma cells, breast cancer and gastric cancer cells indicating a tumor suppressive nature (Bhaumik et al. 2008; Xia et al. 2009; Ali et al. 2010; Kogo et al. 2011). Matrix metalloprotease 16 (MMP16, MT-MMP3) was implicated as a target of miR-146b in mediating loss of invasiveness in brain glioma cells (Xia et al. 2009).

In a high throughput study, out of 156 miRNAs, miR-146 was reported to be one of the most upregulated miRNAs in differentiated Caco-2 cells (Hino et al. 2008). The Caco-2 cell line has been shown to undergo spontaneous differentiation upon reaching confluency in vitro (Pinto 1983) and is widely used as a model for intestinal differentiation. The differentiated cells are characterized by the development of microvilli (brush border membranes) and high levels of alkaline phosphatase, sucrase and lactase (Simon-Assmann et al. 2007).

We have examined the expression of miR-146a/b in the course of spontaneous differentiation in Caco-2 cells. As miR-146a/b have been implicated in reducing cellular invasiveness, we have examined the potential regulation of MMP16, a predicted target of miR-146a, in this model of epithelial differentiation. We believe that investigating the role of miRNAs and their target proteins in epithelial differentiation will help us better understand cellular characteristics in this very complex process.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Cell culture

Caco-2 and HT-29 cells were obtained from ŞAP Enstitüsü (Ankara, Turkey). The Caco-2 cells (between passage no. 8–11) were cultured and maintained in Earl’s Minimal Essential Medium (EMEM) supplemented with 20% fetal bovine serum (FBS). HT-29 cells were propagated in RPMI-1640 medium containing 10% FBS. Cell culture media were supplemented with 1% penicillin/streptomycin, 2 mmol/L L-glutamine, 0.1 mmol/L non essential amino acids, 1.5 g/L sodium bicarbonate and 1 mmol/L sodium pyruvate. The cells were grown in a humidified atmosphere containing 5% CO2 at 37°C. All cell culture media and supplements were purchased from Biochrom (Berlin, Germany).

Caco-2 cells were grown until confluency in 6-well plates and the 100% confluent cells were considered to be at Day 0 for differentiation. The cells were collected at different intervals until Day 30 after reaching confluency and processed for transfections or RNA/protein isolation.

RNA extraction and RT–PCR

Total cellular RNA was isolated from Caco-2 cells using the RNEasy RNA Extraction kit (Qiagen, USA). The quantity and purity of RNA were determined using a Nanodrop reader. After isolation, 2 μg RNA was treated with Dnase I enzyme (Fermentas, Vilnius, Lithuania) according to the manufacturers’ instructions and then reverse transcribed by using Revert Aid First Strand cDNA Synthesis kit (Fermentas) using random hexamers.

MMP16 and pre-miR-146a expression was analyzed semiquantitatively by co-amplification of each of the transcripts with GAPDH in duplex polymerase chain reaction (PCR) reactions. PCR was performed with 1 U Taq polymerase in 1× buffer with ammonium sulfate (Fermentas), 2.5 mmol/L MgCl2, 0.2 mmol/L dNTP mix, 0.5 μmol/L of each primer. The primer sequences are shown in Table 1.

Table 1.   Oligo and primer sequences used in the study
GeneSequence
  1. MMP16, matrix metalloprotease 16. Lower case letters indicate the nucleotides that were mutated.

MMP16 3′ UTR5′-CTCCAGTGCATTACCTATTGcatgtCCACCATagttctcaAAGGGTTAGTGTGGCTTCTGG-3′
MMP16 3′UTR mutated5′-CTCCAGTGCATTACCTATTGcccccCCACCATaaaaaaaAAGGGTTAGTGTGGCTTCTGG-3′
miR-146a5′-TGTATCCTCAGCTTTgagaactgaattccatgggttGTGTCAGTGTCAGAC-3′
miR-146a mutated5′-TGTATCCTCAGCTTTtttttttttttttttttttttGTGTCAGTGTCAGAC-3′
MMP16 (NM_022564.3)Forward 5′-GCTGACCCAAGGAAAAATGA-3′ (Amplicon size 270 bp)
Reverse 5′-CACAAAATTCCCGTCGCTAT-3′
pre-miR146a (NR_029701.1)Forward 5′-CCGATGTGTATCCTCAGCTTTGA-3′ (Amplicon size 99 bp)
Reverse 5′-ACGATGACAGAGATATCCCAGCTG-3′
pre-miR146b (NR_030169.1)Forward 5′–CCTGGCACTGAGAACTGAATTCC-3′ (Amplicon size 76 bp)
Reverse 5′-CCGGGCACCAGAACTGAGTCCAC-3′
GAPDH (NM_002046.3)Forward 5′-GGTGAAGGTCGGAGTCAACG-3′ (Amplicon size 496 bp)
Reverse 5′-CAAAGTTGTCATGGATGACC-3′
GAPDH (NM_002046.3)Forward 5′-CGACCACTTTGTCAAGCTCA-3′ (Amplicon size 238 bp)
Reverse 5′-CCCCTCTTCAAGGGGTCTAC-3′
Sucrase isomaltase (NM_001041.3)Forward 5′-CAAATGGCCAAACACCAATG-3′ (Amplicon size 159 bp)
Reverse 5′-CCACCACTCTGCTGTGGAAG-3′

Mature miRNA detection

Total RNA was isolated from undifferentiated and differentiated Caco-2 cells at predefined days after reaching 100% confluency using the RNeasy RNA extraction kit (Qiagen) and verified for purity using the Nanodrop. Thirty nanograms of the RNA was reverse transcribed using the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems, USA). Mature miR-146a and miR-146b were detected with their specific Taqman MicroRNA assay kits according to manufacturer’s instructions using a Corbett Rotor-Gene 6000 real time PCR machine and its associated software (Qiagen). The miRNA expression was determined by the delta-delta Ct method normalized to the small nuclear RNA U6B (RNU6B) (Applied Biosystems). The experiment was independently repeated six times.

Plasmids and transfections

In order to determine miRNA targeting of the 3′ UTR region of MMP16, a 489 bp portion of the MMP16 3′ UTR region (bases 1251–1740) including one predicted miR-146a binding site was cloned into the pmiR REPORT luciferase vector (Promega, USA) at the SacI and HindIII sites. A mutated vector was generated by replacing the miR-146a binding site nucleotides to T using a commercial site directed mutagenesis kit (Stratagene, USA). For the ectopic expression of miR-146a, the full-length precursor miR-146a was cloned into the pSUPER vector (OligoEngine, USA) at the Hind III and Sal I sites. The mutated miR-146a overexpression vector was generated by replacing the mature miR-146a sequence with T using Pfu and Dpn1 (Fermentas) according to the protocol outlined in a commercial site directed mutagenesis kit. The original and mutated sequences are shown in Table 1. All constructs were confirmed by sequencing.

Transfections were carried out for 24 h in 6-well tissue culture plates in 2 mL serum free OptiMEM (Invitrogen) using Lipofectamine and Plus reagents (Invitrogen) at a ratio of 10:10 per μg of vector used (Cui et al. 2009).

Western blot

Total protein extracts were prepared by lysing the cells with 1× Cell Lysis Buffer (CLB), (Promega) containing 1% NP-40, 1× Protease Inhibitor cocktail (Roche, Germany). The cells were centrifuged at 14 000 g for 2 min at 4°C and the supernatant was collected as the total protein extract. For western blotting, 40 μg protein was loaded in a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel, electrophoresed under reducing conditions and electroblotted onto PVDF membranes (Roche). The membranes were blocked in 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.1% Tween20 (PBST) for 1 h at room temperature with constant agitation. Following this, the membrane was incubated overnight with an anti-MMP16 primary antibody (1:100) (Santa Cruz, USA), washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:1666; Santa Cruz) for 1 h at room temperature. The bands were visualized by using the enhanced chemiluminescence (ECL) Western Blotting Substrate (Pierce) according to the manufacturer’s instructions. To ensure equal protein loading, the membranes were stripped with a stripping buffer (1.5% Glycine, 0.01% SDS, and 1% Tween 20, pH 2.2) and probed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000; Santa Cruz).

Alkaline phosphatase activity assay

Protein extracts were prepared from Caco-2 cells collected at various days after reaching 100% confluency using the 1× CLB (Stratagene) according to the manufacturer’s instructions. Phosphatase inhibitors were omitted from the extraction buffer owing to potential interference with the alkaline phosphatase activity. Twenty μg of protein was added to the substrate p-nitrophenyl phosphate (Sigma) in 96-well plates and then incubated at 37°C for 30 min. The reactions were stopped by the addition of 150 μL 3N NaOH and enzymatic activity was measured spectrophotometrically at 405 nm using the CLB as blank. Specific activity was determined by dividing the activity with the respective protein amount.

Reporter gene assays

MicroRNA binding to the 3′UTR of MMP16 was confirmed by reporter gene assays. For this purpose, the pmiR-REPORT vector containing the MMP16 3′UTR region was transfected to the 100% confluent Caco2 cells on Day 0 of differentiation. After 24 h, the cells were washed with ice cold PBS and harvested using 1× CLB buffer (Stratagene). Proteins were collected from the supernatant after centrifugation at 14 000 g for 2 min at 4°C and assayed for luciferase activity using a luciferase substrate (Roche) according to the manufacturer’s instructions. For all reporter gene assays, co-transfection with a pSV-βgal vector was carried out. Normalization of relative light units was conducted by measuring the β-galactosidase activity spectrophotometrically at 415 nm by using ortho-nitrophenyl-β-galactoside (ONPG) as a substrate.

Zymogram

Caco-2 cells at 100% confluency and Day 0 of differentiation were transfected with the miR-146a overexpression plasmid and its mutated counterpart. The transfection was continued for 48 h after which the culture medium was collected. For the control cells that were not transfected, the 100% confluent cells were grown for 48 h after which the culture medium was collected. Gelatinase activity in the conditioned media was examined by gelatin zymography as described previously (Nakamura et al. 1999). Briefly, two volumes of ice cold acetone was added to the medium and centrifuged at 14 000 g for 30 min at 4°C. Total precipitated protein (1 mg) from the conditioned medium was dissolved in a native loading buffer and separated on a 10% SDS–PAGE gel containing 1 mg/mL gelatin under non-reducing conditions. Following electrophoresis, the gel was washed and incubated with a renaturation solution (2.5% v/v Triton-X-100) at room temperature, washed with distilled water and incubated for 16 h at 37°C in a developing buffer (50 mmol/L Tris-HCl, pH 7.8, 0.2 mol/L NaCl, 5 mmol/L CaCl2, 1 mmol/L ZnCl2 and 0.02% v/v Brij 35). Following this, the gel was washed with distilled water, stained with a staining solution (0.5% w/v Coomassie blue R-250, 5% v/v methanol and 10% v/v acetic acid) for 1 h at room temperature followed by destaining with a destaining solution (10% v/v methanol, 5% v/v acetic acid).

Invasion assay

The invasive capacity of HT-29 cells transfected with miR-146a was determined by an in vitro Boyden chamber assay, as described previously (Cimen et al. 2009). HT-29 cells (5 × 104 cells) in 0.5 mL serum-free RPMI-1640 medium were added to the upper wells of Boyden chambers containing membranes with 8 μm pores (Sigma Aldrich Chemie GmbH, Germany), coated with Matrigel (1:5) (BD Biosciences). Cells were allowed to invade for 96 h and the non-invaded cells were removed by scrubbing with sterile cotton swabs. The chambers were fixed in 100% methanol for 10 min, stained with modified Giemsa staining solution for 2 min and washed twice in distilled water. The invasion of HT-29 cells was quantified under a Leica light microscope with 4× objective by counting five fields per membrane.

Statistical analyses

Each experiment was repeated at least three times and represented as mean ± SD. Data analysis and graphing was performed using the GraphPad Prism 5 software package (Prism, USA). Statistical analyses between experimental results were based on Student’s t-test. Significant difference was statistically considered at the level of P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

miR-146a/b expression in the course of spontaneous differentiation of Caco-2 cells

Given the emerging interest in miR-146a/b in inflammation, development and differentiation, we analyzed the expression of mature miR-146a and miR-146b in the course of differentiation of Caco-2 cells. An increase in the activity of alkaline phosphatase and the expression of sucrase isomaltase confirmed the induction of differentiation in confluent Caco-2 cells cultured over a period of 30 days (Fig. 1a,b).

image

Figure 1.  The expression of miR-146a but not miR146b increases in the course of differentiation in Caco-2 cells. Caco-2 cells were collected at defined days after reaching 100% confluency and processed for protein or mRNA isolation. (a) Alkaline phosphatase activity (***P < 0.0001, Student’s t-test) and (b) sucrase isomaltase expression (SI) was seen to increase in the course of differentiation, indicating the induction of differentiation. (c) Real time quantitative polymerase chain reaction (PCR) was used to determine the expression of mature miR-146a (grey bars) and miR-146b (black bars) in the course of differentiation in Caco-2 cells. The numbers represent the days that the cells were collected after reaching 100% confluence.

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Using mRNA isolated from Caco-2 cells at various time points between 0 and 30 days after reaching 100% confluency, we amplified the mature miR-146a and miR-146b by real time PCR using the Taqman probe based miRNA assay. Our data (Fig. 1c) indicate that the expression of mature miR-146a increased dramatically in the course of differentiation, especially in the later days, with a nearly 65-fold increase after 14 days into the differentiation process. The expression of miR-146b, however, did not increase in the course of differentiation. The precursor form of miR-146a and miR-146b showed the opposite trend in expression as well, with the expression of pre-miR-146a increasing in the course of differentiation, while the expression of pre-miR-146b remained steady (Fig. S1). This indicated that miR-146a could be involved in post-transcriptional regulation of genes during colonic differentiation.

MicroRNA mediated regulation of MMP16 in spontaneously differentiating Caco-2 cells

In order to determine the possible mRNA targets for miR-146a, TargetScan 5.0 (Friedman et al. 2009) and Probability of Interaction by Target Accessibility (PITA) (Kertesz et al. 2007) were used. Both algorithms predicted two binding sites of miR-146a containing one conserved 8-mer exact seed match at positions 1479–1485 (Fig. 2a) and one poorly conserved 7-mer seed match at position 724–730 on the 3′UTR of MMP16.

image

Figure 2. miR-146a mediated regulation of matrix metalloprotease 16 (MMP16) expression in Caco-2 cells (a) Bioinformatic analysis of the predicted interaction between miR-146a and the 3′UTR of MMP16. (b) Duplex reverse transcription–polymerase chain reaction (RT–PCR) shows a decrease in mRNA expression of MMP16 in the differentiated cells. M, marker; NC, negative control. The numbers represent that days that the cells were collected after reaching 100% confluence. The graph shows a densitometric analysis of the RT–PCR data (c) Western blot shows a decrease in protein expression of MMP16 in differentiated cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed as a loading control. (d) Luciferase reporter gene assay showing a significant decrease in luciferase expression when the pMIR REPORT vector containing the 3′UTR of MMP16 with the miR-146a binding site was transfected in Day 0 (D0) or Day 10 (D10) postconfluent Caco-2 cells (n = 3, ***P < 0.001, Students t-test). Empty vector stands for the empty pmiR REPORT vector. β-galactosidase activity was used for the normalization of data.

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In order to determine whether the expression of MMP16 was altered in the course of differentiation, Caco-2 cells were collected on Days 0, 2, 4, 7, 14, 30 after reaching 100% confluency and processed for mRNA and protein isolation. When analyzed for expression at the mRNA level by duplex RT–PCR, MMP16 was expressed in early days of post confluent growth, after which the expression progressively decreased from Day 14 onwards, with very low expression at Day 30 (Fig. 2b). When analyzed for protein expression by western blot, the MMP16 protein levels were seen to be lost after Days 14 and 30 (Fig. 2c). The dramatic decrease in protein levels of MMP16 on Day 14 and Day 30 coincided with a sharp increase in the expression of miR-146a.

To confirm that MMP16 is a target of miR-146a, we cloned a 489 bp region from the 3′UTR of MMP16 containing the predicted binding site of miR-146a or its mutated sequence into the pmiR REPORT vector (please see Table 1 for the sequences). These vectors were separately transfected into Caco-2 cells that were at Day 0 (undifferentiated) or Day 10 (differentiated) after reaching 100% confluency. A significant decrease in luciferase activity was observed in the Day 10 cells compared with the Day 0 cells transfected with the pmiR reporter vector (**P < 0.001), possibly owing to increased endogenous levels of miR-146a in the differentiated cells (Fig. 2d). We have also observed a reversal of the decreased luciferase activity when the pmiR REPORT vector containing the mutated miR-146a binding sequence was transfected in the Day 0 or Day 10 cells. A considerable decrease in the luciferase signal was observed when the Day 0 cells were transfected with the pmiR reporter vector containing the 3′UTR of MMP16 when compared with the empty vector (EV) transfected cells (Fig. 2d). Since most mRNAs are regulated by multiple miRNAs, the decrease in luciferase activity observed in the undifferentiated cells could be due to the targeting of the 3′UTR of MMP16 by other, as of yet unknown, microRNAs. Taken together, our data suggest that MMP16 is regulated by miRNAs in spontaneously differentiating Caco-2 cells and that miR-146a is a strong candidate for this regulation.

miR-146a ectopic expression in spontaneously differentiating Caco 2 cells decreases MMP16 expression

In order to further verify the regulation of MMP16 by miR-146a, we cloned the miR-146a precursor and its mutated counterpart into the pSUPER expression vector and transfected it in Caco-2 cells immediately after reaching 100% confluency (Day 0). At this stage, the MMP16 expression was found to be high (please see Fig. 2b,c). When the Caco-2 cells ectopically expressing miR-146a were transfected with the pMIR REPORT vector containing the 3′UTR of MMP16, a significant decrease in the luciferase activity was observed (***P < 0.0001) (Fig. 3a). No significant change in luciferase activity was observed in Caco-2 cells expressing the mutated miR-146a, indicating that it was necessary to overexpress the intact miR-146a in order to regulate MMP16. Additionally, when the cells were transfected with the empty pmiR REPORT vector, or the reporter vector cloned with the mutated miR-146a seed sequence in cells ectopically expressing miR-146a or its mutated version, no change in the luciferase activity was observed, further emphasizing the necessity for intact binding between miR-146a and the 3′UTR of MMP16 for successful regulation.

image

Figure 3. miR-146a forced expression in undifferentiated Caco-2 cells decreases matrix metalloprotease 16 (MMP16) expression (a) Undifferentiated confluent Caco-2 cells ectopically expressing miR-146a when transfected with a reporter vector containing the 3′UTR of MMP16 showed a significant decrease in luciferase activity (***P < 0.0001, n = 3, Students t-test). This indicates that miR-146a may bind to the 3′UTR and regulate the expression of MMP16. β-galactosidase activity was used for the normalization of data. (b) Transfection of undifferentiated confluent Caco-2 cells with a miR-146a expression vector resulted in a threefold increase in expression (*P < 0.01, Student’s t-test) (c) miR-146a ectopic expression but not its mutated counterpart was associated with a parallel decrease in the mRNA levels of MMP16 in undifferentiated confluent Caco-2 cells. No such changes were observed in the empty pSUPER vector (EV), mock and untransfected (UT) control cells. This decrease in MMP16 mRNA level was similar to its expression in Day 10 differentiated cells (D10, lane 7). M, marker; NC, negative control. (d) Western blot indicates that overexpression of miR-146a, but not the mutated counterpart or the empty pSUPER vector resulted in a decrease in the protein levels of MMP16 in confluent undifferentiated Caco-2 cells. UT, untransfected cells; Mock, mock transfected cells. β-actin was probed as a loading control. (e) miR-146a forced expression does not lead to differentiation of Caco-2 cells as indicated by a lack of increase in expression of sucrase isomaltase (SI), a marker of differentiation.

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We next wanted to determine whether the forced expression of miR-146a affected the expression of MMP16 in Caco-2 cells. We first confirmed the overexpression of miR-146a and its mutated counterpart in Day 0 postconfluent Caco-2 cells by measuring the mature miR-146a levels using qRT–PCR. As can be seen from Fig. 3b, miR-146a could be expressed around threefold more in Caco-2 cells, compared with the control cells. We did not observe any amplification of the mutated miR-146a with the Taqman assay. This was most likely because the probe could not bind to the mutated sequence. Instead, the expression of the mutated miR-146a was confirmed by semiquantitative RT–PCR by using primers that could amplify the pre-miR-146a (Fig. S2). When the MMP16 mRNA expression was analyzed in these cells, the forced expression of miR-146a was observed to accompany a dramatic decrease in the expression of MMP16 mRNA (Fig. 3c). However, no such decrease in MMP16 expression was observed when the Caco-2 cells were transfected with the mutated miR-146a expression vector, further confirming the specificity of the regulation. No change in expression of MMP16 was observed in the pSUPER empty vector transfected, mock transfected or untransfected control cells.

Consistent with the mRNA data, a loss of MMP16 protein was seen in the miR-146a overexpressing cells, but not in the control cells expressing mutated miR-146a or cells transfected with the empty pSUPER vector (Fig. 3d). Taken together, we established that miR-146a could regulate the expression of MMP16 during spontaneous differentiation of Caco-2 cells.

Since neither miR-146a, nor MMP16 have any known function in the process of colonocyte differentiation, we believe that the putative regulation of MMP16 by miR-146a was a consequence of the process of differentiation. To further confirm this, we determined the expression of the differentiation marker Sucrase isomaltase (SI) in Caco-2 cells ectopically expressing miR-146a, its empty vector or the mutated counterpart (Fig. 3e). We have not observed any increased expression of SI in the miR-146a expressing or control cells, indicating that forced expression of miR-146a does not induce differentiation in the Caco-2 cells. An increase in SI expression can be seen in the Day 10 differentiated cells as expected.

miR-146a expression decreases gelatinase activity in Caco-2 conditioned medium

Matrix metalloprotease 16 (MT3-MMP) is a membrane type metalloprotease that functions in activating proMMP-2 (gelatinase A) into its active form as the zymogen is excreted out of the cell (Nakada et al. 1999). Therefore, a zymogram depicting the gelatinase activity of activated MMP2 would be an indirect mechanism of determining the activity of MMP16. We therefore transfected the miR-146a expression vector or its mutated counterpart in undifferentiated confluent (Day 0) Caco-2 cells for 48 h and collected the conditioned medium. The medium was then concentrated and prepared for zymography. Our data (Fig. 4) indicate that the cells ectopically expressing miR-146a exhibited lower gelatinase activity (lane 3) with respect to the cells transfected with the mutated plasmid, the mock transfected or the untransfected counterparts. Additionally, we have also shown that the gelatinase activity was considerably low on the Day 10 differentiated cells, which corroborates with a loss of MMP16 owing to an increase in miR-146a levels.

image

Figure 4.  Gelatinase activity in miR-146a overexpressing Caco-2 cells Total protein (1 mg) from conditioned media obtained from miR-146a overexpressing, mutated vector overexpressing, mock transfected or untransfected undifferentiated confluent Caco-2 cells were separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel containing gelatin under non-reducing conditions. Decreased gelatinase activity, most likely resulting from a loss of activation of MMP2 by matrix metalloprotease 16 (MMP16) owing to miR-146a overexpression resulted in less digestion of the gelatin when compared with the control cells. A comparable low gelatinase activity owing to loss of MMP16 can also be seen in the zymogram of Day 10 differentiated Caco-2 cells where miR-146a is expressed endogenously (lane 6).

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miR-146a expression decreases the invasion of HT-29 cells through Matrigel

In order to validate that the regulation of MMP16 by miR-146a was not restricted to the Caco-2 cells, we expressed miR-146a in another colon cancer cell line, HT-29. This cell line can also undergo differentiation with glucose deprivation; however, unlike Caco-2 cells which spontaneously differentiate into enterocyte-like cells, HT-29 cells generally differentiate into mucus producing goblet cells (Simon-Assmann et al. 2007). Possibly owing to this difference, we did not observe any increase in the expression of miR-146a or a decrease in the expression of MMP16 in the course of differentiation of these cells (data not shown). However, in undifferentiated HT-29 cells ectopically expressing miR-146a (Fig. 5a), a decrease in the mRNA expression of MMP16 was observed (Fig. 5b) indicating the likelihood that miR-146a could regulate the expression of MMP16 in this cell line as well. Moreover, since miR-146a expression has been associated with decreased invasiveness in a number of epithelial cancers (Bhaumik et al. 2008; Xia et al. 2009; Ali et al. 2010; Kogo et al. 2011), we determined the invasiveness of the miR-146a expressing HT-29 cells through Matrigel, using a Transwell assay (Fig. 5c, Fig. S3). The data indicate that a significant (**P < 0.001) decrease in the invasion of the HT-29 cells could be observed when the cells were expressing miR-146a. No such decrease in the invasiveness was observed in the control cells, which include cells transfected with a construct containing mutated miR-146a or the empty vector as well as the untransfected parental cells.

image

Figure 5. miR-146a expression decreases invasion through Matrigel in HT-29 cells. (a) Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) showing that miR-146a overexpression resulted in an increase in the mature miR-146a levels in HT-29 cells. (b) Semi-quantitative RT–PCR showing that miR-146a expression resulted in a decrease in matrix metalloprotease 16 expression in HT-29 cells. M, marker; NC, negative control. (c) miR-146a expression in HT-29 cells significantly decreased the invasion of cells through Matrigel in a Boyden chamber type Transwell invasion assay.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Differentiation of epithelial cells is accompanied by numerous structural and cellular changes that characterize a functional specialization of cells. During neoplastic transformation, cells often become de-differentiated resulting in increased motility, migration and resistance to apoptosis (Polyak & Weinberg 2009). Thus, regulation of the differentiation program and molecules involved in differentiation and de-differentiation is critical. The Caco-2 cell line, which was isolated from a well differentiated tumor, is able to undergo spontaneous differentiation into enterocyte type cells upon reaching in vitro confluency and is frequently used as a model for colonocyte differentiation (Simon-Assmann et al. 2007). In this study we have used Caco-2 cells as a model of re-differentiation from a de-differentiated transformed state. We have elucidated how a microRNA (miR-146a) expression dramatically changes during the process of re-differentiation and regulates a matrix metalloprotease (MMP16) that is frequently overexpressed in many epithelial tumors and is involved in the degradation of the extracellular matrix (Hotary et al. 2006).

MicroRNA mediated posttranscriptional regulation of genes contribute significantly to modulating cellular behavior and function (Erson & Petty 2008). miR-146 is one such candidate that is predicted to regulate a number of different genes (Table S1) and was shown to be highly expressed in differentiated Caco-2 cells by microarray analysis (Hino et al. 2008). Using the Caco-2 cell line, we observed a substantial increase in the expression of miR-146a, but not miR-146b in the course of differentiation. An in silico analysis using available bioinformatic resources revealed that miR-146a/b are candidate microRNAs that can bind to the 3′UTR of MMP16. Concurrently, we observed a decrease in the mRNA and protein expression of MMP16 in the course of differentiation of Caco-2 cells. Further confirmation for the regulation of MMP16 by miR-146a was observed by reporter gene assays using the pmiR REPORT vector containing the 3′UTR of MMP16 with the miR-146a binding site. A significant decrease in luciferase activity was observed in the differentiated postconfluent Caco-2 cells when compared with the undifferentiated confluent cells, indicating that the differentiated cells, expressing more miR-146a, showed a greater level of regulation of MMP16. Moreover, undifferentiated Caco-2 cells ectopically expressing miR-146a also displayed similar regulation of MMP16, as seen with decreased luciferase activity in reporter gene assays as well as decreased mRNA and protein expression of MMP16.

Based on histological classifications, poorly differentiated colorectal neoplasms are usually more metastatic than their well-differentiated counterparts (Hermanek 1995). Invasiveness of cancer cells is, to a large extent, controlled by the extracellular matrix degrading proteins such as MMPs (Kessenbrock et al. 2010) and MMP16 is already known to be overexpressed in many cancers, which by definition are cells that have dedifferentiated (Hotary et al. 2006). MMP16 is a membrane type metalloprotease that can activate the zymogen of MMP2 as the latter is extruded out of the cell. MMP2 can cleave collagen IV of the basement membrane and is implicated in cancer metastasis (Rowe & Weiss 2008). It is therefore not surprising that high MMP16 expression has been associated with increasing invasiveness in gastric cancer (Lowy et al. 2006), hepatocellular carcinoma (Arai et al. 2007), prostate cancer (Daja et al. 2003) as well as melanoma cells (Ohnishi et al. 2001).

In order to determine whether the regulation of MMP16 by miR-146a was functionally relevant, we carried out gelatin zymography, which determines the gelatin degrading enzymes (most commonly MMP2) in the conditioned medium of cells. As MMP2 is activated by MMP16, this would be an indirect indication of the function of MMP16. In undifferentiated confluent cells ectopically expressing miR-146a, as well as in postconfluent differentiated (Day 10) cells that naturally express more miR-146a, a decrease in gelatinase activity was observed. This is most likely due to a loss of MMP16 in these cells, and thereby a lower activation of the zymogen of MMP2 and its gelatinase activity.

The regulation of MMP16 by miR-146a was not restricted solely to Caco-2 cells, since forced expression of this miRNA in HT-29 cells resulted in a lower expression of MMP-16. Moreover, HT-29 cells ectopically expressing miR-146a showed significantly reduced invasiveness through Matrigel. The expression of miR-146a was found to be missing in hormone-refractory prostate cancer and ectopic expression of miR-146a in PC3 cells resulted in a reduction in metastasis to bone marrow, proliferation and invasion by suppression of its target ROCK1, a kinase affecting focal adhesion formation and cellular motility (Lin et al. 2008). Additionally, miR-146b, which has a seed sequence very similar to miR-146a, was found to decrease invasion and migration in brain glioma cells by targeting matrix metalloproteases (Xia et al. 2009). Thus, it is possible that the reduced invasion observed in HT-29 cells expressing miR-146a could stem, at least in part, from the suppression of its target MMP16.

In conclusion, using Caco-2 cells as a model of epithelial differentiation, we have shown that the expression of miR-146a, but not miR-146b, increases dramatically in the course of differentiation. miR-146a also binds to the 3′UTR of its predicted target MMP16, a critical matrix metalloprotease, and suppresses its expression and activity in the differentiated cells. These data highlight the importance of miRNA mediated regulation of genes in the process of epithelial differentiation and could account in part for lower motility of well-differentiated cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

The authors would like to thank Dr Cetin Kocaefe for helpful comments and suggestions and sharing resources. This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK, Project no. 110S165 to S.B.) and the Turkish Academy of Sciences Young Investigators Award (TÜBA-GEBİP to S.B.). E.A. is supported by ÖYP (Osmaniye Korkut Ata University).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
  • Ali, S., Almhanna, K., Chen, W., Philip, P. A. & Sarkar, F. H. 2010. Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer. Am. J. Transl. Res. 3, 2847.
  • Arai, I., Nagano, H., Kondo, M., Yamamoto, H., Hiraoka, N., Sugita, Y., Ota, H., Yoshioka, S., Nakamura, M., Wada, H., Damdinsuren, B., Kato, H., Marubashi, S., Miyamoto, A., Takeda, Y., Dono, K., Umeshita, K., Nakamori, S., Wakasa, K., Sakon, M. & Monden, M. 2007. Overexpression of MT3-MMP in hepatocellular carcinoma correlates with capsular invasion. Hepatogastroenterology 54, 167171.
  • Beaurepaire, C., Smyth, D. & Mckay, D. M. 2009. Interferon-gamma regulation of intestinal epithelial permeability. J. Interferon Cytokine Res. 29, 133144.
  • Bhaumik, D., Scott, G. K., Schokrpur, S., Patil, C. K., Campisi, J. & Benz, C. C. 2008. Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells. Oncogene 27, 56435647.
  • Chassin, C., Kocur, M., Pott, J., Duerr, C. U., Gütle, D., Lotz, M. & Hornef, M. W. 2010. miR-146a mediates protective innate immune tolerance in the neonate intestine. Cell Host Microbe 8, 358368.
  • Cimen, I., Tuncay, S. & Banerjee, S. 2009. 15-Lipoxygenase-1 expression suppresses the invasive properties of colorectal carcinoma cell lines HCT-116 and HT-29. Cancer Sci. 100, 22832291.
  • Cui, M., Zhao, Y., Hance, K. W., Shao, A., Wood, R. J. & Fleet, J. C. 2009. Effects of MAPK signaling on 1,25-dihydroxyvitamin D-mediated CYP24 gene expression in the enterocyte-like cell line, Caco-2. J. Cell Physiol. 219, 132142.
  • Daja, M. M., Niu, X., Zhao, Z., Brown, J. M. & Russell, P. J. 2003. Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines. Prostate Cancer Prostatic Dis. 6, 1526.
  • Dalmasso, G., Nguyen, H. T., Yan, Y., Laroui, H., Srinivasan, S., Sitaraman, S.V. & Merlin, D. 2010. MicroRNAs determine human intestinal epithelial cell fate. Differentiation 80, 147154.
  • Erson, A. E. & Petty, E. M. 2008. MicroRNAs in development and disease. Clin. Genet. 74, 296306.
  • Friedman, R., Farh, K., Burge, C. & Bartel, D. 2009. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92105.
  • Hermanek, P. 1995. pTNM and residual tumor classifications: Problems of assessment and prognostic significance. World J. Surg. 19, 184190.
  • Hino, K., Tsuchiya, K., Fukao, T., Kiga, K., Okamoto, R., Kanai, T. & Watanabe, M. 2008. Inducible expression of microRNA-194 is regulated by HNF-1alpha during intestinal epithelial cell differentiation. RNA 14, 14331442.
  • Hotary, K., Li, X. Y., Allen, E., Stevens, S. L. & Weiss, S. J. 2006. A cancer cell metalloprotease triad regulates the basement membrane transmigration program. Genes Dev. 20, 26732686.
  • Humphries, A. & Wright, N. A. 2008. Colonic crypt organization and tumorigenesis. Nat. Rev. Cancer 8, 415424.
  • Kertesz, M., Iovino, N., Unnerstall, U., Gaul, U. & Segal, E. 2007. The role of site accessibility in microRNA target recognition. Nat. Genet. 39, 12781284.
  • Kessenbrock, K., Plaks, V. & Werb, Z. 2010. Matrix metalloproteinases: Regulators of the tumor microenvironment. Cell 141, 5267.
  • Kogo, R., Mimori, K., Tanaka, F., Komune, S. & Mori, M. 2011. Clinical significance of miR-146a in gastric cancer cases. Clin. Cancer Res. 17, 42774284. United States.
  • Lin, S. L., Chiang, A., Chang, D. & Ying, S. Y. 2008. Loss of mir-146a function in hormone-refractory prostate cancer. RNA 14, 417424.
  • Lowy, A. M., Clements, W. M., Bishop, J., Kong, L., Bonney, T., Sisco, K., Aronow, B., Fenoglio-Preiser, C. & Groden, J. 2006. Beta-Catenin/Wnt signaling regulates expression of the membrane type 3 matrix metalloproteinase in gastric cancer. Cancer Res. 66, 47344741.
  • Mckenna, L. B., Schug, J., Vourekas, A., McKenna, J.B., Bramswig, N.C., Friedman, J.R. & Kaestner, K.H. 2010. MicroRNAs control intestinal epithelial differentiation, architecture, and barrier function. Gastroenterology 139, 16541664.
  • Nakada, M., Nakamura, H., Ikeda, E., Fujimoto, N., Yamashita, J., Sato, H., Seiki, M. & Okada, Y. 1999. Expression and tissue localization of membrane-type 1, 2, and 3 matrix metalloproteinases in human astrocytic tumors. Am. J. Pathol. 154, 417428.
  • Nakamura, H., Ueno, H., Yamashita, K., Shimada, T., Yamamoto, E., Noguchi, M., Fujimoto, N., Sato, H., Seiki, M. & Okada, Y. 1999. Enhanced production and activation of progelatinase A mediated by membrane-type 1 matrix metalloproteinase in human papillary thyroid carcinomas. Cancer Res. 59, 467473.
  • Ohnishi, Y., Tajima, S. & Ishibashi, A. 2001. Coordinate expression of membrane type-matrix metalloproteinases-2 and 3 (MT2-MMP and MT3-MMP) and matrix metalloproteinase-2 (MMP-2) in primary and metastatic melanoma cells. Eur. J. Dermatol. 11, 420423.
  • Pinto, M. 1983. Enterocyte like differentiation and polarization of the human colon carcioma cell line Caco-2. Biol. Cell 47, 323330.
  • Polyak, K. & Weinberg, R. A. 2009. Transitions between epithelial and mesenchymal states: Acquisition of malignant and stem cell traits. Nat. Rev. Cancer 9, 265273.
  • Rowe, R. G. & Weiss, S. J. 2008. Breaching the basement membrane: Who, when and how? Trends Cell Biol. 18, 560574.
  • Simon-Assmann, P., Turck, N., Sidhoum-Jenny, M., Gradwohl, G. & Kedinger, M. 2007. In vitro models of intestinal epithelial cell differentiation. Cell Biol. Toxicol. 23, 241256.
  • Taganov, K., Boldin, M., Chang, K. & Baltimore, D. 2006. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl Acad. Sci. USA 103, 1248112486.
  • Xia, H., Qi, Y., Ng, S. S., Chen, X., Li, D., Chen, S., Ge, R., Jiang, S., Li, G., Chen, Y., He, M. L., Kung, H. F., Lai, L. & Lin, M. C. 2009. microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs. Brain Res. 1269, 158165.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Fig. S1. Expression of the precursor forms of miR-146a and miR-146b in the course of differentiation.

Fig. S2. pre miR-146a expression in Caco-2 cells transfected with a miR-146a overexpressing plasmid.

Fig. S3. miR-146a ectopic expression reduces invasion of HT-29 cells through Matrigel in a Transwell assay.

Table S1. Predicted targets of miR-146a in Homo sapiens by PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_dyn_data.html) and TargetScan (http://www.targetscan.org/).

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DGD_1324_sm_FigsS1-S3-TableS1.pdf1104KSupporting info item

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