The human non-small cell lung cancer cell line NCI-H460 was obtained from the American Type Culture Collection (LGC Promochem, Molsheim, France). Cells were grown in Roswell Park Institute Medium 1640 medium (Invitrogen, Cergy-Pontoise, France) supplemented with 2 mM L-glutamine, 25 mM sodium bicarbonate, 2 mM glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazi-neethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 100 μg/ml streptomycin, 100 U/ml penicillin and 10% endotoxin-free heat inactivated foetal calf serum (FCS, ATGC Biotechnologie, Noisy le Grand, France). The human fibroblast cell line CCD19-Lu (LGC Promochem) derived from adult normal lung tissue was grown in MEM/Earle's/Glutamax medium supplemented with non-essential amino acids, 25 mM sodium bicarbonate, 1 mM sodium pyru-vate, 100 μg/ml streptomycin, 100 U/ml penicillin and 10% FCS. All cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.

Two different sequences of pre-miRNA targeting the human TFPI-2 transcripts [22] were designed using Invitrogen's RNAi design algorithm and BLAST to avoid off-target gene silencing. Pre-miRNA-1 sequence targets the nucleotides 858 to 878 of the 3′UTR region. The sequence of pre-miRNA-2 was designed according to the TFPI-2 siRNA we used in a previous study [23] and targets the nucleotides 581 to 601 of the TFPI-2 K3 domain.

The pcDNA 6.2-GW/EmGFP-miR plasmid (Invitrogen) with blasticidin resistance gene and expressing the EnGFP (emerald green fluorescent protein) was used for the synthesis of pre-miRNA. This pre-miRNA is based on the murine miR-155 sequence [24] and the stem loop structure was optimized by the supplier to obtain a high knockdown rate. Pre-miRNA is then processed by endogenous Dicer enzyme into a 22 nucleotide mature miRNA using cellular machinery. The sequences used were: miRNA-1, top strand 5′-tgctgatgatttgtttcctcatgctggttttggccactgactgaccagcatgaaaacaaatcat-3′; bottom strand 5′-cctgatgatttgttttcatgctggtcagtcagtggccaaaaccagcatgaggaaacaaatcatc-3′; miRNA-2, top strand 5′-tgctgaataatagcgagtcacattgggttttggccactgactgacccaatgtgtcgctattatt-3′; bottom strand 5′-cct-gaataatagcgacacattgggtcagtcagtggccaaaacccaatgtgactcgctattattc-3′. Sequences were annealed and ligated using 1 U/μl T4 DNA ligase (Invitrogen). A control plasmid vector expressing miRNA not directed against a known mammalian gene was used as a negative control (miRNA-Neg). The recombinant plasmids were recovered by PCR using the forward sequencing primer (5′-ggcatggacgagctgtacaa-3′) and the reverse sequencing primer (5′-ctctagatcaaccactttgt-3′) (Invitrogen). The PCR product sequences which contained the miRNA inserts were sequenced (Perkin Elmer AbiPrism™ 377 DNA sequencer).

Confluent NCI-H460 cells were washed with Ca²⁺ and Mg²⁺-free Hank's balanced solution and harvested using 0.05% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA). Cell viability was determined by Trypan blue dye exclusion test and ranged between 90% and 95%. For miRNA transfection, 10⁵ cells were seeded in 24-well plates for 24 hrs in RPMI-1640 10% FCS without antibiotics. Purified pcDNA 6.2GW/EmGFP-miR expression vectors (0.8 μg) containing either the TFPI-2 pre-miRNA insert (pcDNA-TFPI-2 pre-miRNA-1, pcDNA-TFPI-2 pre-miRNA-2) or a negative-control mismatch sequence (pcDNA-TFPI-2 pre-miRNA-Neg) were transfected into 75–80% confluency NCI-H460 cells with 2 μl of Lipofectamine 2000 reagent (Invitrogen). Six hours after transfection, the medium was replaced by fresh complete medium containing 10% FCS. After 24 hrs, cells were plated in 6-well plates with selection medium, containing 6 μg/ml blasticidin. Transfection efficacy was checked by fluorescence microscopy 48 hrs after transfec-tion by measuring EmGFP expression. Successfully transfected cell clones were then obtained by 3 weeks culture in the selection medium and TFPI-2 knockdown was assessed by reverse transcriptase real-time PCR and Western blotting.

Total mRNA was extracted from 10⁶ cells using the Dynabeads mRNA Direct Kit (Invitrogen) according to the manufacturer's instructions. Total mRNA was then reverse transcribed for 1 hr at 42°C in incubation buffer containing 250 μM of each deoxynucleotide triphosphate, 5 μM oligo (dT)₂₀, 24 units RNase inhibitor, and 20 units of avian myeloblastosis virus reverse transcriptase (Roche Diagnostics, Meylan, France).

The amounts of TFPI-2, MMP-1, -2, -3, -7, -9, -13, -14 and EMMPRIN transcripts within cells were assessed by real-time PCR using the icycler iQ detection system (Bio-Rad, Ivry sur Seine, France). PCR was performed in a total reaction volume of 25 μl containing cDNA obtained from mRNA of 2 × 10⁴ cells, 2-fold dilution of Platinum Quantitative PCR SuperMix-UDG (Invitrogen), 0.32 μM of each primer (Eurogentech, Angers, France, Table 1) and a 50,000-fold dilution of Sybr Green solution (Roche Diagnostics). To study gene expression, PCR were initiated by decontamination (50°C for 2 min.) and denaturation steps (95°C for 2 min.), followed by n cycles (Table 1) at 95°C for 20 sec. and at hybridization T°C for 40 sec. The melting curve was analysed for each sample to check PCR specificity. For each gene studied, specific standard curves were established using decreasing amounts of purified PCR products (from 10⁷ to 5 × 10¹ copies). mRNA copies of the gene of interest were then normalized to 10⁶ copies of β-actin mRNA used as gene control.

Transfected cells (miRNA-1 and -2, miRNA-Neg clones and parental NCI-H460) were grown in complete medium to 70–80% confluency in 6-well plates. Cells were eliminated by trypsinization and proteins from the ECM were solubilized in TNC buffer (50 mM Tris-HCl pH 7.5, 0.15 M NaCl, 10 mM CaCl₂ and 0.05% Brij 35) and then centrifuged at 15,000 × g for 5 min. Total protein concentrations of the supernatants were measured using the Lowry method (Total Protein Kit, Sigma Aldrich, Saint Quentin Fallavier, France). Proteins (3 μg) were separated on 12% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were then saturated for 2 hrs at room temperature in TNT buffer (10 mM Tris-HCl and 150 mM NaCl pH 7.4, 0.1% Tween-20) with 5% non-fat dried milk, incubated overnight at 4°C with polyclonal rabbit anti-TFPI-2 antibody (generous gift of W. Kisiel) diluted 1/3000 in TNT buffer with 5% non-fat milk and for 1h with peroxidase-labelled anti-rabbit IgG (Sigma Aldrich) after washing with TNT buffer. Following exposure for 1 min. to the Chemiluminescence Reagent Plus (Perkin Elmer Biosystems, Courtaboeuf, France), membranes were drained, wrapped in a plastic bag and exposed to autoradiography film (Sigma Aldrich) for 10 min. in the dark.

Pathogen-free male BALB/c nude mice 4 weeks old (Charles River laboratories, Lyon, France) were acclimatized for 2 weeks before starting the study in a sterile environment. All animals were handled and cared in accordance with the national and institutional guidelines. Protocols were conducted under the supervision of an authorized investigator with the approval of the institutional ethic committee where experiments are performed (CIPA, TAAM-UPS44 Orléans, France). Mice were maintained in sterilized filter-stopped cages throughout the experimentation. They were examined daily and monitored for signs of distress, decreased physical activity and weight.

Before implantation, confluent miRNA-1 and -2 and miRNA-Neg NCI-H460 cells were harvested using 0.05% trypsin and 0.02% EDTA and washed twice in FCS-free medium. Cells were then resuspended in RPMI-1640 containing 10 mM EDTA (Sigma Aldrich) added immediately before implantation. Trypan Blue dye exclusion test was used to assess cell viability >95% for implantation.

The intrabronchial tumoral cell implantation procedure was derived from that previously described [25]. Mice (nine animals for each miRNA clones) were anesthetized and the surgery area prepared with a skin disinfection using betadine swabs. Immediately before transplantation, a ^99mTc-labelled tin colloid, used as tracer, was added to cell suspension containing 10 mM EDTA. A 0.5 cm ventral incision was made over the region of the trachea superior to expose the trachea that was punctured using a 23-gauge needle. Cell inoculum (7.5 × 10⁵ tumour cells in 25 μl) was aspirated in a 1.9 Fr × 50 cm blunt-ended catheter (Beckton Dickinson, Le Pont de Claix, France) that was inserted and advanced preferentially into the right main bronchus. Position of the catheter was monitored using high resolution radiological imaging (MX-20, Faxitron X-ray Corporation, Wheeling, IL, USA). Tumour cells were slowly injected into the lung and the scintigraphic assessment of cell deposition into lung was performed (Gamma Imager, Biospace Mesures, Paris, France). The catheter was removed, the incision closed and the animals were placed on a heating pad (37°C) until fully awake. Animal reactions were observed to ensure recovery from the anaesthesia. To document tumour location and measurement, computed tomography was performed on anesthetized animals using a small animal imager (eXplore Locus, General Electric Healthcare, Velizy, France). Width (W, in axial), height (H, in mid sagittal) and length (L, in mid sagittal) were measured and the volume was calculated using the ellipsoid formula 4/3π (W/2 ×H/2 ×L/2).

Cell migration was assessed using a model based on the Boyden chamber (8 μm pore size, BD Biosciences). Briefly, transfected cells were harvested by trypsinisation, then washed with phosphate-buffered saline (PBS) and 2 × 10⁵ cells were suspended in 400 μl of serum-free medium and seeded on the upper chamber of the inserts. The lower chamber of a 24-well cell culture plate was filled with 600 μl of culture medium containing 10% FCS used as chemoattractant. Plates were incubated for 48 hrs at 37°C in a humidified atmosphere containing 5% CO₂. The cells remaining in the insert were removed by aspiration and wiping with cotton swabs. Migrated cells on the lower surface of the filter and adhering to the plate were detached by 0.05% trypsin-0.02% EDTA and counted in a Malassez chamber. The results were expressed as the percentage of migrating cells ± S.E.M.

To study the cell invasion through the basement membrane components, cell culture inserts were coated with a thin layer of 0.8 mg/ml Matrigel™ (BD Biosciences) according to the manufacturer's instructions.

Tumour cells (1.25 ± 10⁴) were seeded in 24-well plates and cultured in 700 μl complete medium containing 10% FCS. After 24, 48, 72 and 96 hrs of culture, 140 μl of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium, inner salt) and an electron coupling agent, phenazine ethosulphate (CellTiter 96® AQ_ueousOne Solution, Promega, Charbonnières les Bains, France) were added. Cells were incubated for 1 hr at 37°C in a humidified atmosphere containing 5% CO₂ and absorbance was then measured on an ELISA plate reader (Thermo_max Molecular Devices, St Grégoire, France) at a wavelength of 490 nm.

Adhesion of stably pre-miRNA-transfected NCI-H460 cells to laminin, vitronectin, fibronectin and collagen I and IV was studied using the ‘CytoMatrix SCREEN Kit’ (AbCys, Paris, France) according to the manufacturer's instructions. Cells were serum-starved overnight, detached with 0.05% trypsin-0.02% EDTA and allowed to express novel integrins in the shaking incubator at 37°C in 5% CO2 for 2 hrs in complete medium containing 1% bovine serum albumin (BSA). After counting cells in the presence of Trypan blue using a Malassez chamber, 5 × 10⁴ viable cells in 100 μl complete medium with 1% BSA were plated in triplicate on ECM protein-coated wells, or on wells coated with BSA used as control, for 2 hrs at 37°C in a humidified CO2 atmosphere. Cells were then rinsed three times with PBS containing Ca²⁺ and Mg²⁺ and adhering cells were fixed and stained in 100 μl of 0.2% crystal violet in 10% ethanol over 5 min. of gentle shaking at room temperature. Relative attachment of cells was determined using absorbance readings at 570 nm in a microplate reader (Thermo_max Molecular Devices). Readings measured with BSA for each cell type were subtracted from ECM protein readings.

Identification of cell surface integrins was performed by using the fluorimetric Alpha/Beta Integrin-Mediated Cell Adhesion Assay Combo Kit (Chemicon Millipore, Saint Quentin en Yvelines, France). Briefly, 3 × 10⁶ tumour cells (miRNA-Neg, miRNA-1b or miRNA-2b NCI-H460 clones) were seeded in medium complemented with 1% BSA and incubated overnight at 37°C in 5% CO₂. Cells were then detached with 0.05% to 0.02% EDTA and washed twice in Hank's Buffered Salt Solution (HBSS) (without Ca²⁺ and Mg²⁺). After centrifugation at 800 × g, the pellet was resuspended in 2 ml medium supplemented with 1% BSA and the cells were incubated rocking for 2 hrs at 37°C in 5% CO₂. The cells were then spun down and the pellet was resuspended in 2 ml Assay Buffer. 100 μl of the cell suspension in Assay Buffer were distributed in a microtitre plate coated with anti-human Integrin mouse monoclonal antibodies and incubated for 2 hrs at 37°C in 5% CO₂. The unbound cells were washed away and adhering cells were lysed and detected with the patental CyQuant GR dye (Molecular Probes, Invitrogen, Carlsbad, CA, USA), which binds to nucleic acids, by reading fluorescence with a Wallac 1420 Victor² apparatus (excitation: 485 nm; emission: 535 nm). Results are expressed in relative fluorescence units.

The measurement of 10 signal transduction pathways in the tumour cell clones was performed with the Cignal Finder™ Cancer 10-Pathway Reporter Array (SA Biosciences, TEBU, Le Perry en Yvelines, France). Briefly, 5 × 10⁴ NCI-H460 miRNA-Neg, miRNA-1b or miRNA-2b cells were reverse-transfected with SureFECT reagent directly in a 96-well plate containing a mixture of a pathway-focused transcription factor-responsive. Each of the 10-pathway reporter assays contains an inducible transcription factor responsive firefly luciferase reporter and constitutively expressing Renilla construct (20:1). The cells were incubated for 16 hrs in the trans-fection medium and then cultured 24 hrs in complete medium to allow the transcription factor activity on the reporters. The reporter activity is measured using the Dual Glo® Luciferase Assay kit from Promega with a Wallac 1420 Victor² apparatus (10 sec. reading). Results are expressed in Relative Luciferase Activities (ratio firefly/Renilla).

Tumour cells (miRNA-Neg, miRNA-1b or miRNA-2b NCI-H460 clones) were grown in complete medium to confluency. Cells were then washed twice with HBSS medium without Ca²⁺ and Mg²⁺ and cultured with serum-free medium supplemented with 1% Nutridoma® (Roche Diagnostics) for 24 hrs at 37°C. The conditioned medium was collected, centrifuged at 1600 × g for 10 min. and concentrated 10-fold (Amicon concentrator, Millipore, Saint Quentin en Yvelines, France). Fibroblast cells seeded overnight in 6-well plates (10⁶ cells) in complete medium with 10% FCS were washed twice with PBS, and fresh concentrated conditioned medium equivalent to 10⁷ tumour cells (ratio 10:1) was then applied for 24 hrs. A control condition was assayed by using non-conditioned medium complemented with 1% Nutridoma® and concentrated 10-fold. Twenty-four hours later, fibroblast cells were lysed, total mRNA was extracted and the amounts of MMP-1, -2, -3, -7, -9, -13, -14 and EMMPRIN transcripts assessed by reverse transcription and real-time PCR. MMP-1, -3 and -7 protein expression was evaluated by immunofluorescence assay.

Tumour cells or fibroblasts were seeded in 8-well chamber slides (LabTek, Dominique Dutscher, Issy les Moulineaux, France) 24 hrs prior to immuno-fluorescence staining in complete medium with 10% FCS or with fresh concentrated conditioned medium from tumour cells. Cells were then washed twice with PBS and fixed in 4% paraformaldehyde solution for 10 min. at room temperature. After two washings in PBS, slides were blocked with a fresh saturation solution (PBS/BSA 2%) for 1 hr and then incubated at room temperature with primary antibodies, i.e. polyclonal rabbit anti-TFPI-2 antibody (1/3 000), rabbit monoclonal anti-MMP-1 (RB-9225-P, 1/100) (Microm Microtech France, Francheville, France), anti-MMP-3 (MS-810-P, 1/100) or mouse monoclonal anti-MMP-7 (MS-813-P1, 1/200) (Microm Microtech France) for 1 hr. Cells were washed three times, followed by 1 hr incubation with AlexaFluor 488-labelled anti-rabbit IgG or antimouse IgG secondary antibodies at 1/1 000 (Molecular Probes, Invitrogen) at room temperature in the dark. For the negative control, cells were incubated with isotype-matched control antibody or secondary antibody only. Finally, four washings were performed with PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and cells were viewed under a fluorescence microscope (Leica Microsystems, Nanterre, France) using Nikon Lucia G v.5.0 software.

Data were expressed as means ± S.E.M., statistical analysis was carried out using Student's t-test (two tailed) and P < 0.05 indicates statistical significance.

NCI-H460 cells from a human non-small cell lung cancer were stably transfected with two recombinant plasmids encoding pre-miRNA, i.e. pre-miRNA-1 and pre-miRNA-2, targeting respectively a region of the 3′UTR and the K3 domain of the human TFPI-2 transcript, or with non-silencing miRNA showing no known homology to mammalian genes and used as negative control (miRNA-Neg). First, and in order to identify successful construction of recombinant plasmids, PCR assays were performed and the PCR product sequences containing those of the pre-miRNA inserts were verified by DNA sequencing. According to the computer analysis, these inserted nucleotides should specifically bind to homologous sites of TFPI-2 mRNA, and thus knockdown TFPI-2 expression in the NCI-H460 cells. Efficacy of transfection of NCI-H460 cells with the recombinant plasmids was evaluated with EmGFP fluorescence after cell culture for 3 weeks in medium with 6 μg/ml blasticidin in order to isolate stably transfected clones. For each miRNA, two cell clones (a and b) with the highest GFP expression rate were retained for the next experiments.

To examine miRNA-induced gene down-regulation, total mRNA and protein from the ECM of NCI-H460, miRNA-Neg, miRNA-1a and -1b, and miRNA-2a and -2b cells were extracted. Although the miRNA-Neg and miRNA-1a clones exhibited no interference effect, significant inhibition of TFPI-2 transcripts was achieved in the miRNA-1b, -2a and -2b cell clones as demonstrated by RT and real-time PCR, with 91%, 73% and 93.5% inhibition, respectively, compared to parental NCI-H460 cells (Fig. 1A). As shown in Fig. 1B, TFPI-2 protein level was strongly decreased in the ECM of miRNA-1b and -2b cells, and to a lesser extent in miRNA-2a cell ECM. The presence of TFPI-2 triplets was demonstrated in the ECM of untransfected NCI-H460 and in miRNA-Neg and miRNA-1a transfected cells. Two clones, miRNA-1b and -2b, with the highest TFPI-2 inhibition were thus selected for further experiments, and immunofluores-cence staining clearly showed the reduced expression of TFPI-2 in both these cell clones (Fig. 1C). In contrast, no variation in TFPI-2 expression was observed in miRNA-Neg cells compared to NCI-H460 parental cells. Therefore, miRNA-Neg was used as negative control. Transfection stability with miRNA was checked every month by measuring EmGFP fluorescence and the inhibition of TFPI-2 transcripts was monitored by real-time RT-PCR. No detectable loss of TFPI-2 inhibition was observed over a year.

miRNA-Neg, miRNA-1b and -2b NCI-H460 cell suspensions containing a ^99mTc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea. The catheter position into the right main bronchus was checked using X-ray imaging and the punctual deposition of cells into the lung assessed by scintigraphy. Only one mouse died immediately after surgery and another one died during experimentation due to tumour progression. When cells were orthotopically implanted with 10 mM EDTA, lung tumours were obtained in 95% of mice after a 4-week period. Tumours were located in the lower and middle parts of the lung as demonstrated by tomography scanning (Fig. 2). Lung tumour volumes were calculated on axial transverse sections. The results showed an increase in tumour volumes with both miRNA-1b (P= 0.05) and -2b (P= 0.02) clones compared to miRNA-Neg cells.

The migratory ability of miRNA-1b and -2b NCI-H460 clones through insert filters of 8 μm pore size was significantly increased 1.7-fold (P < 0.001) and 1.4-fold (P < 0.05) respectively), compared to those of the miRNA-Neg clone. Down-regulation of TFPI-2 was also associated with a significant increase (P > 0.001) in the invasion of miRNA-1b (2.5-fold) and of -2b (1.8-fold) cell clones through Matrigel™, which mimics basement membrane components (Fig. 3A). In both cases, the miRNA-1b cell clone exhibited greater migratory and invasion ability than miRNA-2b.

Growth of miRNA clones and miRNA-Neg cells seeded at low density was then measured every 24 hrs over 96 hrs. As shown in Fig. 3B, cell proliferation was not affected by TFPI-2 down-regulation and no significant difference among miRNA-Neg, miRNA-1b and -2b cell clones was detected (P < 0.05). Moreover, cell viability was similar, as trypan blue staining detected no more than 10% dead cells in all experiments.

To explore further the mechanisms by which TFPI-2 is able to regulate cell invasion, we investigated whether cell attachment to different ECM components was affected when this serine protease inhibitor was down-regulated. The results showed increased adhesion to fibronectin, vitronectin, laminin and collagen IV with both miRNA-1b and -2b clones compared to miRNA-Neg cells (Fig. 3C). Attachment to laminin was particularly marked, with a 2.2-fold and a 2-fold increase for miRNA-1b and -2b when compared to miRNA-Neg cells, respectively, and to collagen IV, with a 1.8-fold and a 1.7-fold for miRNA-1b and -2b, respectively. In contrast, adhesion to collagen I was similar for all cells and no significant differences were observed between miRNA clones and miRNA-Neg cells.

To investigate whether the expression of integrins on cell surface could mediate cell-matrix adhesion and thus invasion process, we determined the integrin profiles of cells using various α and β subunit antibodies. Our data clearly demonstrated that down-regulation of TFPI-2 could promote expression of α₁ subunit (P < 0.01) while β4 and α₅β₁ were significantly decreased (P < 0.01) (Fig. 4). In contrast, the expression of the other α and β integrins remained unchanged in miRNA-Neg, mi-RNA-1b and -2b clones.

Levels of MMP-1, -2, -3, -7, -9, -13, -14 and EMMPRIN transcripts were quantified in miRNA-Neg, miRNA-1b and -2b clones using RT and real-time PCR (Fig. 5A). Varying amounts of MMP transcripts were quantified in all cell clones, with fewer than 10 copies for MMP-9, -13 and -14 to more than 10⁵ copies for MMP-1 and EMMPRIN. A 5.5-fold and an 8.6-fold increase in MMP-1 transcripts were found for miRNA-1b and -2b clones, respectively, when compared to miRNA-Neg cells (P < 0.001). Similar results were observed with MMP-3 since mRNA levels were significantly increased (P < 0.01) in miRNA-1b and -2b cell clones (11.3-fold and 10.4-fold, respectively) compared to the miRNA-Neg clone. Immunocytochemical staining also clearly showed that this increase in MMP-1 and -3 transcript levels was associated with enhanced protein expression (Fig. 5B). No significant differences were found in mRNA expression for MMP-9, -13, -14 and EMM-PRIN in either miRNA-1b or -2b compared to the control clone (Fig. 3A). In contrast, MMP-7 and MMP-2 transcript levels were significantly decreased in miRNA-1b (P < 0.01) and -2b (P < 0.001 and P < 0.01, respectively) clones. As shown in Fig. 5(B), MMP-7 protein expression was slightly decreased in both miRNA clones.

To determine whether various signalling pathways were activated in TFPI-2 down-regulated non-small lung cancer cells, we measured the activities of 10 signal transduction pathways involved in cancer biology using a dual-luciferase reporter assay. As shown in Fig. 6, an increase in relative luciferase activity was observed for extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK) and NF-κB pathways in NCI-H460 clones when compared to negative control. In addition, down-regulation of TFPI-2 in miRNA-1b and -2b clones was also associated with a slight increase in NF-κB pathway (P > 0.05) and a significant increase in the ERK and JNK pathways compared to the miRNA-Neg clone (P < 0.05). Other signalling pathway activities remained unchanged in all NCI-H460 clones compared to negative control.

In order to examine the effects of soluble factors produced by tumour cells expressing (or not) TFPI-2 on metalloproteinases and EMMPRIN synthesis by fibroblast cells, conditioned serum-free media were obtained from miRNA-Neg, miRNA-1b and -2b clones. Fresh conditioned media equivalent to 10⁷ tumour cells were applied to 10⁶ fibroblasts for 24 hrs. Controls were performed by incubating fibroblasts with non-conditioned medium concentrated 10-fold. MMP-1, -2, -3, -7, -9, -13, -14 and EMMPRIN transcript levels in fibroblast cells, quantified by RT and real-time PCR, were not affected when cells were incubated with concentrated conditioned medium from miRNA-Neg compared to concentrated medium used as control (Fig. 7A). In contrast, a significant increase in MMP-1, -3 and -7 transcript levels were observed when normal lung fibroblasts were stimulated by concentrated conditioned medium obtained from miRNA-1b (P < 0.01) and -2b (P < 0.05) clones. The levels of MMP-2, -9, -13, -14 and EMMPRIN transcripts were also increased with both miRNA 1b and miRNA 2b conditioned media but differences were not significant. As shown in Fig. 7B, the increase in mRNA levels demonstrated by immunofluorescence assay was correlated with the protein expression by fibroblasts, particularly with MMP-1 and MMP-3.