The A549 cell line was obtained from ATCC (Rockville, MD, USA). Cells were maintained at 37°C in a 5% CO₂ incubator in RPMI-1640 medium (Sigma-Aldrich, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin.

This study was approved by China Medical University Ethics Committee and was conducted according to regulations of the Helsinki Declaration. All patients provided written informed consent to participate in the present study. Fifty-six patients who were diagnosed with primary non-small cell lung cancer (NSCLC) at the Department of Thoracic Surgery, The First Affiliated Hospital of China Medical University from January 2006 to December 2010 were included in the present study. Twenty-eight patients had stage IIIB NSCLC (locally advanced) and 28 patients had stage IV NSCLC (metastatic) according to the International Association for the Study of Lung Cancer (IASLC) staging committee (13). All diagnoses were based on standard laboratory tests (cytology and histology), and confirmed by computerized tomography of the thorax. None of the patients underwent radiotherapy or chemotherapy prior to surgery.

Total RNA was isolated from the tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. First-strand cDNA was reverse transcribed with 1 μg of total RNA, using Takara Reverse Transcription kit and oligo(dT)15 primers (both from Takara, Dalian, China). The resultant cDNA was then used for quantitative PCR reactions. The fibulin-3 primers were: 5′-GAGCAGCTCCAGGGGACCGCCGCG-3′ (sense) and 5′-TCCCCGACACGCTACCTTCG-3′ (antisense). The housekeeping gene, GAPDH, was used as an internal control for normalization of the results. The GAPDH primers were: 5′-AGAAGGCTGGGGCTCATTTG-3′ (sense) and 5′-AGGGGCCATCCACAGTCTTC-3′ (antisense). Amplification of fibulin-3 and GAPDH was performed with 1 cycle at 95°C for 10 min, and 40 cycles at 95°C for 15 sec and 60°C for 60 sec. Calculation of the relative expression of each transcript was performed using the 2^−ΔΔCt method.

Genomic DNA was extracted from lung cancer specimens using a TissueGen DNA kit (CWbiotech, Beijing, China). Genomic DNA (2 μg) was denatured with 0.2 M NaOH. Then, 10 mM hydroquinone and 3 M sodium-bisulfite (both from Sigma) were added. The solution was incubated at 55°C for 16 h. DNA samples were then purified using a Wizard^® DNA Purification Resin (Promega Corporation, Madison, WI, USA). In this procedure unmethylated (but not methylated) cytosines are converted to uracil, which is then converted to thymidine during subsequent PCR to give sequence differences between methylated and unmethylated DNA. The modified DNA was used as a template both for MSP and USP. The primer sequences for the methylated fibulin-3 gene were: 5′-GTAGTTTTAGGGGATCGTCGC-3′ (sense) and 5′-TCCCCGACACGCTACCTTCG-3′ (antisense); and for the unmethylated allele were: 5′-GAGTAGTTTTAGG GGATTGTTGT-3′ (sense) and 5′-TCCCCAACACACTACCT TCA-3′ (antisense). The PCR products were separated on 2% agarose gel with ethidium bromide and visualized under UV illumination.

The tissues or cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail (Sigma). The extracted protein amounts were quantified using the BCA protein assay kit (CWbiotech). Equivalent amounts of protein (40 μg) were separated using 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Western blotting was performed using the following primary antibodies: fibulin-3 (sc-365224), MMP-2 (sc-8835), MMP-9 (sc-12759), phospho-JNK (sc-12882), p38 (sc-7149), phospho-p38 (sc-166182), ERK 1/2 (sc-135900), phospho-ERK 1/2 (sc-16982) and β-actin (sc-130657) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The binding for each specific antibody was detected with horseradish peroxidase (HRP)-conjugated respective secondary antibodies and ECL solutions (both from Amersham Biosciences, Amersham, UK).

Tissues were fixed with 10% buffered formalin, embedded in paraffin, and decalcified in 10% EDTA solution. Representative blocks were then cut into 4-μm sections, deparaffinized with xylene and rehydrated in a series of ethanol washes (100, 90, 80 and 70%). The sections were then incubated with 3% H₂O₂ and 5% serum to block endogenous peroxidase activity and non-specific binding. For the fibulin-3 protein, sections were incubated with the anti-human fibulin-3 antibody. The sections were then incubated with biotinylated secondary antibodies and visualized by DAB. Counterstaining was carried out with hematoxylin. The sections were dehydrated in alcohol and coverslipped. For the negative controls, PBS replaced the primary antibody.

Levels of fibulin-3 were measured in peripheral blood of the patients with NSCLC and quantified in nanograms/milliliter with the use of the human fibulin-3 enzyme-linked immunosorbent assay (Uscn Life Science Inc., Wuhan, China).

Blood samples (7.5 ml) from the patients with NSCLC were drawn into CellSave^® tubes (Veridex LLC, Warren, NJ, USA), which were maintained at room temperature (RT) and processed within 72 h of collection. CTCs were defined as nucleated EpCAM-positive cells, lacking CD45 but expressing cytoplasmic cytokeratins 8, 18 and 19. All CTC evaluations were performed by qualified and trained personnel.

The plasmid pEGFP-C1-fibulin-3 (gifted by Li Yin, China Medical University) was transfected into A549 cells by Lipofectamine™ 2000 (Invitrogen Life Technologies) according to the manufacturer's protocol. Twenty four hours post-transfection, drug-resistant clones were isolated and expanded. A549 cells transfected with pEGFP-C1 were used as a control. All gene expression studies were conducted using pools of colonies (n=150) to avoid a clonal bias.

Cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized in 1% Triton X-100 for 5 min, and blocked with 5% bovine serum albumin (BSA) in PBS containing 0.5% Triton X-100 for 1 h. Fibulin-3 expression was detected using the anti-fibulin-3 antibody for 1 h at room temperature. Cells were washed with PBS and incubated with the appropriate secondary fluorophore-conjugated antibody for 1 h at RT, washed with PBS, and mounted using SlowFade^® Gold Antifade reagent (Invitrogen Life Technologies). The secondary antibody used for detection of fibulin-3 was Alexa Fluor^® 594 rat anti-mouse IgG (H+L) (Invitrogen Life Technologies).

Cells were plated in 96-well plates (1,500 cells/well). After 24 h, 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) was added to each well. Four hours later, cells were lysed with dimethyl sulfoxide (DMSO), and the absorbance rates were measured at 550–560 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Cells were grown in a 6-well dish. A confluent monolayer of cells was scratched with a 200-μl pipette tip to simulate a wound. Cells were washed twice with PBS and then supplemented with medium and incubated for 4 h at 37°C. Cell migration into the wounded area was monitored microscopically. Images were captured at the interface of the unwounded and wounded areas.

Cells were plated at 2×10⁵ cells/well in 0.5 ml of serum-free medium in 24-well Matrigel-coated Transwell units with polycarbonate filters (8-μm pore size, Costar Inc., Milpitas, CA, USA). The lower chamber was loaded with 600 μl of RPMI-1640 containing 10% FBS. After incubation for 24 h in normal culture conditions, we did not see any cells floating in the upper chamber, indicating that the cells had not undergone apoptosis at this time-point. The top surface of the membrane was gently scrubbed with a cotton bud and fixed in 4% paraformaldehyde (Sigma) and stained with crystal violet. The cells that had invaded through the membrane filters were counted using a light microscope. Ten microscopic fields (x400) were randomly selected to count the cells.

All experiments were performed in triplicate, and the results are expressed as the means ± standard deviation (SD). Kaplan-Meier survival plots were generated and comparisons were made with log-rank statistics. P-values <0.05 were considered to indicate statistically significant results. All the statistical analyses and graphics were performed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

Real-time PCR and western blot assays were performed to detect the levels of fibulin-3 mRNA and protein. As shown in Fig. 1A and B, the levels of fibulin-3 mRNA and protein were low in the low-stage NSCLC (IIIB) tissues and undetectable in the advanced stage (IV) tissues (P<0.05). Immunohistochemistry was then performed on 56 NSCLC tissue samples and the corresponding non-tumor tissue samples. Representative examples of fibulin-3 protein expression in NSCLC tissue samples showed that fibulin-3 protein was weakly expressed in the lung cancer specimens, but was highly expressed in the normal areas of the specimens (Fig. 1C). A correlation was noted between fibulin-3 promoter methylation and downregulation of fibulin-3 mRNA levels in the tumor samples. Methylated tumor lung tissue samples showed loss of fibulin-3 mRNA (Fig. 1D). Taken together, the loss of fibulin-3 gene expression in NSCLC may be correlated with the methylation of the fibulin-3 gene promoter.

A statistically significant difference was observed between circulating fibulin-3 levels in the low stage NSCLC (IIIB) patients and the advanced stage (IV) patients (P<0.05, Fig. 2A). In the present study, we isolated and enumerated the CTCs in patients using the CellSearch^® system (Fig. 2B). Less CTCs were noted in the peripheral blood of patients with low stage NSCLC (IIIB) when compared with the patients with advanced stage NSCLC (IV) (P<0.05, Fig. 2C). The plasma levels of fibulin-3 and the number of CTCs were significantly negatively correlated between stage IIIB and IV NSCLC patients (r=−0.585, P<0.05, Fig. 2D or r=−0.933, P<0.05, Fig. 2E), respectively.

We analyzed the potential relationship between the expression of fibulin-3 and the clinicopathological characteristics of the NSCLC patients. Fibulin-3 expression was not associated with any of the clinicopathological characteristics of the patients with stage IIIB or IV NSCLC (Table I). However, the serum level of fibulin-3 was associated with lymphatic invasion (P<0.05, Table II). To investigate the correlation of serum level of fibulin-3 with patient survival, the survival data from 50 patients with NSCLC (6 missing at follow-up) were assessed. In patients with stage IIIB and IV NSCLC, comparison by the Kaplan-Meier method according to low vs. high fibulin-3 expression showed a significant difference in the 5-year survival rate (P<0.05, Fig. 3A–C). The patients with <5 CTCs showed a higher survival rate when compared with the patients with 5–10 CTCs or with >10 CTCs. A significantly different survival rate was confirmed between the patients with 5–10 CTCs and the patients with >10 CTCs in the Cox model (P<0.05, Fig. 3D).

A549 cells were transfected with the pEGFP-C1-fibulin-3 expression vector, and the levels of fibulin-3 protein and mRNA were measured by western blotting, RT-PCR and immunofluorescence. As shown in Fig. 4A, the results of RT-PCR and western blot analysis confirmed exogenous expression of fibulin-3 in A549 cells after transfection. Immunofluorescence analysis showed the localization of GFP and fibulin-3 in the fibulin-3-expressing A549 cells (Fig. 4C). We observed that the long form of fibulin-3 was largely secreted into the culture medium, whereas the short form was expressed at lower levels, accumulated in the total cell lysate, and was virtually absent from the culture medium (Fig. 4B). We next assessed the effect of fibulin-3 expression on the growth of A549 cells. The growth curves obtained by MTT assays demonstrated that fibulin-3 inhibited the growth of A549 cells (P<0.05, Fig. 5A). Next, the mobility of fibulin-3-expressing A549 cells was determined using wound-healing and Transwell assays. As shown in Fig. 5B, the percentage of wound closure of A549 cells (16.7%) was decreased when compared to the untreated (100.0%) and mock transfected (100.0%) cells (P<0.05). Overexpression of the fibulin-3 gene significantly inhibited the invasion of A549 cells into the Matrigel as detected using Transwell assay (P<0.05, Fig. 5C). Furthermore, the activities of MMP-2 and −9 were significantly decreased in cells transfected with pEGFP-C1-fibulin-3 when compared to the untreated cells (Fig. 4A).

To identify the mechanism of fibulin-3, western blot assays were performed to detect changes in possible signaling pathway proteins. Expression of MMP-2 and −9 was downregulated in fibulin-3-expressing A549 cells. While total levels of p38 and ERK1/2 showed no changes, the levels of phospho-p38 and phospho-ERK1/2 were observed to be significantly higher in the fibulin-3-positive cells when compared to that in negative cells (Fig. 6). Notably, phospho-JNK was also increased in the fibulin-3-positive cells (Fig. 6). In combination, these results suggest that fibulin-3 inhibited the mobility of A549 cells via the p38 and ERK 1/2 proteins.