Cells and culture conditions

The human lung cancer cell lines XWLC-05 were supplied by the Institute of Clinical Cancer, Kunming Medical College, China. These cell lines were cultured in RPMI 1640 (Sigma, St. Louis, MO, USA) at 37°C in an atmosphere of 5% CO₂ in the air with 10% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA).

Construction of pAQP3-siRNA

DNA sequence of AQP3 (GenBank no: BC013566) was inputed into Dharmacon web-based software for selecting target sequences. The following criteria were used to identify targets for siRNAs from the AQP3 cDNA coding sequence: (a) start with an AA dinucleotide; (b) 21 nucleotides in length; (c) G/C content of < 50% and (d) no sequence homology to other coding sequences on BLAST search. Using these criteria, two siRNA sequences from human AQP3 were chosen as follows: 5′-CGAGGAAGAGAAUGUGAAG-3′ (A1), 5′- GAGAAUGUGAAGCUGGCCC-3′ (A2). One negative control siRNA containing a scrambled sequence with the same nucleotide composition was also selected. Sense and antisense primers containing the sense siRNA sequence, 9 bp loop sequence, antisense siRNA sequence, and RNA polymerase III terminator sequence were created at BamH I and Hind III restriction sites on the 5′ and 3′ ends, respectively. These primers were annealed and inserted into pSilencer 4.1-CMV neo (Ambion, Inc., Austin, TX) downstream of the H1RNA polymerase III promoter following the manufacturer’s   instructions.   The   resulted   plasmids   containing siRNA sequences A1, A2 and negative control sequence NC were named pAQP3-siRNA1, pAQP3-siRNA2 and pAQP3-N.

Cell transfection

Cells were cultured in the RPMI 1640 medium, seeded at 1×10⁶ /well in 6 well plates. When the cells reached 80 to 90% confluence, transfection of dsRNA was performed with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, one day before transfection, the attached cells in logarithmic growth phase were respectively implated into 12-well plates at density of 1×10⁵ /well. Transfection mixes containing Solution A (5 mg plasmid + 100 μl OptiMEM medium) and Solution B (10 µl Lipofectamine^TM 2000 + 100 μl OptiMEM medium) were prepared in 96-well plates. When cells reached 70% confluence, the plasmid DNA was transfected into the cells. The transfection efficiency was observed by fluorescence microscopy after 24 h. Cells were transfected for 6 h per day, lasted for 3 days and were collected in the 4th day. The transfection concentration of the experimental group shRNA was 1.0, 2.5 and 5.0 μg/L respectively.

RNA isolation and RT-PCR analysis

Total RNA was extracted with Trizol reagent according to the protocol described by the supplier (TakaRa, Dalian, China). cDNA was obtained from the total RNA by reverse transcriptase polymerase chain reaction (RT-PCR) according to the standard protocols (Wang et al., 2009). PCR primers were designed as follows: (300bp) upstream: 5'- AGGCGATTAAGTTGGGTA-3' and downstream: 5'- CGGTAGGCGTGTACGGTG-3'; Amplification reactions (25 μl) contained 1μl (50 ng) of cDNA, 2 μl (10 μmol/L) of each primer, 0.5 μl (10 μmol/L) of probe, 12.5 μl of PCR mix and 7 μl of deionized water. Mixed them gently and amplified by real time RT-PCR, cycling parameters were 55°C for 2 min, then 35 cycles of 93°C for 10 min, 93°C for 10 min and 60°C for 55 min. β-actin was used as the control. The targeted DNA amplified specifically was confirmed by electrophoresis and sequencing. PCR products were analyzed using GelWorks software, after the ethidium bromide-stained 1.5% agarose gel was scanned.

Western blot

Cells transfected with various vectors were lysed in RIPA buffer (1% NP-40, 50 mmol/L Tris, 150 mmol/L NaCl) 48 h after transfection. Proteins in an equal amount were separated by gel electrophoresis and transferred into a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk and then incubated with 1:1000 dilution of primary antibody overnight at 4°C. Subsequently, the membrane was washed and incubated with a secondary peroxidase-conjugated antibody for 1 h after being washed. Antibody binding was detected by using an enhanced chemiluminescence detection system. Western immunoblotting films were digitized, and band intensities were quantified by a Millipore Digital Bioimaging System (Bedford, MA). β-actin protein ratio was calculated for each protein.

MTS assay

Cell proliferation was assayed by the CellTiter 96 AQ non-radioactive cell proliferation assay (MTS) from Promega (Madison, WI). After 24, 48 and 72 h, 20 μl of freshly prepared MTS/PMS solution was added to each well, and the mixture was incubated for 2 h at 37°C. Optical density was read directly at 490 nm using the ELISA plate reader. All samples were assayed at least in quadruplicate with an appropriate blank.

Matrigel invasion assay

Matrigel matrix (Becton Dickinson Labware, Franklin Lakes, NJ) was applied and polymerized in 24-well 9-mm inserts containing polyethylene terephtphalate (PET) membranes with 8-µm pores to create invasion chambers as directed by the supplier (Becton Dickinson). Matrigel was thawed out at 4°C overnight on ice. Pipettes, plates and tips were chilled at -20°C. Matrigel (5 mg/ml) in serum-free cold DMEM was diluted with pre-cooled pipettes. 100 μl (h = 100 ul/200 mm 2 = 0.5 mm) of the diluted matrigel per well may be gently pipetted using a pre-cooled pipette to ensure homogeneity. The transwell was incubated at 37°C for 30 min to 5 h for gelling.

Cells were harvested when they grew to near confluence by trypsinization, which was inactivated with medium containing bovine calf serum, and cells were subsequently washed twice in DMEM without added serum or proteinase inhibitor. 100 μl of the cell suspension was put onto the matrigel. Lower chamber of the transwell was filled with 600 μl (3 mm) of culture medium containing 5 μg/ml fibronectin, as an adhesive substrate. Incubated at 37°C for 48 h, then removed transwells from 24-well plates and stained with Diff-Quick solution. Noninvaded cells were scraped off on the top of the transwell with a cotton swab, and invaded cells were counted on the transwell under a light microscope.

Statistical analysis

Experiments were performed at least in duplicate, and a typical set of data was indicated. Differences were statistically evaluated by Student’s t-test. A P-value of less than 0.05 was considered to be statistically significant.