RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco, (Gaithersburg, MD, USA) and Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). Plasmids for pLVX and pLVX encoding the full length of acetylcholine receptor α7 gene (pLVX-CHRNA7, NM_001190455) were constructed by GenScript Corporation (Nanjing, China). The antibody against nicotinic acetylcholine receptor α7 (anti-α7nAChR) was purchased from Abcam (Oxford, UK) and antibodies against matrix metalloproteinase (MMP)-1, -2, -3, -9 and -10 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies for phospho-ERK1/2, phospho-Akt (Ser473), phospho-JNK, phospho-p38, phospho-p65 (NF-κB), phospho-PI3K (p55/p85), ERK1/2, Akt, JNK, p38 and p65NF-κB were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody for β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). PI3K inhibitor LY294002 was purchased from Calbiochem (La Jolla, CA, USA). Goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) and goat anti-rat IgG antibody conjugated to HRP were purchased from Santa Cruz Biotechnology, Inc.

LoVo human colorectal adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC CCL-229™; Manassas, VA, USA) and were grown in RPMI-1640 medium containing 10% heat-inactivated FBS, 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin and maintained at 37°C in a of 5% CO₂/95% air atmosphere. Cell viability was determined by trypan blue exclusion assay. When growing at an exponential phase, the cells were transfected with pLVX-CHRNA7 or pLVX-vector using Lipofectamine 2000 according to the manufacturer's instructions. After 48 h, the cells were harvested for subsequent experiments.

Cells were lysed in 1X RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (all from Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined by the Bradford protein assay. Total proteins (40 µg) were separated by SDS-PAGE and blotted onto PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight on ice with the primary antibodies against α7nAChR (1:5,000), MMP-1, -2, -3, -9, -10, phospho-ERK1/2, phospho-Akt, phospho-JNK, phospho-p38, phospho-p65 (NF-kB), phosph-PI3K (55/85), ERK1/2, Akt, JNK, p38, p65NF-κB and β-actin (all dilutions 1:1,000). The next day, the membranes were washed with TBST and incubated with HRP-conjugated anti-rabbit or anti-rat secondary antibodies for 2 h at room temperature. After the final washing, the membranes were incubated with ECL reagent mixture and the signal was developed on a digital image system (FluorChem E; Proteinsimple, Santa Clara, CA, USA).

Cells were seeded into 96-well plates at a density of 2,000 cells per well, grown at 37°C overnight and then transfected with pLVX-CHRNA7 or pLVX-vector. Cell growth was analyzed using a WST-8 Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) at a frequency of every 24 h until 5 days after transfection. CCK-8 solution (10 µl) was added to each well, and the cultures were incubated at 37°C for 1 h. Absorbance at 450 nm was measured using an ultra microplate reader (SpectraMax; Molecular Devices Corp., Sunnyvale, CA, USA). Experiments were set in triplicates and repeated three times.

Transwell assays was used to estimate the effect of CHRNA7 on migration and invasion of colon cancer cells. Cell invasion and migration assays were carried out using modified Boyden chambers consisting of Transwell (8-µm pore size; Corning Costar Corp., Cambridge, MA, USA) membrane filter inserts in 24-well tissue culture plates. For the invasion assay, 48 h after transfection, the cells were trypsinized and resuspended in serum-free RPMI-1640 medium at the density of 5×10⁵ cells per ml. A total of 200 µl of cells were seeded into each upper chamber of the Transwells, and 500 µl of medium with 20% FBS was added in the lower chamber. The chambers were incubated at 37°C in a humid atmosphere with 5% CO₂. After 48 h, the non-migrating cells on the upper surface of the insert were removed with a cotton swab, and the cells that migrated to the underside of the membrane were fixed with ice-cold methanol and stained with crystal violet (0.1%). The cell surface of the insert was then photographed under an inverted microscope (Olympus CKX31; Olympus, Tokyo, Japan) for capturing images and quantified by counting the number of cells in five fields at ×100 magnification per filter. For the invasion assay, an additional 50 µl of BD Matrigel (BD Biosciences, San Jose, CA, USA) was added onto each upper chamber of the Transwells and the Transwells were placed in a 37°C incubator for 6 h to solidify the Matrigel. The tumor cell invasion capacity was then assessed in a way similar to the migration assay.

Gelatin zymography was used to determine the activity of MMP-2 and MMP-9. Forty-eight hours after gene transfection, cell culture supernatants of the LoVo cells were collected and concentrated in Amicon Ulta-4 Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Equal amounts of total protein were then mixed with SDS loading buffer and electrophoresed on a 10% SDS polyacrylamide gel polymerized with 5 mg/ml gelatin. After electrophoresis, the gels were re-natured by soaked for 30 min at room temperature in 2.5% Triton X-100. The gels were then incubated in a developing buffer [50 mM Tris-HCl buffer (pH 7.4), 10 mM CaCl₂] overnight at 37°C. The gels were stained with 0.5% Coomassie Brilliant Blue R-250 and de-stained in washing solution without dye. Gelatinolytic bands were observed as clear zones against a blue background.

All assays were performed in triplicate, and experiments were repeated at least three times. Data are presented as mean ± SE. Statistical analysis was performed using the Student's t-test to identify significant differences unless otherwise indicated. P<0.05 was considered to indicate a statistically significant difference. ImageJ software was used to quantify the results of the western blotting. GraphPad Prism 5.0 software was used to create graphics.

In order to evaluate the influence of CHRNA7, a plasmid of pLVX-CHRNA7 was constructed and transfected into LoVo cells. The transfection efficiency was ~80% as indicated by a GFP-expression vector (data not shown). Western blotting revealed the abundant expression of α7nAChR in the total cell lysate of the LoVo cells 48 h after transfection (Fig. 1A).

Physiologically, we firstly measured the cell proliferation of LoVo cells with or without CHRNA7 overexpression. A CCK-8 cell proliferation assay was performed at different times after LoVo cells were transfected with pLVX-CHRNA7 or pLVX-vector. There was no difference between the two groups of cells from day 1 to 5 after transfection (Fig. 1B).

We then detected the influence of CHRNA7 overexpression on colon cancer cell metastasis. A Transwell assay was conducted and BD Matrigel™ was used to imitate the extracellular matrix. The migratory and invasive abilities were evaluated based on the number of LoVo cells that passed through the polycarbonate membrane of the Transwell invasion chamber. As shown in Fig. 1C, the number of LoVo cells that passed through the polycarbonate membrane in the pLVX-CHRNA7 transfection group was significantly lower than that in the pLVX-vector control group (P<0.01). The invasion assay (Fig. 1D) showed similar results. The number of LoVo cells in the pLVX-CHRNA7 transfection group was also significantly lower than that in the pLVX-vector control group (P<0.01).

Next, we assessed whether the inhibition of metastatic ability by CHRNA7/LoVo was associated with the production of MMPs. Western blot analysis showed that levels of MMP-1 and MMP-9 expression in the CHRNA7-transfected cells were evidently less than these levels in the control vector-transfected cells (Fig. 2A). There was no major difference in MMP-2, -3 and -10 expression with or without CHRNA7 transfection. We also determined the enzymatic activities of the MMPs by gelatin zymography using conditioned medium. Gelatin zymography showed weaker lytic zones at the molecular masses corresponding to MMP-9 in cells with elevated CHRNA7 expression and had no effect on MMP-2 (Fig. 2B). Consistently, the activity of MMP-9 was markedly decreased when CHRNA7 was overexpressed. These data were consistent with the results observed in the cell migration and invasion assays, indicating that CHRNA7 inhibited the expression levels and activities of MMP-1 and MMP-9, and thus suppressed the invasion of colon cancer cells.

We then investigated the activation of relevant intracellular MAPK (JNK, p38 and ERK), NF-κB and PI3K/Akt signaling pathways (19,20) in cells overexpressing CHRNA7. Western blot assays were performed to examine the phosphorylation of JNK, p38, ERK, NF-κB (p65), Akt and PI3K (p55/p85). As detailed in Fig. 3, following comparison of the CHRNA7-transfected cells and the control vector-trans-fected cells, the CHRNA7-transfected cells exhibited higher phosphorylation levels of Akt and PI3K p55. The phosphorylation of ERK, JNK, P38 and NF-κB showed no difference between the CHRNA7-overexpressing cells and the control cells.

To determine whether enhanced PI3K/AKT activation may be involved in the inhibitory effect of CHRNA7 on the invasion of LoVo cells through Matrigel, we included the PI3K/AKT pathway inhibitor LY294002 into the experimental setting. Our results indicated that LY294002 significantly suppressed the phosphorylation of AKT in the CHRNA7-transfected cells (Fig. 4A), and reversed the inhibitory effect of CHRNA7 on the expression of MMP-1 and MMP-9 (Fig. 4B). Furthermore, LY294002 significantly blocked the negative effects of CHRNA7 on cell invasion of LoVo cells (Fig. 5). Taken together, these results suggest that the CHRNA7 inhibition of the invasion of colon cancer LoVo cells and MMP expression were PI3K/AKT activation-dependent.