All of the tissue specimens were obtained between November 2011 and September 2012 from 50 patients who underwent surgery for therapeutic treatment at Tongji Hospital. Immediately after the surgery, samples were snap-frozen in liquid nitrogen and stored at -80°C. There were 45 bladder cancer and 5 normal bladder tissues in all of the specimens. As controls, biopsies of normal bladder samples were obtained from 5 patients who underwent transvesical prostatectomy. No treatment was given to the patients before surgery. The samples were sectioned for hematoxylin and eosin (H&E) staining for histological confirmation by the Department of Pathology of Tongji hospital. Tumor staging was determined according to the sixth edition of the tumor node metastasis (TNM) classification of the International Union Against Cancer. This study was approved by the ethnics committee of Huazhong University of Science and Technology. All patients provided informed consent.
Reagents and cell culture
The plasmid p3XFLAG-CMV9-LRIG1 and rabbit antihuman LRIG1 polyclonal antibodies were generous gifts from Hakan Hedman (Umea University, Sweden). Two human aggressive bladder cancer cell lines(T24 and 5637) were used in this study. All of this cell lines were obtained from the American Type Cell Collection(ATCC), and grown in complete growth medium supplemented with 10% fetal bovine serum(FBS) and maintained in a humidified 5% CO2 atmosphere 37°C.
The plasmid p3XFLAG-CMV9-LRIG1 was transfected into the two bladder cancer cells by using Lipofectamine2000 reagent (Invitrogen, Groningen, the Netherlands) according to the manufacturer’s instructions. For control experiments, the vector p3XFLAG-CMV9-EGFP was also transfected into the two bladder cancer cells. All transfected cells were exposed to G418 (800 μg/mL, Sigma Chemical Co., St. Louis, USA) for 3 weeks of selection. Resistant clones representing stably transfected cells were ring-cloned and expanded for further experiment.
siRNAs against EGFR were transfected into T24 and 5637 cells according to the transfection protocol of Lipofectamine2000 (Invitrogen). A nonspecific control siRNA strand was used as a negative control. Seventy-two hours after transfection, knockdown was assessed by western blot from a parallel transfection. After downregulation of EGFR, we detected the effect of LRIG1 cDNA on cell proliferation and EGFR signaling pathway by CCK-8 assays and western blot respectively.
Quantitative real-time RT-PCR
Total RNA was extracted from 45 cases of bladder cancer and 5 cases of respective non-neoplastic tissue samples and 2 bladder cancer cell lines with Trizol reagent. The expression of LIG1 and EGFR mRNA was done using quantitative real-time RT-PCR. RNA samples were run in triplicate using 20 ng of RNA perreaction. The resulting cDNA samples were amplified by real-time PCR using gene-specific primer sets in conjunction with the SYBR Premix Ex Taq (TaKaRa) in a Mx3000p instrument. The qPCR was performed with the following conditions: activation at 95°C for 5 min followed by 40 cycles of denaturation at 94°C for 15 s, amplification at 60°C for 30 s, elongation at 72°C for 30 s. In the last, a cycle of solubility curve was added to examine the amplification quality. Expression of mRNA for GAPDH was used as an internal standard. Reverse transcription products were amplified by PCR using specific primers for human LRIG1 (forward 5′-GGTGAGCCTGGCCTTATGTGAATA-3′; reverse 5′-GGTGAGCCTGGCCT TATGTGAATA-3′) and human EGFR (forward 5′-TCCCTCAGCCACCCATAT GTAC-3′; reverse 5′-TCCCTCAGCCACCCATATGTAC-3′).
Formalin-fixed and paraffin-embedded tissue sections (5 mm) were dewaxed with xylene and rehydrated through an ethanol gradient into water. Following blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide for 10 min, the sections were washed with phosphate buffered saline(PBS) and incubated over-night with rabbit LRIG1 antibody or EGFR antibody at the dilution of 1:100 in a humidified chamber at 4°C. After washing with PBS, sections were incubated with biotinylated secondary antibody for 30 min at 37°C and then with horseradish peroxidase labeled streptavidin for 30 min at 37°C. Diaminobenzidine(DAB) was used as chromogen and the sections were subsequently counterstained with hematoxylin, then dehydrated, cleared and mounted.
Western blotting analysis
The transfected bladder cancer cells were collected and washed with 0.01 mol/L PBS for three times. Then the cells were added into 200ul pre-cold RIPA-PICT cell disruption liquor and centrifuged. All subsequent manipulations were performed on ice. After centrifugation, the supernatant was collected. The protein concentration of each sample was measured with micro-BCA protein assay reagent. The mixture was heated to 100°C for 5 min to denature the proteins. The protein from each sample was subjected to electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gel. Then protein was transferred to nitrocellulose membrane, which were blocked with PBS containing 5% non-fat milk for 2 h and then incubated with anti-LRIG1 (1:5,000), anti-EGFR(1:2,000), anti-p-EGFR(1:2,000), anti-MAPK(1:2,000), anti-p-MAPK(1:2,000), anti-AKT(1:2,000), anti-p-AKT(1:2,000), anti-caspase-8(1:1,000), anti-MMP-2(1:2,000), anti-MMP-9(1:2,000) and β-actin(1:2,000) at 4°C overnight. Then secondary antibody labeled with alkaline phosphatase were added at room temperature. One hour later, the samples were washed for three times with TBST, and then visualized using DAB detection system.
The total protein was prepared using M-PERTM mammalian protein extraction reagent (Pierce). For each sample, 10 μL of anti-LRIG1 antibody or control IgG was added to 1 mg of protein in 200 μL of lysis buffer and placed on a rocker overnight at 4°C. Twelve microliters of protein G beads was added to each sample, which was placed on a rocker at 4°C for 1 h. The beads were washed three times with 1 ml of lysis buffer and then boiled in 50 μL of SDS sample buffer; 20 μL was then loaded per lane and subjected to Western blotting.
Annexin V-PE/7-aad double staining assay was used to detect cell apoptosis. After transfected and incubated for 3 days, cells were collected, centrifuged and washed with phosphate—buffered saline(PBS) for two times. Binding buffer was then added to each tube and cells were re-suspended. The cells were incubated with 5 μL of annexin V-PE and 5 μL of 7-aad for 15 min at room temperature in the dark. Then, the apoptotic analyses were done by flow cytometry within one hour.
Survival assay by CCK-8
The growth of T24 and 5637 cells after LRIG1 gene transfection were evaluated by Cell Counting Kit-8 assays. Untreated cells, cells treated with liposome alone and cells treated with the vector control were used for comparison. Cell suspensions (at 1 × 103/mL) were transferred to 96-well plates in triplicate and incubate for 24, 48 and 72 hours. Subsequently, CCK-8(10 μL) was added to each well, cells were incubated for an additional 4 h. Then, The values of each well was measured by microplate reader at 450 nm.
Clonal forming assay
T24 and 5637 cells were infected with LRIG1 cDNA and cultured for 24 h, then plated in 6-well plates at 200 cells/well. Plates were subsequently incubated for 14 days in a humidified incubator at 37°C, and the colonies were stained with 0.5 ml of 0.0005% crystal violet solution for 1 h and counted by using a microscope. Five random fields were counted from each sample and average values presented ± the SD.
Matrigel invasion assays
The in vitro invasive ability of bladder cancer cells was measured in transwells chambers assay. 100ul matrigel was put into upper chambers of the transwell insets. Incubated the inserts at 37°C for 4 h for gelling and then pretreated with serum-free medium at 37°C for 1 h before seeding cells at a density of 2 × 104 /ml with 1% FCS. The lower chambers of the transwells were filled with 600 ul medium containing 10% FCS. Then the transwell were incubated at 37°C with 5% CO2 for 24 h to allow cells to migrate. After that, removed the cells on the upper side by wiping with cotton swab. Cells that had invaded through matrigel were fixed in paraformaldehyde and crystal violet stained according to the manufacture’s instruction. Cells that had invaded the matrigel and reached the lower surface of the filter were counted under a light microscope at a magnification of 200×. We chose five fields of vision and counted the numbers of the invaded cells and the results from three separate chambers were then averaged. The experiment was performed in triplicate.
The cell culture data from at least three independent experiments were expressed as means ± SD and examined by one-way analysis of variance followed by the Student–Newman–Keuls test. A Pearson’s correlation test was performed to examine the relationship of LRIG1 and EGFR expression in bladder cancer and non-neoplastic tissues. All P-values were two-sided, and values less than 0.05 were considered significant. SPSS v16.0 software was used for all statistical procedures.