Cell lines and culture conditions
The HepG2, Hep3B and sk-Hep1 HCC cell lines were obtained from the American Type Culture Collection (ATCC). The LO2, Bel7402 and HEK293 cell lines were obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The Huh7 cell line was obtained from the RIKEN cell bank (Ibaraki, Japan). The LO2, Bel7402 and sk-Hep1 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS (fetal bovine serum) and 1%
penicillin-streptomycin. The HepG2, Hep3B, Huh7 and HEK293 cells were cultured in DMEM (Dulbecco modified Eagle medium) supplemented with 10% heat-inactivated FBS (fetal bovine serum) and 1%
penicillin-streptomycin. All cells were incubated at 37°C in a humidified chamber containing 5% CO2.
Patients and tumor tissue samples
A total of 126 human primary HCC tissues and matched control tissues were obtained from patients who underwent hepatectomy at the Sun Yat-sen University Cancer Center between 2001 and 2004. None of these patients had received preoperative chemotherapy or radiotherapy. The follow-up data from the HCC patients in this study were available and complete. The postoperative follow-up occurred at our outpatient department and included clinical and laboratory examinations every 3 months for the first 2 years, every 6 months during the third to fifth years, and annually for an additional 5 years or until patient death, whichever occurred first. Overall survival, which was defined as the time from the operation to patient death or the last follow-up, was used as a measure of prognosis. Both the tumor and the corresponding non-tumor tissues not less than 2 cm away from the HCC were sampled, and the diagnosis was confirmed by pathological examination. After surgical resection, the matched fresh tissues were immediately immersed in RNAlater (Ambion, Inc., USA), kept at 4°C overnight, then stored at −80°C until the RNA isolation. All the tissue samples were fixed in 10% formalin and embedded in paraffin, and consecutive 2 µm sections were cut. Histological types were assigned according to the WHO classification criteria. This study was approved by the Ethics Committee of the Sun Yat-sen University Cancer Center, and written informed consent was obtained from each patient.
RNA extraction and real-time quantitative PCR
Total RNA was extracted using TRIzol solution (Invitrogen, USA) according to the manufacturer's instruction. RNase-free DNase I was used to remove DNA contamination. The total RNA concentration and quantity were assessed by absorbency at 260 nm using a Nanodrop spectrophotometer (ND-1000, Thermo Scientific, USA). The first-strand cDNA synthesis was performed using 2 µg of total RNA and M-MLV reverse transcriptase according to the manufacturer's instructions (Promega, USA). The resulting cDNAs were subjected to real-time PCR analysis to evaluate the relative expression levels of LZAP and GAPDH (an internal control) using the following primers: 5′- TCTGGGTCCTACATTCACTACTTTC-3′ (F) and 5′-CTCCTGCCAATCCTTCATCC-3′ for LZAP; and 5′-CTCCTCCTGTTCGACAGTCAGC-3′ (F) and 5′- CCCAATACGACCAAATCCGTT-3′ for GAPDH. Each 15 µl of reaction volume contained 0.5 µl of cDNA that was synthesized as above, 7.5 µl of 2× SYBR Green master mix (Invitrogen, USA), and 200 nM of each pair of oligonucleotide primers described above. The cycling parameters began with 95°C for 10 minutes, followed by 45 cycles of 90°C for 30 seconds and 60°C for 60 seconds, followed by a melting curve analysis. Ct was measured during the exponential amplification phase, and the amplification plots were analyzed using the software provided with the instrument (SDS 2.0). The relative expression levels of the target gene were normalized to that of the internal control gene, GAPDH. The data were analyzed using the comparative threshold cycle (2−ΔΔCT) method.
Protein extraction and western blotting analysis
The frozen HCC samples, including the tumor tissue, non-tumor control tissue and cells from the HCC cell lines (LO2, HepG2, Hep3B, Huh7, Bel7402 and sk-Hep1), were homogenized in a RIPA lysis buffer, and the lysates were cleared by centrifugation (14,000 rpm) at 4°C for 30 minutes. Approximately 40 µg of protein sample were run on a 12% SDS-PAGE gel and transferred to PVDF membranes. After blocking the non-specific binding sites for 60 minutes with 5% non-fat milk, the membranes were incubated with primary monoclonal antibodies against LZAP (at a 1
1000 dilution) or GAPDH (at a 1
10000 dilution) overnight at 4°C. Next, the membranes were subjected to three 15 minute washes with TBST and then incubated with HRP-conjugated secondary antibody (at a 1
2000 dilution) for 45 minutes at room temperature. The membrane was washed three more times with TBST and developed using an enhanced chemiluminescence system (ECL, Cell Signaling Technologies).
Immunohistochemistry and immunofluorescence
Paraffin-embedded tissue blocks were sectioned for immunohistochemistry. The sections were deparaffinized and rehydrated with graded ethanol. For the antigen retrieval, the slides were immersed in EDTA (1 mmol/L, pH 8.0) and boiled for 15 minutes in a microwave oven. After rinsing with PBS, the endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 15 minutes at room temperature. The slides were incubated with the primary antibody (mouse anti-LZAP monoclonal antibody, Santa Cruz Biotechnology, USA, at a 1
500 dilution) overnight in a humidified chamber at 4°C. The sections were washed three times with PBS, incubated with horseradish peroxidase-conjugated secondary antibody (Envision™ Detection Kit, GK500705, Gene Tech) at 37°C for 30 minutes, and then washed three more times with PBS. Finally, 3, 3′-diaminobenzidine tetrahydrochloride (DAB) was used for signal development, and the sections were counterstained with 20% hematoxylin. The slides were dehydrated, cleared and evaluated. Each sample was incubated with an isotypic antibody dilution under the same experimental conditions as the negative control.
The HepG2 or sk-Hep1 cells infected with the Ad-control or Ad-LZAP were plated on glass coverslips. At 48 hours post-infection, the coverslips were washed extensively in PBS and fixed with 4% paraformaldehyde. After rinsing with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. The coverslips were then washed and blocked with 1% BSA in PBS for 60 minutes. The slides were incubated with mouse anti-LZAP monoclonal antibody (Santa Cruz Biotechnology, USA, at a 1
100 dilution) in PBS with 0.2% Triton X-100 and 0.1% BSA at room temperature for 1 hour. The slides were then washed extensively with PBS and treated with fluorescein (FITC)-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, USA) for 30 minutes. After further washing, the slides were labeled with DAPI and images were acquired using a Laser Scanning Confocal Microscope.
The total LZAP immunostaining score was calculated as both the percentage of positively stained tumor cells and the staining intensity. The percent positivity was scored as “0” (<5%, negative), “1” (5%–25%, sporadic), “2” (25%–50%, focal), or “3” (>50%, diffuse). The staining intensity was scored as “0” (no staining), “1” (weakly stained), “2” (moderately stained), or “3” (strongly stained). Both the percentage of positive cells and the staining intensity were evaluated under double-blind conditions. The LZAP immunostaining score was calculated as the percentage positive score × the staining intensity score and ranged from 0 to 9. We defined the LZAP expression levels as follows: ‘−’ (score 0–1), ‘+’ (score 2–3), ‘++’ (score 4–6) and‘+++’ (score >6). Based on the LZAP expression levels, the HCC patients were divided into two groups: the low LZAP expression group (LZAP− or LZAP+) and the high LZAP expression group (LZAP++ or LZAP+++).
RNA oligonucleotides and cell transfections
The siRNAs for the LZAP knockouts were synthesized by GenePharma (Shanghai, China). The siRNA sequences were as follows: siLZAP-1#, sense
=5′- AUACAGAGCUAUAAUCUCCTT- 3′
; siLZAP-2#, sense
=5′- GAGAUCCCCUCACUGAAGATT -3′
=5′- UCUUCAGUGAGGGGAUCUCTT -3′
; siLZAP-3#, sense
=5′- CCCUGACACUGCUUGAAUATT- 3′
=5′- UAUUCAAGCAGUGUCAGGGTT -3′
; and negative control (NC), sense
=5′- ACGUGACACGUUCGGAGAATT -3′
The HepG2 and sk-Hep1 cells were infected with Ad-LZAP and transfected with 20 µM siLZAP or NC 48 hours later. The transfection was performed using the Lipofectamine RNAi MAX reagent (Invitrogen, USA) according to the manufacturer's protocol.
Recombinant adenovirus construction and tumor cell infection
The LZAP-recombined adenoviral expression vector and the control vector were constructed by the rapid BP/LR reaction in the Gateway cloning system (Invitrogen, USA) according to the manufacturer's instructions. PacI enzyme-linearized adenoviral vectors were transfected into the HEK293 cells using Lipofectamine 2000 (Invitrogen, USA). At 10–13 days after the transfection, when an approximately 80% cytopathic effect (CPE) was observed in the HEK293 cells, the adenovirus-containing HEK293 cells and media were collected. Three freeze/thaw cycles followed by centrifugation were used to prepare the viral lysates. The Ad-LZAP and Ad-control titers were measured with an Adenovirus Titer Immunoassay Kit (Innogent, China). The recombinant adenoviruses were stored at −80°C for use. The HepG2 and sk-Hep1 cells were cultured in 6-well plates and infected with adenovirus (Ad-LZAP and Ad-control) at a multiplicity of infection (MOI) of 200.
Colony formation assay
The cells infected with Ad-LZAP and Ad-control (1×103) were plated in each well of a 6-well plate. The surviving colonies (>50 cells) were counted with crystal violet staining after two weeks of culture. Colony-forming efficiency (CFE %) was defined as the ratio of the number of colonies formed in culture to the number of cells inoculated. This experiment was performed in triplicate.
The MTS cell proliferation assay was used to evaluate the growth rate of the cells infected with Ad-LZAP and Ad-control. The cells were seeded in 96-well plate at a density of 5×103 per well. The growth rate was detected using the MTS cell proliferation kit according to the manufacturer's instructions (Promega, USA). Three independent experiments were performed.
Cell cycle assay
The cell cycle analysis was performed at 48 hours after the cells were infected. The HepG2 or sk-Hep1 cells infected with Ad-LZAP and Ad-control were washed twice with ice-cold PBS and fixed with ice-cold 75% ethyl alcohol at −20°C for one hour. After two PBS washes, the cells were resuspended in 400 µl of ice-cold PBS and incubated with RNase in a 37°C water bath for 30 minutes. The cells were subsequently incubated with propidium iodide at 4°C in the dark for 30–60 minutes and analyzed using a flow cytometer (Beckman, USA).
The apoptosis assays were performed at 48 hours, 72 hours and 96 hours after the cells were infected with the adenovirus. The HepG2 and sk-Hep1 cells infected with Ad-LZAP or Ad-control were washed twice in ice-cold PBS, resuspended in 400 µl of 1× Binding Buffer and incubated with Annexin V-FITC (Bestbio, China) for 15 minutes at 4°C in the dark, according to the manufacturer's instructions. After staining, the cells were incubated with propidium iodide for 5 minutes at 4°C in the dark and then analyzed using a flow cytometer (Beckman, USA).
Cell migration assay
The cell migration assays were performed in a chamber system consisting of polycarbonate membrane inserts with an 8-µm pore size (Corning, USA) placed in 24-well cell culture insert companion plates. The migration assay was conducted at 48 hours after the HepG2 and sk-Hep1 cells were infected with Ad-control or Ad-LZAP. The cells (5×104 in 100 µl of growth medium without FBS) were placed in the upper chamber and 500 µl of growth medium with 10% FBS was placed in the lower chamber. The cells were incubated at 37°C for 12 hours. Following the incubation, the insert membranes were fixed with 75% methanol for 30 minutes. The cells on the upper surface were removed with cotton-tipped swabs, and the migrated cells on the lower surface were stained with 0.5% crystal violet containing 20% methanol for 60 minutes. The stained cells were counted under an inverted microscope (10 fields per membrane). Each experiment was performed in triplicate.
Matrigel invasion assay
The Matrigel invasion assay was performed in a chamber system consisting polycarbonate membrane inserts with 8-µm pores (Corning, USA) placed in 24-well cell culture insert companion plates. The inserts were coated with a thin layer of 0.5 mg/ml Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA). The invasion assay was conducted at 48 hours after the HepG2 and sk-Hep1 cells were infected with Ad-control or Ad-LZAP. The cells (1×105 in 200 µl of growth medium without FBS) were placed in the upper chamber, and 0.5 ml of growth medium containing 20% FBS was placed in the lower chamber. The cells were incubated at 37°C and allowed to invade through the Matrigel layer for 48 hours. After incubation, the insert membranes were fixed with 75% methanol for 30 hours. The cells on the upper surface were removed with cotton-tipped swabs, and the invading cells on the lower surface were stained with 0.5% crystal violet containing 20% methanol for 60 minutes. The stained cells were counted under an inverted microscope (10 fields per membrane). Each experiment was performed in triplicate.
Tumorigenicity assays in nude mice
Female BALB/c athymic nude mice (4–5 weeks old) were obtained from the Medical Experimental Animal Center of Guangdong Province. The mice were randomly divided into 4 groups of 8 mice each. Group 1 was injected with HepG2 cells that had been infected with Ad-control; Group 2 was injected with HepG2 cells that had been infected with Ad-LZAP; group 3 was injected with sk-Hep1 cells that had been infected with Ad-control; group 4 was injected with sk-Hep1 cells that had been infected with Ad-LZAP. For the injections, 8×106 tumor cells were suspended in 200 µl PBS and then subcutaneously injected into the posterior flank of the mice. The tumor size was monitored every 3 days by measuring the length (L) and width (W) of the tumor with calipers. The tumor volume was calculated according to the following formula: (L×W2)/2. At 4–5 weeks after inoculation, all the mice were sacrificed, and the tumors were harvested and photographed. The weight of the tumors was also measured. All the experimental procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publications Nos. 80–23, revised 1996) and the institutional ethical guidelines for animal experiments.
The statistical analyses were performed using the Statistical Package for the Social Sciences, version 16.0 (SPSS Inc., Chicago, IL, USA). A paired-samples t-test was used to compare LZAP mRNA and protein expression in the HCC tumors with that of their paired adjacent non-cancerous tissue samples. The correlation between tumor LZAP expression and the clinical and pathological features was performed using χ2 tests. Overall survival curves were calculated with the Kaplan-Meier method and were analyzed with the log-rank test. A Cox proportional-hazards analysis was used in univariate and multivariate analyses to explore the effects of LZAP expression and HCC clinicopathological variables on survival. The results were expressed as mean ± SD and analyzed using the Student's t-test. Differences were considered significant at p<0.05.