The highly invasive human bladder cancer EJ-M3 cell line was established and preserved at the Department of Urology of the Second Affiliated Hospital of Kunming Medical University (Yunnan Institute of Urology, Kunming, China) (11). Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO₂. All reagents were purchased from EMD Millipore (Billerica, MA, USA).

EJ-M3 cells were transfected with a LASS2 shRNA plasmid (cat no. HSH008629-CU6) or a LASS2 overexpression plasmid (cat no. EX-T3019-M98-5) (both GeneCopoeia, Inc., Rockville, MD, USA). Briefly, the plasmids were amplified in DH5α Escherichia coli cells (Takara Biotechnology Co., Ltd., Dalian, China), purified using an E.Z.N.A.^® Endo-Free Plasmid Mini Kit I (Omega Bio-Tek, Inc., Norcross, GA, USA), and transfected into 80% confluent EJ-M3 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A lentiviral vector expressing green fluorescent protein (GFP) alone was used as the control. At 72 h following transfection, LASS2 overexpression and LASS2 shRNA positive cells were selected using 1 µg/ml neomycin or puromycin, respectively, and the transfection efficiency was examined by fluorescence microscopy (Olympus BX53; Olympus Corporation, Tokyo, Japan).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and reverse transcribed into cDNA using Quant Reverse Transcriptase (Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer's protocol. β-Actin was used as the internal control. RT-qPCR was performed in a 20 µl reaction containing 1 µl cDNA template, 1 µl primer and 10 µl FastStart Universal SYBR Green Master (ROX) (Roche Ltd., Mannheim, Germany), using an Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific Ltd., Waltham, MA, USA). The primers sequences were as follows: β-actin forward, 5′-GAAGGTGAAGGTCGGAGTC-3′; and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; LASS2 forward, 5′-TCTCCTGGTTTGCCAATTACG-3′; and reverse, 5′-CCGGGCAGGGACCCTCATCA-3′. All the primers were synthesized by GeneCopoeia, Inc. The amplification program consisted of denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 30 sec, and then extension at 60°C for 30 sec. All experiments were repeated at least three times. The relative expression of LASS2 mRNA was calculated using the 2^−ΔΔCq method (14).

Cell lysates were extracted using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). Total proteins were separated via SDS-PAGE on a 10% gel, transferred onto 0.45 µm polyvinylidene difluoride membranes (EMD Millipore), and blocked with 5% skimmed milk. Using β-actin as the internal control, the membranes were incubated with a mouse anti-LASS2 (cat. no. sc-390745; dilution, 1:500) or anti-β-actin monoclonal antibody (cat. no. sc-47778; dilution, 1:500) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (cat. no. sc-2005; dilution, 1:2,500; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Protein bands were visualized through enhanced chemiluminescence by using Immobilon Western Chemiluminescent HRP substrate (EMD Millipore Corporation, Billerica, MA, USA) and captured using a MicroChemi 4.2 system (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Relative protein quantitative quantification was performed using ImageJ image analysis software (version 1.34; National Institutes of Health, Bethesda, MD, USA).

The animal experiments were approved by the Institutional Animal Care and Use Committee of Kunming Medical University (Kunming, China) (approval no. KYLL20140071), and were performed in compliance with all regulatory institutional guidelines for animal welfare (the National Institutes of Health Publications no. 80-23). Fifteen 6-week-old female nude mice (BALB/c-nu/nu) weighing 16–20 g, were purchased from Beijing HFK Bioscience Company (Beijing, China), and were randomly divided into the following 3 groups (5/group): A non-transfected EJ-M3 cell group [control (Con)]; a LASS2-overexpression EJ-M3 cell group (LASS2-over); and a LASS2-shRNA EJ-M3 cell group (LASS2-shRNA). All nude mice were housed in a sterile environment with a room temperature of 20–25°C, a relative humidity of 30–70% and a 12 h light/dark cycle. All mice were given free access to food and water, and the bedding materials, drinking water, feeding cages and other items in contact with the animals were all autoclaved prior to use. Cell suspensions (200 µl, 2×10⁶/ml) of non-transfected, LASS2-over or LASS2-shRNA EJ-M3 cells were subcutaneously injected into the right side of the dorsal aspect of the nude mice using a 6-gauge needle. The tumor size and body weight of each mouse were measured once a week. The tumor volumes were calculated using the following formula: V=π/6 × largest diameter × smallest diameter² (15), and tumor growth curves were plotted. The tumor inhibition rate (TIR) was determined using the formula: TIR (%)=[1-(mean tumor volume of experimental group/mean tumor volume of control group)]x100. All mice were sacrificed 6 weeks following injection, and the tumors were resected. One part of each tumor was fixed in 4% formalin, dehydrated with gradient alcohol, cleared with xylene, embedded in paraffin, sectioned serially (4-µm-thick sections), and then mounted on a slide for hematoxylin and eosin staining and immunohistochemical analysis. Another part of each tumor was stored at −80°C for the detection of matrix metalloproteinase (MMP)-2/−9 activity. The slides were stained with hematoxylin and eosin (H&E), and the properties of the xenograft tumors were assessed under an optical microscope (Eclipse E200; Nikon Corporation, Tokyo, Japan).

The slides were heated and subsequently put in xylene, washed with gradient alcohol for deparaffinization, and then rehydrated with distilled water. After that, antigen retrieval was performed in 1 mM boiling EDTA buffer (pH 8.0; EMD Millipore) for 15 min at 92–100°C. The slides were incubated with 3% hydrogen peroxide for 30 min, washed, and blocked with 5% goat serum (EMD Millipore) for 60 min at room temperature. A mouse monoclonal anti-LASS2 antibody (cat. no. sc-390745; dilution, 1:100, Santa Cruz Biotechnology, Inc.) was applied to each slide overnight at 4°C. Following washing with PBS (pH 7.4), the slides were incubated for 30 min with a rabbit anti-mouse secondary antibody (cat. no. A-11062, dilution, 1:400; Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the slides were stained with diaminobenzidine, counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, mounted on coverslips and examined under an optical microscope (Eclipse E200; Nikon Corporation, Tokyo, Japan). The cells with yellow-brown staining in their cytoplasm were considered LASS2-positive. The mean density of staining, calculated using Image Pro-Plus (Media Cybernetics, Inc., Rockville, MD, USA) as previously described (16), was used to quantify the LASS2 staining.

To investigate MMP-2 and MMP-9 activity in the xenografts expressing different LASS2 levels, gelatin zymography was used. SDS-PAGE on a 10% gel containing 0.1 mg/ml gelatin was used for electrophoresis. The protein samples (20 µg of each) were loaded into each lane, and electrophoresis was performed at 100 V for 1.5 h at 4°C. Subsequently, the gel was washed twice with 100 ml solution containing 2.5% Triton X-100 on a rotary shaker for 40 min at room temperature, then incubated in 100 ml reaction buffer (50 mmol/l Tris-HCl, 5 mmol/l CaCl₂, 0.02% NaN₃, pH 7.6) for 42 h at 37°C. Following staining with Coomassie brilliant blue and destaining with methanol and acetic acid, the gels were scanned using the BioSpectrum Imaging System (BioSpectrum 510; UVP, Inc., Upland, CA, USA), and the relative activities of MMP-2 and MMP-9 were quantified by densitometric analysis of the zymograms using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

All data were expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). A one-way analysis of variance followed by a Tukey's multiple comparison test was used to evaluate the differences between the groups. P<0.05 was considered to indicate a statistically significant difference.

To confirm transfection efficiency, fluorescence microscopy was used to detect the expression of GFP in EJ-M3 cells transfected with a LASS2 shRNA construct or a LASS2 overexpression plasmid 72 h following transfection (Fig. 1A). Non-transfected EJ-M3 cells expressing GFP alone were used as the Con group. Subsequently, the cells were collected to detect the expression of LASS2 at the mRNA and protein levels by RT-qPCR and western blot analysis, respectively. β-Actin was used as the internal control for normalization. The RT-qPCR results demonstrated that LASS2 expression in the LASS2-shRNA group was significantly decreased by 39% (P<0.001), while that in the LASS2-over group was significantly increased by 263% (P<0.001), compared with the Con group (Fig. 1B). A western blot analysis demonstrated similar results at the protein level (Fig. 1C and D). These results demonstrate that the two different recombinant LASS2 plasmids were successfully transfected into the EJ-M3 cells.

To determine the functional role of LASS2 in human bladder cancer initiation and progression, EJ-M3 cells transfected with LASS2 shRNA constructs or LASS2 overexpression plasmids were subcutaneously injected into the right side of the dorsal aspect of the neck of nude mice. Non-transfected EJ-M3 cells were injected as the Con group. Tumor growth and body weight were closely monitored during the following weeks (Fig. 2). Ten days following injection, palpable subcutaneous xenografts were observed in all nude mice, and the tumor incidence rates in the three groups were 100%. Tumor growth curves demonstrated that the xenografts grew significantly faster in the LASS2-shRNA group compared with both the LASS2-over and Con groups (both P<0.001); however, no significant difference was observed between the LASS2-over and Con groups (P>0.05) (Fig. 2A). No animals died during the observation period. Six weeks following injection, all mice were sacrificed and the tumors were removed. All subcutaneous xenografts exhibited oval or nodal shapes with a smooth surface (Fig. 2C and D). The tumor volumes of the LASS2-shRNA, LASS2-over and Con groups were 222.32±124.97, 41.01±15.97 and 69.60±31.37 mm³, respectively. Compared with the Con group, the TIRs in the LASS2-shRNA and LASS2-over groups were −219.42 and 41.08%, respectively (data not shown). These results indicate that LASS2 knockdown significantly increases tumor volume, while LASS2 overexpression does not significantly affect tumor growth when compared with the Con group, despite a trend for LASS2 overexpression to decrease tumor volume (P=0.821; Fig. 2E). During the experimental period, LASS2 knockdown had a tendency to reduce the body weight of nude mice; however, there was no significant difference in body weight between these three groups (Fig. 2B).

H&E staining confirmed the subcutaneous xenografts as bladder transitional cell carcinoma. The tumor cells exhibited a nest-like distribution, apparent heteromorphism, and large and irregular nuclei with obvious nucleoli, particularly in the LASS2-shRNA group (Fig. 3A). Additionally, immunohistochemical analysis was used to detect LASS2 expression levels in the xenografts. There was a significantly higher mean density of LASS2-positive cells in the LASS2-over group compared with those in the LASS2-shRNA and Con groups (P<0.001; Fig. 3B and C). These results suggest that different LASS2 expression levels regulate the tumorigenicity of bladder cancer EJ-M3 cells, the growth of xenografts and the degree of tumor cell heteromorphism in vivo.

Using gelatin zymography and a quantified analytical system, MMP-2 and MMP-9 activity in the xenografts with different LASS2 expression levels was analyzed. MMP-2 and MMP-9 expression levels were identified as clear bands against a lighter blue background (Fig. 4A). Compared with the Con group, the relative activities of MMP-2 (Fig. 4B) and MMP-9 (Fig. 4C) in the LASS2-shRNA group were significantly higher (P<0.0001), while those in the LASS2-over group were significantly lower (P<0.0001).