A total of 40 pairs of cancer tissues and corresponding normal mucosa for QRT-PCR and Western blot and 98 primary gastric cancers for immunostaining were collected from surgically removed gastric cancer diagnosed at the Department of Surgery, Ruijin hospital, Shanghai Jiao Tong University School of Medicine. Normal mucosa samples were taken from the margin of the resection where the tumors located at least over 6 cm apart from it. This study protocol was approved by the independent ethics committee of Ruijin Hospital, Shanghai Jiaotong University, School of Medicine. The informed consent was written. All samples were snap frozen in liquid nitrogen overnight and kept at −80°C before use. 4% Formaldehyde-fixed tissue sections of both tumor and mucosa were stained with H&E and examined and verified histopathologically by two pathologists. Each specimen was attributed a diagnosis and scored for Lauren classification, differentiation, pTNM stage. Tumor size, location and other clinical pathological characteristics were obtained from clinical records with patient permission.
Gastric cancer cell lines SNU-1 (ATCC: CRL-5971), AGS (ATCC: CRL-1739), NCI-N87 (ATCC: CRL-5822) and KATOIII (ATCC: HTB-103) were obtained from the American Type Culture Collection (Manassas, VA, USA). Another 3 gastric cancer cell lines, MKN-45, MKN-28 and SGC-7901, were preserved at our institute. All cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS).
Primary antibodies used for staining included human specific anti-vimentin (Abcam, Cambridge, UK), anti-fibronectin (Abcam, Cambridge, UK), anti-α-smooth-muscle actin (Sigma Chemical, St Louis, MO, USA), anti-pan-cytokeratin (Abcam, Cambridge, UK), anti-SM22 (Abcam, Cambridge, UK), and mouse anti-GAPDH (Kangchen, China).
Isolation of human gastric fibroblasts
Fibroblasts were isolated from cancer- and non-cancer-associated regions of gastric tissues dissected from patients with gastric cancer during radical gastric resection from the Department of Surgery, Ruijin hospital, Shanghai Jiao Tong University School of Medcine. The cancer-associated regions were selected to be minimally necrotic regions of the tumor mass. Non-cancer-associated stroma, which was isolated from tissue at least 2 cm distal to the outer margin of the cancer mass. Normal mucosa samples were taken from the margin of the resection where the tumors located at least over 6 cm apart from it. Tissues were minced into organoids of approximately 1 mm3 and seeded on uncoated plastic material in RPMI 1640 medium containing human basic fibroblast growth factor, 20% fetal calf serum (FCS) and penicillin and streptomycin as antibiotics. These conditions produced a homogenous fibroblastic cell population after 7 days of culture. Each fibroblast was then expanded at a 1:3 ratio by trypsin-EDTA into 10 cm petri dishes. We used fibroblasts passaged for up to 8 population doublings (PDs) for subsequent experiments, in order to minimize clonal selection and culture stress which could occur during extended tissue culture.
Immunohistochemical staining (IHC)
Cells or tissues were fixed for 30 min with 4% formalin and rinsed with phosphate buffered saline (PBS). After endogenous peroxidase activity was quenched with 0.3% H2O2, cells were blocked with normal nonimmune serum for 30 min prior to being incubated with primary antibody at 37°C for 2 hrs. Cells or tissues were then labeled with streptavidin-biotin-horseradish peroxidase staining kit (Dako, Carpinteria, CA, USA). The presence of labeled peroxidase on the sections was visualized by incubation with 3,3′-diaminobenzidine, DAB + substrate chromogen and the sections were counterstained with hematoxylin. Negative control slides were performed by omission of the primary antibody.
TAGLN expression was assessed by the intensity of stained cells, and determined in two categories (weak positive and strong positive). The staining intensity was classified according to four grades (intensity scores): no staining (grade 0), light brown staining (grade 1), brown staining (grade 2), and dark brown staining (grade 3). Grade 0 and grade 1 was defined as weak positive, grade 2 and grade 3 were defined as strong positive.
Quantitative real-time PCR (QRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was obtained with 1 μg RNA using Reverse Transcription System Kit (Promega, Madison, WI, USA) and QRT-PCR was performed by the SYBR Green PCR core Reagent kit (Applied Biosystems, Warrington, UK). Primers specific to TAGLN were as follows: 5′-GAGCAAGCTGGTGAACAGCC-3′ (upper), 5′-GACCATGGAGGGTGGGT TCT-3′ (lower). Glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference. Its sequences were 5′-GGACCTGACCTGCCGTCT AG-3′ (upper), 5′-GTAGCCCAGGATGCCCTTGA-3′ (lower). QRT-PCR for quantitation of the mRNA levels of TAGLN was done on an ABI Prism 7000 (Applied Biosystems, Foster City, CA, USA). Data were analyzed by using the comparative Ct method. Specificity of resulting PCR products was confirmed by melting curves.
Cells were harvested and lysed with mammalian protein extraction reagent (Pierce, Rockford, IL, USA). Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Samples containing 50 μg of total protein were loaded onto each lane of 12% acrylamide gel in a minigel apparatus (Bio-Rad, Richmond, CA, USA). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Richmond, CA, USA). After being incubated with primary antibody (1:1,000) and HRP-conjugated secondary antibody (1:5,000) respectively, immune complexes were detected by the enhanced chemiluminescent (ECL) system (Millipore, Bedford, MA, USA).
Enzyme-linked immunosorbent assay (ELISA)
The protein levels of MMP-2 in supernatants were measured by an ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.
Cell growth assay
The different groups of cells (1 × 103) were seeded into 96-well plates in triplicate. The number of viable cells was determined daily using the Cell Counting Kit (CCK-8) by utilizing a highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo, Japan). Briefly, 20 μl of CCK-8 solution was added into plates, absorbance at 450 nm was measured after 4 hrs incubation.
Assessment of tumor invasion and migration
Tumor invasion and migration assay were performed with QCMTM24-well invasion kit (Millipore, Bedford, MA, USA) and QCMTM24-well migration kit (Millipore, Bedford, MA, USA) according to the protocol provided by the manufacturer, respectively. Add prepared MKN-45 cell suspension (2 × 105 cells/well) to the upper chamber and add pre-suspended fibroblasts (5 × 104 cells/well in RPMI 1640 medium containing 20% FBS) to the lower chamber. The TAGLN of some fibroblasts in lower chambers was inhibited using siRNA 24 hrs later. MKN-45 and fibroblasts were co-cultured non-contactedly for 48 hrs, then lysised the cells and determined RFU values with a fluorescence plate reader using 480/520 nm filter set.
The Qiagen software was used to design RNAi sequences targeting human TAGLN (Accession no. NM_003186), and the siRNA sequence with the highest putative efficacy (sense: 5′-AAAUCGAGAAGAAGUAUGAdtdt-3′, antisense: 5′-UCAUAC UUCUUCUCGAUUUdtdt-3′) was synthesized by Shanghai GeneChem Co, Ltd. siRNA with randomized sequence (sense: 5′-UUCUCCGAACGUGUCACG Utt-3′, antisense: 5′-ACGUGACACGUUCGGAGAAtt-3′) against no gene (scrambled siRNA group) was transfected as internal control. For experiments, cells were transfected with the DOTAP liposomal transfection reagent (Roche, Germany). Cells were seeded at 2 × 105 cells/well in 6-well plates. After 24 hrs when cells were in the phase of log growth, 250 μl Opti-MEM I was mixed with 5 μl of 20 μM siRNA duplex, while another 250 μl Opti-MEM I was separately incubated with 11.88 μl of DOTAP liposome. The two mixtures were gently mixed, and incubated for about 30 min at room temperature. For transfection, the entire mixture was added to each well in 1.5 ml of fresh medium without antibiotics. The final transfected concentration of siRNA was 20 nM. Cells were collected for further assay at 24, 48 and 72 hrs after transfection.
The culture supernatants were harvested at 24 hrs and mixed with a gel sample buffer (0.5 M Tris–HCl, glycerol, 10% sodium dodecyl sulfate [SDS], β-mercaptoethanol, and 0.5% bromophenol blue). Ten micrograms of protein were taken by SDS-PAGE separation; the SDS-PAGE gels contained 0.1% gelatin (Sigma Chemical, St Louis, MO, USA). After electrophoresis, the gels were washed in 50 mM Tris buffer containing 2.5% Triton X-100. The gels were incubated for an additional 24 hrs in incubation fluid (50 mM Tris buffer [pH 7.6], 10 mM CaCl2, and 200 mM NaCl). The gels were stained with 0.5% Coomassie blue, white bands (72 kDa) on a blue background indicated zones of digestion corresponding to the presence of MMP-2.
Female BALB/c nu/nu nude mice, age-matched between 4–5 weeks (Institute of Zoology Chinese Academy of Sciences), were housed at a specific pathogen-free environment in the Animal Laboratory Unit, Shanghai Jiao Tong University School of Medicine, China. Fibroblasts and MKN-45 were mixed at the ratio of 1:4 within 0.1 ml PBS and injected into the lateral tail vein. Six mice were included in each group in all experiments, and each experiment was performed twice. Animals were sacrificed and lung metastatic nodules were recorded 6 weeks after tumor cell implantation. Tumor specimens were collected, mixed in formalin, embedded in paraffin, and subjected to H&E staining. Mice were manipulated and cared according to the guidelines and protocols approved by the Medical Experimental Animal Care Commission of Shanghai Jiaotong University School of Medicine.
All experiments were repeated at least three times. Results were summarized as means ± standard deviation (SD). The correlation between TAGLN expression and clinicopathological parameters was calculated with two-sided chi-square test. P < 0.05 was selected as the statistically signignificant value. SPSS version 11.0 software was used for all analyses.