Frozen clinical gastric cancer tissue specimens and matched non-tumorous gastric tissue specimens were collected from 63 gastric cancer patients from at the Department of Gastrointestinal Surgery (Zhengzhou, China). None of the patients received radiotherapy, chemotherapy or biotherapy prior to surgery. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Written informed consent was provided by the patients prior to commencement.

The SGC-7901 and MGC-803 human gastric cancer lines were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). The two cell lines were cultivated in RPMI 1640 (cat no. 21870-076; Gibco-BRL, Invitrogen Life Technologies, Inc., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 4 mM glutamine (cat no. 25030-149; Invitrogen Life Technologies), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/ml streptomycin (Sigma-Aldrich) in an incubator with a humidified atmosphere containing 5% CO₂ at 37°C (12).

NMIIA isoform-specific small interfering (si)RNA and control siRNA were smart pools from Qiagen (Hilden, Germany). siRNA transfection was performed using HiPerfect transfection reagent (Qiagen) according to the manufacturer's instructions. Cells were plated in 12-well plates 72 h after transfection with 2 µM siRNA. Following incubation for 24 h, cells were harvested and subjected to subsequent experiments.

Total RNA was extracted from minced tissues using TRIzol reagent (Invitrogen Life Technologies, Inc.), followed by cDNA synthesis using the TaqMan reverse transcription kit (cat. no. 4304134; Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primers used for the amplification of the cDNAs were as follows: NMIIA forward, 5′-AGA GCT CAC GTG CCT CAACG-3′ and reverse, 5′-TGA CCA CAC AGA ACA GGC CTG-3′; β-actin forward, 5′-ATT GCC GAC AGG ATG CAGA-3′ and reverse, 5′-GAG TAC TTG CGC TCA GGA GGA-3′ (Sangon Biotech, Shanghai, China). The PCR mixture (10 µl) was comprised of 5 µl TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Inc.), 1 µl cDNA (1:50 dilution) and 2 µl of each forward and reverse primer (1 mM). PCR was performed using the following cycling parameters: Denaturation at 95°C for 5 min in the first cycle and for 30 sec in the second cycle, annealing at 57°C for 30 sec and elongation at 72°C for 30 sec, with a final extension at 72°C for 5 min. The β-actin gene was used as an internal control. The PCR products were separated using agarose gel (Beyotime Institute of Biotechnology, Haimen, China) electrophoresis. Data were analyzed by 2^{−[∆Ct sample − ∆Ct control]}.

The expression of NMIIA in the frozen clinical tissue specimens and cultured cells at 24 h after siRNA transfection was examined using western blot analysis. The tissue specimens and cultured cells were homogenized and lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology), containing 100 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1mM EDTA, 10mM glycerophosphate, 2 mM sodium vandate and protease inhibitor. Protein concentration was determined using a Micro-BCA protein kit (Pierce Biotechnology, Inc., Rockford, IL, USA), The protein (40 µg/lane) was then resolved by 12% SDS-PAGE (Beyotime Institute of Biotechnology). Following electrophoresis, the blots were transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were incubated with rabbit polyclonal NMIIA antibody (1:500; cat. no. ab24762; Abcam, Cambridge, UK) at 4°C overnight or anti-β-actin mouse monoclonal antibody (1:1,000; cat. no. A1978; clone AC-15; Sigma-Aldrich) at 37°C for 2 h. After washing with Tris-buffered saline containing Tween 20, the blots were visualized using an enhanced chemiluminescence kit (cat. no. sc-2048; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). In addition, the levels of JNK and c-Jun in SGC-7901 cells were detected by western blot analysis. In this assay, antibodies against phosphorylated (p)-JNK (rabbit monoclonal; cat. no. 4668; clone 81E11; 1:1,500; Cell Signaling Technology, Inc., Danvers, MA, USA) and p-c-Jun (mouse monoclonal; cat. no. 2315; clone L70B11; 1:1,000; Cell Signaling Technology, Inc.), JNK (mouse monoclonal; cat. no. 610627; clone 37/pan-JNK/SAPK1; 1:1,000; BD Transduction Laboratories, Franklin Lakes, NJ, USA) and c-Jun (mouse monoclonal; cat. no. 610327; clone 3/Jun; 1:1,000; BD Transduction Laboratories) were used, which were incubated at 4°C overnight. β-actin (Sigma-Aldrich) were used. All procedures were repeated at least three times. Following antibody incubation, the membranes were washed with Tris-buffered saline containing Tween 20 (pH 7.4) three times. Following enhanced chemiluminescence, the blots were exposed to Kodak X-OMAT BT film (Kodak, Rochester, NY, USA). The bands were visualized using densitometry with Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA)

Briefly, 1×10⁴ cells/well in serum-free medium were seeded into the upper chamber of a Transwell plate (cat no. 3422, Corning Inc., Corning, NY, USA) that was filter-coated with Matrigel (cat no. E1207; Sigma-Aldrich). At the same time, the bottom compartment of the chamber was filled with medium containing 10% FBS as a chemoattractant. After 24 h of incubation at 37°C with 5% CO₂, the cells remaining in the upper chamber were carefully removed using a cotton swab and the cells that had transgressed through the Matrigel and were located at the bottom of the membrane were fixed with 100% methanol and stained with hematoxylin. Quantification was performed by counting the number of cells transgressed through the matrigel using an inverted microscope (GX41; Olympus Corporation, Tokyo, Japan) at 200× magnification.

Cells were seeded into six-well tissue culture plates at 2×10⁶ cells per well and grown in serum-free RPMI 1640 medium for 24 h to form a confluent monolayer. A wound across the cell monolayer was created using a 100-µl pipette tip (Sinoland, Qingdao, China). Cell migration into the wound area was then inspected under an IX70 inverted phase-contrast microscope (Olympus Corporation) at 100× magnification. The distance of wound closure was calculated for quantitative analysis.

Values are expressed as the mean ± standard deviation. Student's t-test was used for comparing significant differences between the means of the two groups. Statistical analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). A P-value from a two-tailed test of <0.05 was considered to indicate a statistically significant distance between values.

To investigate the levels of NMIIA expression in gastric cancer, clinical gastric cancer tissue specimens and matched non-tumorous gastric tissue specimens were subjected to western blot analysis. The results showed that the levels of NMIIA protein expression in 51 out of 63 gastric cancer tissue specimens were increased compared to those in their matched non-tumorous gastric tissue specimens. Statistical analysis showed that differences in NMIIA expression between gastric cancer tissues and matched non-tumorous gastric tissues were statistically significant (P<0.001) (Fig. 1).

As shown in Fig. 2, the MYH9 gene was silenced in SGC-7901 and MGC-803 with specific siRNAs and the levels of NMIIA protein decreased compared with those in the control group (P<0.05). A wound-healing assay was performed to assess the migration capacity of gastric cancer cells. As shown in Fig. 3, knockdown of NMIIA inhibited the migratory capacity of SGC-7901 and MGC-803 cells, as indicated by a decreased amount of wound closure of gastric cancer cells following knockdown of the MYH9 gene (P<0.01), indicating that NMIIA is involved gastric cancer cell migration.

A Matrigel invasion assay was performed to observe the effects of NMIIA knockdown on the invasive capacity of gastric cancer cells. As shown in Fig. 4A and B, knockdown of NMIIA inhibited the number of SGC-7901 and MGC-803 cells transgressing though the Matrigel. Quantification of the results showed that NMIIA knockdown significantly reduced the invasive capacities of the two cell lines (P<0.01) (Fig. 4C).

The levels of p-JNK and p-c-Jun in SGC-7901 cells were detected using western blot analysis prior to and following knockdown of NMIIA expression by siRNA. The results showed that the levels of p-JNK and p-c-Jun in SGC-7901 cells were high. However, after inhibition of NMIIA expression by siRNA, the levels of p-JNK and p-c-Jun were significantly decreased (P<0.05), which indicated that NMIIA is an activator of JNK and c-Jun (Fig. 5).