Human gastric cancer cell line HGC-27 (derived from a lymphatic metastasis) and primary gastric cancer cell lines AGS and SNU-1 were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The cells were incubated in RPMI-1640 medium (Cytiva; cat. no. SH30809.01B) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.; cat. no. 10270-106) at 37˚C in an environment containing 5% CO₂. The protein and mRNA expression levels of KLK6 in HGC-27, AGS and SNU-1 cells were measured using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively.

pSICOR interference vectors and pCDH-CMV- MCS-EF1-CopGFP-T2A-Puro overexpression vectors were supplied by Addgene, Inc. KLK6 mRNA was amplified via PCR (forward, 5'-GCTCTAGATGAAGAAGCTGATGGTG-3'; reverse, 5'-CGGGATCCTCACTTGGCCTGAATGGT-3') and inserted into the pCDH vector to produce pCDH-KLK6 overexpression (OV) plasmids. The short hairpin (sh) interference fragments (shKLK6-1, 5'-AGAATAAGTTGGTGCATGG-3'; shKLK6-2, 5'-CAGATGGTGATTTCCCTGAC-3'; shKLK6-3, 5'-GATCAAAGGAGAAGCCAGGA-3'; and shKLK6-4, 5'-CAGATACACGAACTGGATCC-3') were inserted into pSICOR vectors to produce pSICOR-shKLK6 plasmids. HGC-27 cells were transfected for 48 h with 0.8 µg pCDH-KLK6 (OV-KLK6), pSICOR-shKLK6 (shRNA) or their corresponding negative controls (OV-NC and sh-NC, respectively) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 11668-027). Non-transfected cells served as the control (CON).

Cell viability was measured using the MTT assay. HGC-27 cells were seeded in a 96-well plate at a density of 5x10³ cells/well and maintained overnight at 37˚C with 5% CO₂. A 20 µl volume of MTT reagent (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd.; cat. no. C1736) was added to each well after 24, 48 and 72 h of transfection. The cells were incubated at 37˚C for 4 h in an atmosphere containing 5% CO₂ and subsequently incubated with 150 µl of dimethyl sulfoxide for 10 min. The absorbance of the wells was measured using a microplate reader at 570 nm.

Flow cytometry was performed to evaluate apoptosis and cell cycle progression in HGC-27 cells. To assess cell apoptosis, 1x10⁶ HGC-27 cells were resuspended in 1 ml of phosphate-buffered saline (PBS) and centrifuged at 400 x g for 5 min at 4˚C. The cells were then resuspended in 200 µl of PBS, stained with 10 µl of Annexin V-fluorescein isothiocyanate (BD Biosciences), and 10 µl of propidium iodide (PI; BD Biosciences) in the dark at 4˚C for 30 min. Thereafter, the cells were subjected to flow cytometry. To assess cell cycle progression, 1x10⁷ HGC-27 cells were fixed in a mixture of 300 µl of PBS and 700 µl of absolute ethyl alcohol at -20˚C for 24 h. After two washes with PBS, the cells were resuspended in 100 µl of RNase A (BD Biosciences) and maintained at 37˚C for 30 min. The cells were then stained with 400 µl of PI (50 µg/ml) in the dark at 4˚C for 5 min and subjected to flow cytometry (NovoCyte, ACEA Biosciences, Inc.; Agilent Technologies, Inc.). The data were analyzed using NovoExpress software version 1.3.0 (ACEA Biosciences, Inc.; Agilent Technologies, Inc.). For apoptosis analysis, both early- and late-stage apoptosis (quadrants Q2-2 and Q2-4) were evaluated.

HGC-27 cells were cultured overnight in 6-well plates at a density of 1x10⁶ cells/well. After 4 h of transfection, the cells were wounded by scratching the cell monolayer with a sterile 200-µl plastic pipette tip. The scratches were observed and imaged under an inverted fluorescence microscope (magnification, x100; Leica Microsystems, GmbH) after incubation in serum-free medium at 37˚C in an environment containing 5% CO₂ for 0 and 24 h.

Cell migration and invasion were evaluated using two-chamber Transwell inserts (Corning, Inc.). The cells were starved in serum-free medium for 24 h and resuspended in RPMI-1640 medium (Cytiva; cat. no. SH30809.01B) containing 1% FBS. In the upper chambers, 0.5 ml of treated cells were seeded at a density of 1x10⁵ cells/ml and the lower chambers were filled with 0.75 ml of RPMI-1640 medium supplemented with 10% FBS. The inserts for the cell invasion assay were pre-coated for 30 min at 37˚C with Matrigel (BD Biosciences; cat. no. 354230) between the lower and upper chambers. After 24 h of incubation at 37˚C, the cells were fixed at 25˚C with 4% paraformaldehyde for 10 min and stained with 1 ml of 0.5% crystal violet (Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd.; cat. no. C1701) for 30 min at room temperature. Non-invading or non-migrating cells were wiped away using cotton swabs, and the migrated or invaded cells were counted under an inverted fluorescence microscope (magnification, x100; Leica Microsystems GmbH).

RT-qPCR was performed to examine the mRNA expression levels of KLK6 in HGC-27, AGS and SNU-1 cells, as well as EMT-related genes in HGC-27 cells. Total RNA was extracted from HGC-27, AGS and SNU-1 cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and reverse-transcribed into cDNA using the M-MuLV kit (42˚C for 60 min; 70˚C for 15 min and held at 16˚C), according to the manufacturer's instructions (Takara Bio, Inc). The collected cDNA was amplified using a SYBR Green PCR kit (KAPA Biosystems, Inc.; Roche Diagnostics; cat. no. KM4101) following the manufacturer's instructions in a CFX-Connect 96 apparatus (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: 95˚C for 3 min; 39 cycles of denaturation at 95˚C for 5 sec, annealing at 56˚C for 10 sec, and extension at 72˚C for 25 sec; and final extension at 65˚C for 5 sec and 95˚C for 50 sec. The primer sequences were as follows: KLK6 forward, 5'-GAACTCATCCAGCCCCTT-3' and reverse, 5'-CATCCCC AGCACACAACA-3'; epithelial (E)-cadherin forward, 5'-GGC AAGGTTTTCTACAGC-3' and reverse, 5'-ATGTGGCA ATGCGTTCT-3'; vimentin forward, 5'-TTGAACGCAAAGT GGAATC-3'; and reverse, 5'-AGGTCAGGCTTGGAAACA-3'; GAPDH forward, 5'-CCACTCCTCCACCTTTG-3' and reverse, 5'-CACCACCCTGTTGCTGT-3'. GAPDH served as an endogenous control. Relative mRNA expression levels were calculated using the 2^-∆∆Cq method (16).

Western blotting was performed to determine the protein expression levels of KLK6 in HGC-27, AGS and SNU-1 cells and EMT-related markers in HGC-27 cells. Total proteins were collected using radioimmunoprecipitation assay lysis buffer (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd.; cat. no. W1689) and quantified using a bicinchoninic acid assay kit (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd.; cat. no. W1712). Proteins (20 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked and incubated overnight at 4˚C with primary antibodies against epithelial cell adhesion molecules (EP-CAM, Bioswamp, cat. no. PAB30814, 1:1000), E-cadherin (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd.; cat. no. PAB43792, 1:1000), vimentin (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. PAB40646; 1:1,000), phosphorylated (p)-SMAD2 (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. PAB43294-P; 1:1,000), SMAD2 (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. PAB30712; 1:1,000), p-SMAD3 (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. PAB43521-P; 1:1,000), SMAD3 (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. PAB30705; 1:1,000), and GAPDH (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. PAB36269; 1:1,000) and were subsequently incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG secondary antibodies (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. SAB43714; 1:20,000). The membranes were developed using ECL (EMD Millipore) and then observed using a Tanon-5200 apparatus (Tanon Science & Technology Co., Ltd.) and data were analyzed using Tanon GIS software version 4.2 (Tanon Science & Technology Co., Ltd.). GAPDH served as an internal reference.

Female BALB/c nude mice (age, 6 weeks; weight 18-20 g; n=15) were supplied by Changzhou Cavens Experimental Animal Co. Ltd. (ref. no. 1107301911000025) and divided into five groups (n=3 per group): CON, sh-NC, sh-KLK6, OV-NC and OV-KLK6. All mice were injected with 200 µl of HGC-27 cells at a density of 1x10⁶ cells/m in the right axilla. When the tumor size reached 80-100 mm³ (12 days after injection), the mice were treated with 10 µg of PBS, sh-NC, sh-KLK6, OV-NC and OV-KLK6, respectively, by intratumoral injections once every three days for a total of four times. Animal health and behavior were monitored every day. When the tumor could be clearly identified (4 days after injection), tumor volume was measured every two days using the following formula: volume (mm³) = length x width²/2(17). After 10 days of treatment, the mice were euthanized with an intraperitoneal injection of sodium pentobarbital at 100 mg/kg of body weight (death was confirmed by the absence of heartbeat and breath), and the tumors were removed for further analysis. The humane endpoint was when the tumor maximum diameter reached >15 mm. No animals died during the course of the experiment. All animal procedures were approved by the ethics committee of Wuhan Myhalic Biotechnology Co., Ltd. (approval no. HLK-20181102-01), who also conducted the animal experiments.

Pathological changes in the tumors were evaluated by H&E staining. The tissues were fixed in 10% formalin buffer at 25˚C for 48 h, embedded in paraffin and sectioned at a thickness of 4 µm. After dewaxing, the sections were stained with hematoxylin (Bioswamp, cat. no. I1709) at 25˚C for 3 min and then stained with eosin solution (Bioswamp Life Science Lab; Wuhan Beinlai Biotechnology Co., Ltd; cat. no. I1703) for 3 min. Pathological changes were assessed using a light microscope (magnification, x100), Leica Microsystems GmbH).

Data are presented as the mean ± standard deviation (SD). Differences between two groups were analyzed using unpaired Student's t-test and those between more than two groups were analyzed using one-way analysis of variance, followed by Tukey's test. P<0.05 was considered statistically significant.

As indicated in Fig. 1A and B, the expression of KLK6 was significantly higher in metastatic gastric cancer cells (HGC-27) than in primary gastric cancer cells (AGS and SNU-1). Thus, the effect of KLK6 on gastric cancer was investigated in vitro and in vivo using HGC-27 cells. In HGC-27 cells, KLK6 was inhibited by pSICOR-shKLK6 plasmids and overexpressed by pCDH-KLK6 plasmids (Fig. 1C-F). As shRNA3 interference resulted in the greatest reduction in the expression of KLK6, it was selected for subsequent experiments. MTT assay demonstrated that KLK6 overexpression enhanced the viability of HGC-27 cells, whereas KLK6 inhibition attenuated cell viability in a time-dependent manner in comparison with controls (Fig. 2A). After 24 h of transfection, cell viability was significantly different between the CON group and the sh-KLK6 or OV-KLK6 groups. Therefore, subsequent experiments were performed 24 h after transfection. Flow cytometry indicated that KLK6 inhibition promoted apoptosis and G2/M phase arrest in HGC-27 cells, while KLK6 overexpression showed the opposite effect (Fig. 2B).

The wound healing ability of sh-KLK6-transfected HGC-27 cells was poorer than that of control cells, whereas it was increased by KLK6 overexpression (Fig. 3A). In addition, the Transwell assay showed that KLK6 inhibition reduced the number of migratory and invading cells in comparison with the control, whereas numbers were increased by KLK6 overexpression (Fig. 3B and C).

Western blotting and RT-qPCR were performed to evaluate the expression of EMT-related factors. As shown in Fig. 4, compared to the CON group, the protein and mRNA expression levels of E-cadherin were increased by sh-KLK6 but decreased by OV-KLK6 in comparison with controls, whereas vimentin showed the opposite trend as that of E-cadherin. Additionally, KLK6 inhibition suppressed the protein expression of EP-CAM and the phosphorylation of SMAD2 and SMAD3. These results demonstrated the regulatory effect of KLK6 on EMT in HGC-27 cells through modulation of EMT-related proteins.

To investigate the effect of KLK6 on gastric cancer in vivo, a xenograft mouse model was constructed by injecting HGC-27 cells. Mice were treated with sh-KLK6, OV-KLK6 or the corresponding negative controls. The results demonstrated that both the mRNA and protein expression levels of KLK6 in tumor tissues were increased compared to those in normal tissues (Fig. 5). KLK6 overexpression promoted the growth of gastric cancer, while KLK6 inhibition blocked tumor growth (Fig. 6A-C). H&E staining revealed that KLK6 increased tumor progression (Fig. 6D). In addition, the expression of EMT-related proteins in tumors was measured. Compared to the CON and negative controls, the protein expression of EP-CAM and vimentin and the phosphorylation of SMAD2, and SMAD3 was downregulated by sh-KLK6, whereas that of E-cadherin was upregulated, with OV-KLK6 showing the opposite trend (Fig. 6E).