Human gastric cancer MKN-45 cells were obtained from the Chinese Academy of Sciences (Shanghai, China) and cultivated in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS; both purchased from Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin and streptomycin (both from North China Pharmaceutical Co., Inc., Shijiazhuang, China) in a humidified atmosphere containing 5% CO₂ at 37°C.

H. pylori NCTC11637 bacteria were provided by the Key Laboratory of Digestive System Tumors of Gansu Province (Lanzhou, China) and cultured on Columbia agar plates containing 7% defibrinated goat blood (Solarbio Science and Technology Co., Ltd., Shanghai, China) in an anaerobic tank. Next, the bacteria were acquired and resuspended in RPMI-1640 medium without antibiotics, but containing 10% FBS. The optical density (OD) at 600 nm was used to measure the density of the bacteria (one unit of OD₆₀₀ was equal to 1×10⁸ colony-forming units/ml).

Following digestion, the MKN-45 cells were seeded into three culture dishes and cultured in physiological conditions until they reached the logarithmic growth phase. Next, the old medium was discarded and replaced with RPMI 1640 medium without antibiotics supplemented with FBS. H. pylori were then added to the MKN-45 cells in a bacterium to cell ratio of 100:1. The bacteria and cells were co-cultured for 6 h at 37°C in an atmosphere with 5% CO₂ and saturated humidity, at which point HPA expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting.

A small interfering RNA (siRNA) against HPA and scrambled siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The siRNA sense strand for HPA was 5′-AGUCCGUCCAUUCAAAUAGUAGUGA-3′, and the antisense strand was 5′-UCACUACUAUUUGAAUGGACGGACU-3′. The scrambled siRNA sense strand was 5′-UCUAAGCGAGAAUUCGUACGAUAUC-3′, and the antisense strand was 5′-ACGUGACACGUUCGGAUAAUUAUCU-3′. Lipofectamine^™ 2000 (Santa Cruz Biotechnology, Inc.) was used to transfect the siRNA sequences, according to the manufacturer's protocol. SB203580, a p38 MAPK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA). SB203580 (20 µM) was added to MKN-45 cells for 2 h prior to the co-culture with H. pylori to confirm that the MAPK signalling pathway was involved in H. pylori-induced HPA expression in gastric cancer cells. Cells were then used in RT-qPCR, western blotting, Cell Counting kit-8 (CCK-8), the Transwell method and the Scratch test.

Total RNA was extracted from the cells using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) supplemented with trichloromethane, isopropanol and ethyl alcohol (all obtained from Lianchuang Biotechnology Co., Ltd., Lanzhou, China). RNA quality was analysed using spectrophotometry (Nanodrop 2000). Next, total RNA was reverse transcribed into cDNA using the PrimeScript^™ RT Reagent kit (Takara Biotechnology Co., Ltd.) in accordance with the manufacturer's protocols. The RT reaction was performed for 5 sec at 85°C and 15 min at 37°C. The primers used in qPCR were designed by the Key Laboratory of Digestive System Tumors of Gansu Province and synthesised by Lianchuang Biotechnology Co., Ltd. (GenBank accession no. NM002046). The primer sequences were as follows: HPA sense, 5′-CCTCATCCTCCTGGGTTCTC-3′ and antisense, 5′-TATCCTGGTTGACTTGAGATTGC-3′; and GAPDH sense, 5′-AAGGCTGGGGCTCATTTG-3′ and antisense, 5′-AGGAGGCATTGCTGATGATC-3′. The qPCR amplifications were performed using 7500/7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and SYBR^® Premix Ex Taq^™ II (Takara Biotechnology Co., Ltd.). According to the manufacturer's protocol, a 20 µl reaction volume was used, and the thermal cycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. HPA mRNA levels were quantified using ABI Prism 7900HT and the 2^−ΔΔCq method (40) was used to analyze the results.

The treated cells were washed with ice-cold PBS, and total protein was extracted from the cells using radioimmunoprecipitation assay lysis buffer, phenylmethane sulfonyl fluoride (both from Beyotime Institute of Biotechnology, Haimen, China) and protein phosphatase inhibitor (Solarbio Science and Technology Co., Ltd.). The protein concentration was measured using a BCA protein assay (Beyotime Institute of Biotechnology) subsequent to centrifugation at 13,000 × g for 30 min at 4°C. Next, the protein was separated on a 10% gel by SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Solarbio Science and Technology Co., Ltd.), which were subsequently blocked with 5% non-fat milk for 2 h at 4°C. Following the blocking step, the membranes were incubated with the following primary antibodies: anti-HPA1 (1:1,000; cat. no. ab128931), anti-phosphorylated (p)-p38 MAPK (1:1,000; cat. no. ab195049), anti-p38 MAPK (1:1,000; cat. no. ab170099), anti-p-p65 NF-κB (1:1,000; cat. no. ab76302), anti-p65 NF-κB (1:1,000; no. ab32536; all Abcam, Cambridge, UK) and anti-β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc., Beijing, China). Following overnight incubation at 4°C, the membranes were washed three times with Tris-buffered saline/Tween-20 (Solarbio Science and Technology Co., Ltd.) and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. no. ZA-2301; OriGene Technologies, Inc.) at room temperature for 1 h. The SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) was used to visualise the protein bands, which were imaged using the VersaDoc Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometry measurements were performed to analyze protein expression using Bio-Rad Quantity One Software, version 4.62 (Bio-Rad Laboratories, Inc.).

CCK-8 assay is designed to detect cell proliferation. Briefly, cells were seeded in 96-well plates at a density of 1×10⁴ cells per well. SB203580 (20 µM) was added to MKN-45 cells for 2 h prior to the co-culture with H. pylori, and then 100 µl complete medium and 10 µl CCK-8 reagent was added to each well. After the cells were incubated at 37°C in an atmosphere with 5% CO₂ and saturated humidity for 4 h, the absorbance was detected at a wavelength of 450 nm using a multifunctional enzyme marking instrument.

The different groups of cells were cultured for 72 h. Next, cells were seeded into 6-well culture plates at a density of 300 cells per well, and cell culture medium was added to the wells. Following a 14-day incubation, the cells were washed twice with PBS, fixed at room temperature for 15 min using 99% methanol and stained with crystal violet solution for 20 min. Images were captured and the number of clones were counted using a microscope.

A Transwell assay was used to detect the invasion capability of the tumour cells. Briefly, a 1:8 mixture of Matrigel gel to RPMI-1640 medium (100 µl) was added to the lower chamber, which was placed in 24-well plates at 37°C for 24 h. A serum-free cell suspension (200 µl) at a concentration of 2.5×10⁵ cells/ml was added to the upper chamber, while serum-containing medium was added to the lower chamber. Subsequently, the cells were cultured at 37°C in an atmosphere with 5% CO₂ and saturated humidity for 24 h. Following incubation, the cells remaining in the upper chamber were carefully wiped off, and cells in the lower chamber was rinsed twice with PBS and fixed in methanol for 15 min. The cells were stained with crystal violet solution in methanol for 30 min, and then the excess crystal violet was washed off. Finally, the cells were observed and images were obtained using a microscope. The number of invading cells was counted in several fields of view.

The scratch test was used to detect the migration ability of tumour cells. Briefly, horizontal lines were drawn every 0.5–1 cm on the back of a 6-well plate, and 5×10⁵ cells were added to each well. The cells were incubated overnight until 100% confluence was reached, and then a scratch was made across the dish with a 200 ml pipette. Subsequently, the cells were rinsed twice with PBS, serum-free medium was added, and the cells were incubated at 37°C in an atmosphere with 5% CO₂. Following the incubation, images of the cells were obtained, and the migration distance was measured.

IBM SPSS software (version 22.0; IBM Corp., Armonk, NY, USA) was used to analyse all data. The experimental data are expressed as the mean ± standard deviation. SigmaPlot (version 13.0; Systat Software, Inc., Chicago, IL, USA) was used to construct the graphs. P<0.05 indicated that the difference between groups was statistically significant.

According to previously published studies, the ratio of bacteria to gastric cancer cells is ~100:1 (41,42); therefore, this ratio was used in the experiments conducted in the current study. To detect the effects of H. pylori infection on HPA expression in gastric cancer cells, H. pylori and MKN-45 cells were co-cultured at the aforementioned ratio for 0, 6, 12, 24 and 48 h. The mRNA expression levels of HPA in MKN-45 cells were analysed by RT-qPCR assays. The co-culture of H. pylori and MKN-45 cells induced a significant increase in the mRNA expression level of HPA, which reached a peak level at 6 h and then decreased (Fig. 1A). The findings of western blot analysis supported the RT-qPCR results, demonstrating that HPA expression was also enhanced at the protein level (Fig. 1B and C). The HPA protein level peaked at 24 h in H. pylori-infected gastric cancer cells. Taken together, these results indicated that gastric cancer cells infected with H. pylori had increased HPA, in a time-dependent manner.

To illustrate whether MAPK signalling is involved in the H. pylori-induced expression of HPA, the expression of p-p38 MAPK was detected by western blot analysis after H. pylori and MKN-45 cells were co-cultured for 0, 30, 60, 120 and 480 min. The expression of p-p38 MAPK was significantly higher at 30 min and peaked at 60 min, whereas the expression of p38 MAPK remained unchanged (Fig. 2A and B). To further confirm that H. pylori induces the activation of the MAPK signalling pathway, leading to the activation of NF-κB of MKN-45 cells, the expression of p-p65 NF-κB was also detected by western blot analysis following co-culture of H. pylori and MKN-45 cells for 0–480 min. The expression of p-p65 NF-κB gradually increased with the duration of the co-culture, peaking at 240 min (Fig. 2A and C). Furthermore, MKN-45 cells were pre-treated with a MAPK inhibitor, SB203580, for 2 h prior to co-culture with H. pylori. The expression of p65 NF-κB was significantly lower when MKN-45 cells co-cultured with H. pylori were pre-treated with SB203580 (Fig. 2D and E). Therefore, the results revealed that the MAPK/NF-κB signalling pathway may participate in H. pylori-induced HPA expression in gastric cancer cells, which requires further investigation.

To further illustrate whether the H. pylori-induced upregulation of HPA was mediated through the MAPK signalling pathway, 20 µM SB203580 was added to MKN-45 cells for 2 h prior to the co-culture with H. pylori. The HPA mRNA expression was significantly higher when H. pylori infected the MKN-45 cells, but that upregulation was significantly prevented by SB203580 (Fig. 3A). These changes were also reflected at the protein level (Fig. 3B and C). Furthermore, the CCK-8 proliferation assay confirmed that the addition of SB203580 to H. pylori-infected MKN-45 cells significantly reduced the cell proliferation, as compared with that in untreated H. pylori-infected MKN-45 cells (Fig. 3D). In addition, the Transwell invasion (Fig. 3E and F) and scratch test migration (Fig. 3G and H) assays confirmed that the addition of SB203580 to H. pylori-infected MKN-45 cells markedly decreased the invasion and migration abilities of MKN-45 cells, respectively. These results indicated that the MAPK signalling pathway was involved in the H. pylori-induced upregulation of HPA in gastric cancer cells.

When MKN-45 cells were transfected with siRNA against HPA, the HPA mRNA and protein expression levels were significantly decreased (88 and 83%, respectively; Fig. 4A-C). To verify the involvement of HPA in the proliferation of gastric cancer cells induced by a H. pylori infection, the expression of HPA in H. pylori-infected HPA-knockout MKN-45 cells was detected by RT-qPCR analysis and western blotting. The results indicated that the expression level of HPA in H. pylori-infected HPA-knockout MKN-45 cells was significantly lower compared with that in H. pylori-infected MKN-45 cells at the mRNA and protein levels (Fig. 4D-F). Furthermore, it was revealed that proliferation (Fig. 4G) and colony formation (Fig. 4H and I) were significantly attenuated by HPA knockout in H. pylori-infected MKN-45 cells. These results revealed that HPA may have the potential to become a target for the treatment of gastric cancer, especially when H. pylori infection occurs.