Knockdown of TIPE2 increases the proliferation in lipopolysaccharide-stimulated gastric cancer cells

This study was approved by the Ethics Committee of Zhongshan Hospital, Xiamen University (Xiamen, Fujian Province, China). Written consent was obtained from all the participants.

The SGC7901 (TCHu 46) and BGC823 (TCHu 11) cells (purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Shanghai, China) were cultured in RPMI 1640, with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA) and 1% penicillin G/streptomycin at 37 °C in an atmosphere of 95% air and 5% CO₂.

Cells with stable TIPE2 knockdown and the controls were incubated without or with LPS for 2 h, and then were analyzed by western blot. Primary antibodies against AKT (#4691), phospho-AKT (#4060), IκBα (#9242), phospho-IκBα (#2859), ERK (9102), phospho-ERK (9101) were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibody against TIPE2 (15940–1-AP), CDK4 (11026–1-AP), Cyclin D3 (26755–1-AP) and beta-actin (20536–1-AP) were purchased from Proteintech (Wuhan, Hubei, P.R.C).

GC tissue section was deparaffinized, rehydrated and then rinsed with PBS. High-pressure antigen retrieval was carried out in citrate antigen retrieval solution (MVS-0101; Maixin Biotech, Fuzhou, China). Endogenous peroxidase was blocked using endogenous peroxidase blocking solution (SP KIT-A1; Maixin Biotech, Fuzhou, China). The sections were incubated with 1% Triton X-100 for 10 min, followed by 10% normal donkey serum for 15 min at room temperature. Next, the sections were incubated overnight with rabbit anti-human TIPE2 antibody (15940–1-AP, Proteintech, Wuhan, Hubei, China) overnight at 4 °C. Next day, the sections were rinsed with PBS, incubated with biotin-labeled secondary antibody for 20 min at room temperature, rinsed againwith PBS, and incubatedwith horseradish peroxidase polymer conjugate (Elivision™ Super HRP (Mouse/Rabbit) IHC Kit-9922; Maixin Biotech, Fuzhou, China). The sections were stained with the chromogen 3,3-diaminobenzidine from the DAB Detection Kit (DAB-0031; Maixin Biotech, Fuzhou, China) for approximately 3 min and counterstained with hematoxylin.

Cells with stable TIPE2 knockdown and the controls were seeded into 96-well plates at a density of 2 × 10³ cells/well without or with 1 μg/ml LPS for 2 h. After 24, 48, 72, 96 h or 5 d, cells were incubated with Cell Counting Kit-8 solution (DoJinDo, Tokyo, Japan) for additional 1 h. The absorbance was measured using a microplate reader at a wavelength of 450 nm.

The EdU assay was performed according to the manufacturers’ instructions (RiboBio). Cells were seeded at 2 × 10⁴ cells/well in a 6-well plate and then incubated with 1 μg/ml LPS for 2 h. Finally, cells were incubated with 50 μM EdU for 2 h, and then the nuclei were stained with DAPI (Invitrogen). The images were acquired using a Leica SP laser scanning microscope system.

Cells were seeded at 1 × 10⁴ cells/well in a 12-well plate and then incubated with 1 μg/ml LPS for 2 h. Cell monolayers were collected and fixed by the dropwise addition of 70% ethanol at − 20 °C. Then, the fixed cells were washed with PBS and incubated in the dark for 30 min with 50 μg/ml propidium iodide (PI) and 100 μg/ml RNase A and measured with a flow cytometer (BD, Franklin Lakes, NJ) equipped with a 488-nm argon laser. Histograms of the PI intensities were plotted. The percentage of cells in each phase of the cell cycle was analyzed using ModFit software.

Statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-test (means ± standard deviation) and Chi-Square test were used for data analyze according to different data types. p <  0.05 or p <  0.01 was considered to be statistically significant. Graphs were illustrated by GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA).

We collected tumor samples from 46 human GC patients and detected the expression in human GC tissues. TIPE2 expression at mRNA level was significantly lower in gastric tumor lesions than in adjacent non-cancerous tissues (Fig. 1a, P < 0.05). Consistent with mRNA levels, TIPE2 expression at protein levels were significantly lower in GC lesions than in adjacent tissues by immunohistochemistry (Fig. 1b). Additional samples were collected and TIPE2 expression was measured in 68 pairs of GC and normal tissue, the positive expression rate of TIPE2 in GC was 17.65%, significantly lower than that in normal gastric mucosa (72.06%) (Table 1, P < 0.00). Further analysis of clinical characteristics revealed that TIPE2 was closely associated with tumor differentiation, stages and lymph node metastasis. Low expression of TIPE2 indicated the worse differentiation and the poor prognosis of GC patients (Table 2). Thus, these results indicate that TIPE2 expression decreases as GC progresses.

To determine the TIPE2 effects in GC development, we established stable TIPE2 knockdown SGC7901 and BGC823 cell lines from three TIPE2 shRNA sequences (sh213, sh431 and sh523), as well as controls. TIPE2 expression was drastically inhibited in the TIPE2 knockdown SGC7901 (Fig. 2a) and BGC823 (Fig. 2c) cell lines at protein level. Since sh523 showed higher efficacy than sh213 and sh431 in inhibiting TIPE2 expression, we chose sh523 for the subsequent experiments. Then we performed CCK-8 proliferation assays and demonstrated that there are no significant differences of cell proliferation ability between shGFP-control SGC7901 and BGC823 cells with or without 1 μg/ml lipopolysaccharide (LPS) treatment (Fig. 2b and d, shGFP-DMSO group versus shGFP-LPS group, DMSO as control). However, the cell proliferation was significantly increased in stable TIPE2 knockdown SGC7901 (Fig. 2b, P < 0.05) and BGC823 (Fig. 2d, P < 0.05) cells under the stimulation of LPS compared to the DMSO controls treatment.

EdU proliferation assays were also performed in TIPE2-knockdown cell lines. Treated with LPS, stable TIPE2-knockdown SGC7901 and BGC823 cells showed increased cell proliferation compared with corresponding controls (Fig. 3a-d, P < 0.05). All these results collectively indicate that knockdown of TIPE2 promotes proliferation of GC cells under the LPS stimulation.

Cell proliferation and cell cycle changes are closely related, thus, we performed flow cytometry to detect the cell cycle of TIPE2-knockdown GC cells under LPS stimulation. shTIPE2 significantly decreased cell G₀/G₁ phase ratio and increased G₂/M phase in both SGC7901 (Fig. 4a and b) and BGC823 (Fig. 5a and b). Thus, shTIPE2 promoted mitosis of GC cells to increase cell proliferation.

AKT, IκBα and ERK phosphorylation levels were analyzed to identify the molecular signaling pathways of TIPE2-mediated GC cell proliferation. AKT and IκBα phosphorylation was declined in TIPE2 knockdown SGC7901 and BGC823 cells, while no significant differences in ERK phosphorylation were observed (Fig. 6a-d).

Next, we examined cell cycle related proteins and explored that CDK4 and CyclinD3 levels were significantly upregulated in TIPE2 knockdown SGC7901 and BGC823 cells compared with control cells (Fig. 6a-d). Therefore, TIPE2 regulates the proliferation may via AKT and IκBα phosphorylated activation in GC cells.