ZBTB20 promotes cell migration and invasion of gastric cancer by inhibiting IκBα to induce NF-κB activation

Abstract Zinc finger and BTB domain containing 20 (ZBTB20), a sequence-specific transcriptional repressor, has been found to be involved in tumorigenesis. However, its role(s) in gastric cancer and the molecular mechanisms involved are poorly investigated. Here, our data demonstrated that ZBTB20 expression was markedly upregulated in gastric cancer cell lines infected with Helicobacter pylori (H. pylori) and in gastric cancer tumor samples. Loss- and gain-of-function studies showed that ZBTB20 promoted cell proliferation, invasion and migration of gastric cancer cell lines. Mechanistically, the phosphorylation of NF-κBp65 and expression and activity of MMP-2 and MMP-9 were increased, while IκBα expression was decreased by ZBTB20 in gastric cancer cells. We further revealed that IκBα overexpression significantly inhibited NF-κB signaling as well as cell migration, invasion and proliferation in gastric cancer cell lines induced by ZBTB20 overexpression. Therefore, our findings emphasize an important role for ZBTB20 in controlling gastric cancer development, which is helpful to identify potential therapeutic targets for its treatment.


Introduction
Gastric cancer is one of the most common malignancies worldwide with an overall 5-year survival rate lower than 25% [1]. Despite efforts to improve diagnostic technology and patient managements, the prognosis of gastric cancer patients has not been improved significantly [2]. The formation of gastric cancer is a complex process, which eventually develops into gastric cancer from precancerous lesions such as superficial and atrophic gastritis, intestinal metaplasia, and dysplasia. Helicobacter pylori (H. pylori) infection is a common chronic infection that is one of the most important risk factors for gastric cancer compared with others such as genetic factors, high-salt diets, smoked foods, smoking, and alcohol abuse [3]. H. pylori infection is necessary but not sufficient for the development of gastric adenocarcinoma. Globally, the infection rate of H. pylori is about 40%-80%, but only 1% of H. pylori infections progress to gastric cancer [4]. It is suggested that individuals with different genetic backgrounds have different susceptibility to gastric cancer when exposed to the same environment. Given that different DNA, mRNA and protein expressions modulated by H. pylori infection could regulate gastric cancer cell proliferation, apoptosis, migration and invasion, they could serve as therapeutic targets in gastric cancer [5,6].
Zinc finger and BTB domain containing 20 (ZBTB20) has been implicated in developmental neurogenesis [7], glucose and lipid homoeostasis, and the tumorigenesis of glioblastoma [8], liver [9] and lung cancer [10]. Polymorphisms of ZBTB20 appeared to be associated with gastric cancer susceptibility in allelic, homozygous, dominant and recessive models, suggesting its role in gastric cancer [11]. NF-jB is activated in a variety of cancers as detected by phosphorylation and degradation of IjBa proteins, phosphorylation of RelA/p65, and elevated expression of NF-jB target genes. ZBTB20 enhances the innate immune responses induced by Toll-like receptor (TLR) through promoting NF-jB activation by repressing IjBa gene transcription [12]. Moreover, the activation of NF-jB was significantly attenuated by ZBTB20 deficiency after partial hepatectomy and was associated with hepatocyte proliferation in mouse liver regeneration [13]. However, whether and how IjBa/NF-jB signaling pathway involved in the ZBTB20-associated carcinogenesis in gastric cancer is still unknown.
Here, we determine the effect of ZBTB20 on the regulation of cell migration, invasion and proliferation of gastric cancer and explore the possible molecular mechanism involved in NF-jB signaling. Our findings reveal that ZBTB20 promotes cell migration and invasion of gastric cancer by inhibiting IjBa to induce NF-jB activation.

Clinical samples
In total, the primary tumor samples were collected from 37 patients at Xuzhou Central Hospital who underwent surgery for gastric cancer between 2014 and 2017. None of the patients received preoperative radiotherapy or chemotherapy. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Xuzhou Central Hospital and with the 1964 Helsinki declaration. This study protocol was approved by the Ethics Committee of Xuzhou Central Hospital. Written informed consents were provided prior to enrollment of patients.

Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Five-micrometer-thick slices were incubated in 3% H 2 O 2 in methanol and 5% normal horse serum to minimize nonspecific staining. Sections were subsequently incubated overnight with the primary anti-ZBTB20 antibody (1:500; ab127702; Abcam, USA) at 4 C, and then incubated with horseradish peroxidase (HRP)-labeled IgG secondary antibody (Beyotime Institute of Biotechnology, Shanghai, China) for half an hour at 25 C. The section was then stained with diaminobenzidine (DAB) and hematoxylin for 3 min and washed in water for 10 min. Fields from each slide were examined and photographed under a light microscopy (Â200). Quantitative data of positive area for immunocytochemistry (IHC) was analyzed using Image-pro Plus 6.0 software (Media Cybernetic, Silver Springs, MD, USA).
Luciferase reporter gene assay BGC-823 and MKN-45 cells were transfected with the pGL3-ZBTB20 promoter using Lipofectamine 2000 (Invitrogen Life Technologies) following the manufacturer's protocol. After 12 h transfection, cells were infected with H. pylori. Then, the cells were collected, and Luciferase activity was determined using a Luciferase assay kit (Promega).

Wound healing assay
BGC-823 and MKN-45 cells were seed in 35 mm 2 tissue culture dishes at a density of 8 Â 10 5 and further seeded until reached 100% confluence. Then confluence cultures were scratched using a pipette tip. After scratching, gently wash the well twice with medium to remove the detached cells. Scratched cultures were photographed under a microscope at 0 and 24 h. Migration of cells was established by measuring the width of the scratched area at each time point in the scratched area.

Gelatin zymography gel assay
To investigate the MMP-2 and MMP-9 activities in gastric cancer cell lines, proteins were separated on a 10% SDS-PAGE containing 1% gelatin (Sigma-Aldrich, M€ unchen, Germany). The gels were incubated at 37 C overnight, stained with Coomassie Blue, de-stained, and then scanned. Signal intensity was quantified by densitometry using Bio-1D software version 15.01 (Vilber Lourmat, Eberhardzell, Germany).

Western blotting
BGC-823 and MKN-45 cells were lysed by RIPA lysis buffer (Solarbio). After centrifugation, the supernatants were collected and BCA reagent was used to determine the concentration of protein. Next, 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to isolate the proteins. After being transferred to polyvinylidene fluoride membranes, the protein was blocked with nonfat dry milk, added antibodies, and detected with supersignal west picochemiluminescent substrate. Primary antibodies include anti-ZBTB20 (

Statistical analysis
All data were representative of experiments done in triplicate and were expressed as the mean ± SD. Samples were evaluated by the SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA) using the Student 0 s t test for two groups and analysis of variance (ANOVA) for multiple groups. Overall survival of gastric cancer patients from Kaplan-Meier plotter database was analyzed by the Kaplan-Meier survival curves and logrank nonparametric test. When p < .05, the difference between groups was statistically significant.

ZBTB20 expression is increased in human gastric cancer tumors and correlated with overall survival
Analysis of tissues collected from our independent hospital demonstrated that ZBTB20 mRNA expression levels in human gastric cancer tumor samples (n ¼ 37) were higher than those in the paired adjacent normal gastric tissue samples (n ¼ 37) (Figure 1(A)). Similarly, ZBTB20 protein expression measured by immunohistochemistry was also upregulated in human gastric cancer tumor samples compared with the paired adjacent normal gastric tissue samples (Figure 1(B)). Mover, ZBTB20 mRNA expression levels were also increased in patients with H. pylori infection or metastasis (Figure 1(C,D)). According to the Kaplan Meier-plotter database, higher ZBTB20 expression also correlated with a poor overall survival of gastric cancer patients (Figure 1(E)).

ZBTB20 expression is increased by H. pylori infection in gastric cancer cell lines
To assess the role of ZBTB20 in gastric cancer development, the expression of ZBTB20 in gastric cancer cell lines was measured. Our data showed that ZBTB20 expression was increased in gastric cancer cell lines compared with GES-1 cells, with the highest expression detected in BGC-823 and MKN-45 cells (Figure 2(A)). These two cell lines were therefore used for subsequent experiments. Given that H. pylori infection is considered a major risk factor for gastric cancer, the effect of H. pylori infection on the expression of ZBTB20 was further examined in BGC-823 and MKN-45 gastric cancer cell lines. As shown in Figure 2(B,C), H. pylori infection significantly increased the mRNA and protein expression of ZBTB20 in BGC-823 and MKN-45 cells in a dose-dependent manner. Moreover, the transcriptional activation of ZBTB20 upon H. pylori infection was measured by the luciferase reporter gene assay (Figure 2(D,E)). To investigate the role of IjBa/NF-jB signaling pathway in gastric cancer, the effect of H. pylori infection on the expression of IjBa, p-NF-jB and NF-jB was also measured. We found that H. pylori infection significantly decreased IjBa expression, increased p-NF-jB levels, but had no effect on NF-jB expression in BGC-823 and MKN-45 cells (Figure 2(F,G)). Taken together, these data support the notion that ZBTB20 and IjBa/NF-jB signaling pathway may participant in the occurrence and development of gastric cancer.

ZBTB20 silencing represses cell invasion and migration of gastric cancer
Furthermore, siRNA-1 and siRNA-2 also significantly inhibited the migration of BGC-823 cells by 48.2% and 57.2% and the invasion of BGC-823 cells by 57.4% and 58.3%, respectively, compared with siNC ( Figure 4(A)). siRNA-1 and siRNA-2 also significantly inhibited the migration of MKN-45 cells by 41.8% and 50.5% and the invasion of MKN-45 cells by 53.3% and 56.1%, respectively, compared with siNC (Figure 4(B)). Compared with siNC, the scratch wound was wider with fewer migrating cells at 24 h after transfected with siRNA-1 and siRNA-2 ( Figure 4(C,D)). Moreover, the expression of IjBa, p-NF-jBp65 and NF-jBp65 as well as the expression and activity of MMP-2 and MMP-9 were measured by western blotting and Gelatin zymography gel assay. It showed that siRNA-1 and siRNA-2 transfection in BGC-823 and MKN-45 cells significantly reduced the phosphorylation of NF-jBp65 and the expression and activity of MMP-2 and MMP-9, but induced IjBa expression compared with siNC ( Figure 4(E-H)).

ZBTB20 overexpression promotes cell proliferation, invasion and migration of gastric cancer
In addition, recombinant pCDNA3.1(þ)-ZBTB20 or blank pCDNA3.1(þ) as a negative control was also transduced into BGC-823 and MKN-45 cells. Overexpression of ZBTB20 markedly increased the mRNA and protein expression of ZBTB20 by 3.98-fold and 1.10-fold in BGC-823 cells and by 7.12-fold and 1.61-fold in MKN-45 cells, respectively, compared with the blank vector ( Figure 5(A-D)).

The involvement of IjBa/NF-jB signaling in the function of ZBTB20
Give the role of ZBTB20 in the regulation of expression of IjBa, NF-jBp65, MMP-9 and MMP-2, we speculate that inhibition of IjBa expression to induce NF-jB activation may involve in the function of ZBTB20 in gastric cancer. Therefore, IjBa expressing vector (pCDNA3.1(þ)-IjBa) was constructed in BGC-823 and MKN-45 cells and its effect on NF-jB activation, cell proliferation, migration and invasion was also measured. As shown in Figure 7(A-D), overexpression of IjBa markedly increased the mRNA and protein expression of IjBa by 9.43-fold and 7.19-fold in BGC-823 cells and by 9.78-fold and 0.60-fold in MKN-45 cells, respectively, compared with the blank vector. Moreover, our data also demonstrated that IjBa overexpression significantly inhibited the proliferation, invasion and migration of MKN-45 and BGC-823 cells induced by ZBTB20 overexpression (Figures 7(E,F) and 8(A-D)). We further checked up the phosphorylation of NF-jBp65 as well as the expression of IjBa, NF-jBp65, MMP-2 and MMP-9. As shown in Figure 8(E-H), IjBa overexpression significantly inhibited the phosphorylation of NF-jBp65 and the expression and activity of MMP-2 and MMP-9 in BGC-823 and MKN-45 cells induced by ZBTB20 overexpression, suggesting the inactivation of NF-jB signaling. These data indicate that ZBTB20 promotes invasion, migration and proliferation of gastric cancer cell lines by inhibiting IjBa expression to induce NF-jB activation.

Discussion
Although increasing studies have focused on ZBTB20 and cancer [8][9][10], the underlying mechanisms of ZBTB20 in cancer initiation remain largely unknown. In this study, we found that ZBTB20 expression is markedly upregulated in gastric cancer tissues and in gastric cancer cell lines infected with H. pylori. ZBTB20 knockdown significantly inhibits cell proliferation, invasion and migration as well as induces IjBa expression to inactivate NF-jB signaling in gastric cancer, while ZBTB20 overexpression induced the inverse effects were inhibited by IjBa overexpression.
Gastric cancer (GC) is the third leading cause of cancerrelated deaths worldwide, and H. pylori infection is an important factor in the pathogenesis of gastric cancer. The major mechanisms of H. pylori-associated gastric cancer include perturbed multiple signaling pathways, induced mutations, and accumulated aberrant DNA methylation [14]. In this study, we presented that ZBTB20 expression was significantly upregulated in gastric cancer tissues and in gastric cancer cell lines infected with H. pylori, which suggest that ZBTB20 may involve in the initiation and progression of gastric cancer. Our study evaluated for the first time correlations between H. pylori infection and ZBTB20 expression in gastric cancer cell lines. Similar to our findings, previous studies have demonstrated that ZBTB20 is significantly up-regulated in liver and lung cancer tissues compared with adjacent normal tissues [9,10]. ZBTB20 suppressed apoptosis and induced cell invasion, migration and proliferation of glioblastoma [8]. ZBTB20 promoted cell cycle progression and cell proliferation in hepatocellular carcinoma and non-small cell lung cancer [9,10]. Gastric cancer is characterized by poor prognosis, strong invasion, and early metastasis. Identify new and specific markers for gastric cancer that contributes to this neoplasm at an early stage will improve the poor prognosis. A study showed that the rs9841504 polymorphism in ZBTB20 was statistically susceptible to gastric cancer [15]. Consistently, our data showed that ZBTB20 knockdown in gastric cancer cell lines markedly suppressed cell proliferation, invasion and migration while ZBTB20 overexpression significantly promoted those. However, Shi et al., found a significant association between the rs9841504 polymorphism in ZBTB20 and decreased gastric cancer susceptibility, suggested that the ZBTB20 rs9841504 polymorphism is a protective factor for gastric cancer [11]. Meanwhile, no statistical difference was found between the rs9841504 polymorphism in ZBTB20 and gastric cancer risk in other previous studies [16][17][18]. It is likely that the contradictory results were due to the relatively small sample sizes and different genetic backgrounds. Therefore, the role of ZBTB20 in regulating gastric cancer process needs to be further investigated.
In cancer, NF-jB is proposed to contribute to oncogenesis through the induction of genes encoding proteins involved in promoting invasion and migration and suppressing apoptosis. Suppression of NF-jB activity through the inhibition of IjBa degradation contributes to the decreased cell migration in triple negative breast cancer [19]. Activating the NF-jB signaling pathway also promoted cell invasion, migration and proliferation in gastric cancer [20]. The NF-jB protein dimer is complexed with IjB protein under resting conditions. After stimulation with cytokines or lipopolysaccharide, IjBa is phosphorylated by upstream IKK (IjB kinases) and degradation, finally resulting in the activation of NF-jB [21]. Consistently, our loss-and gain-of-function studies showed that ZBTB20 knockdown significantly induced IjBa expression, and then resulted in the decrease in phosphorylation of NF-jBp65 in gastric cancer cell lines. Consistent with our findings, and mechanistically, ZBTB20 promotes NF-jB activation through inhibiting IjBa gene transcription and governing IjBa protein expression [12]. Moreover, ZBTB20 promotes hepatocyte proliferation and activation of NF-jB in regenerating liver after partial hepatectomy [13].
In this study, we also found that the expression and activity of MMP-9 and MMP-2 were markedly downregulated in ZBTB20 knockdown cells but upregulated in ZBTB20 ectopic cells. Matrix metalloproteinases (MMPs) are of great importance in regulating cell migration and proliferation. Dysregulation of MMPs is correlated with many diseases including cancer [22,23]. NF-jB has been shown to directly regulate the transcription of MMP-2 and MMP-9 [24]. The upregulation of MMP-2 and MMP-9 expression by activating NF-jB signaling pathway could promote metastasis in hepatocellular carcinoma [25], induce cell proliferation, invasion and migration in gastric cancer in vitro, and promote tumor growth and metastasis in gastric cancer in vivo [23]. Thus, it is indicates that ZBTB20 regulates cell invasion, Figure 8. IjBa overexpression inhibits gastric cancer cell migration and invasion induced by ZBTB20. BGC-823 and MKN-45 cells were infected with pCDNA3.1(þ)-ZBTB20 and pCDNA3.1(þ)-IjBa. Cell migration and invasion were determined by Transwell (A, B) and Wound healing assay (C, D); the protein expression of IjBa, p-NF-jBp65, NF-jBp65, MMP-2 and MMP-9 (E, G) was determined by western blotting; and the activity of MMP-2 and MMP-9 was determined by Gelatin zymography gel assay (F, H). ÃÃ p < .01, ÃÃÃ p < .001 compared with control; ### p < .001 compared with pCDNA3.1(þ)-ZBTB20. migration and proliferation in gastric cancer through IjBa/ NF-jB signaling pathway.

Conclusion
In summary, we have identified upregulated ZBTB20 expression in gastric cancer cell lines infected with H. pylori and in human gastric cancer tumor samples. ZBTB20 promotes cell invasion, migration and proliferation of gastric cancer through inhibiting IjBa to induce NF-jB activation. These findings demonstrated that ZBTB20 may be an appropriate target for gastric cancer prevention and treatment.

Disclosure statement
There are no competing financial interests in relation to this work.