DEC1 promotes progression of Helicobacter pylori‐positive gastric cancer by regulating Akt/NF‐κB pathway

Abstract Helicobacter pylori (H. pylori) infection plays a crucial role in the initiation and progression of gastric cancer (GC). Differentiated embryo‐chondrocyte expressed gene 1 (DEC1) is dysregulated in some cancers and may regulate cell proliferation in specific contexts. Of note, DEC1 is emerging as one of the important factors regulating cellular responses in microenvironment. However, the triggers and precise regulation mechanism for DEC1 during inflammatory carcinoma transformation of GC are unclear. In this study, we identified DEC1 was upregulated in both H. pylori‐infected gastric tissues and GC cells. DEC1 expression was positively associated with H. pylori infection status and GC progression. DEC1‐positive expression indicated a poorer prognosis in H. pylori‐positive GC. DEC1 was required for H. pylori‐induced GC cells proliferation. Mechanistically, H. pylori infection significantly activated Akt/NF‐κB signal pathway and this induction depend on DEC1 expression level in GC cells. Importantly, their interaction pathway was further verified by H. pylori‐positive gastritis mice model. Taken together, our findings identified a novel function of DEC1 in GC. H. pylori infection induce DEC1 expression, and which leading to the progression of GC through activating Akt/ NF‐κB signalling pathway. Blocking DEC1/Akt/NF‐κB, therefore, presents a promising novel therapeutic strategy for H. pylori‐positive GC.


| INTRODUC TI ON
is a hypoxia-induced gene involved in cell differentiation, proliferation and apoptosis. 7 DEC1 could resist oxidative stress-mediated cell death in skeletal muscle and podocytes. 8,9 Chemotherapy drugs, tumour necrosis factor (TNF)α and transforming growth factor (TGF)β could induce DEC1 expression and regulate apoptosisrelated signalling pathways. [10][11][12] DEC1 also can drive inflammation and enable typical immune responses in autoimmunity and infection. 9,10 DEC1 is a key regulator of cytokine production by human T cells and essential for mitochondrial metabolism in tumour infiltrating lymphocytes. [13][14][15][16][17] H. pylori infection is an intrinsic niche for tumorigenesis and GC progression. However, it is not clear whether the inflammatory environment regulates the expression of DEC1 in cancer cells. Furthermore, in cancer-related studies, the role of DEC1 as a promoter or inhibitor of cancer remains controversial. 3,12,[18][19][20][21][22][23][24] DEC1 expression level also can be used to predict the effect and clinical prognosis of cancer after radiotherapy and chemotherapy. 6,20,25 Our previous studies have shown DEC1 is highly expressed in GC, and it is negatively correlated with the degree of differentiation of tumours. 19,26 However, the original causes that initiate the increased expression of DEC1 in GC during carcinogenesis are still unknown.
It is well established that some oncogenic signalling pathways, including the PI3 kinase-Akt, β-catenin-WNT, Hedgehog/GLI, ERK, JNK and NF-κB pathways, were involved in H. pylori pathogenesis. [27][28][29][30][31] Therefore, there are some key genes could determine the pathogenic effect of H. pylori by regulating these oncogenic signalling pathways. It has been shown DEC1 is upstream of p-Akt, p-GSK3β, p53, caspase-8 and Fas. 11,32,33 Investigation the complex intracellular signalling events related to the role of DEC1 in H. pyloriinfected GC may be important for further elucidation of its role in the pathogenesis of tumours.
In this study, we find that H. pylori induce DEC1 expression in GC. Moreover, we validate DEC1 is required for the oncogenic role of H. pylori by activation p-Akt/ NF-κB signal pathway. The role of DEC1 in the progression of H. pylori-positive GC may imply DEC1 as a potential therapeutic target.

| Bioinformatics analysis
GSE chips were downloaded from the GEO website (https://www. ncbi.nlm.nih.gov/ geo/), and gene expression data of each sample in the chips were normalized using R 3.3.3 software. Bioconductor limma package was used to analyse the differential expression genes.

| Clinical samples
Eighty human gastritis tissues were purchased from Alenabio Biotechnology Co., Ltd (Xi'an China). A total of 80 samples of primary GC and tumour adjacent gastric mucosa were collected from the department of pathology, Jinan central hospital. 32 samples of H. pylori-positive GC were collected. The patients did not receive any chemotherapy or radiotherapy before the operation. Pathological TNM staging is based on the 8th AJCC Cancer Staging System. This study was approved by the ethics committee of Jinan central hospital.

| Immunohistochemical staining of paraffin sections
Immunohistochemical (IHC) staining was performed by the streptavidin-peroxidase method. In brief, after deparaffinization, hy-

| Cell transfection and lentiviral transduction
The DEC1 vector (Shanghai GeneChem, Shanghai, China) was prepared with full-length complementary DNA (cDNA). An empty vector GV492 (EV) was used as the control. shRNAs specific to DEC1 expression were from our previous study. 19 The target sequence of the shDEC1 was 5'-CATTGCCCTGCAGAGTGGTTTACAACTT CC TGTCAGATTGTAAACCACTCTGCAGGGCAATG-3'. The lentiviral vectors were cotransfected with packaging vectors psPAX2 and pMD2G into 293T cells for lentivirus production. Transfection was performed using the Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Puromycin (2 μg/ml, Sigma) was used to select stable clones for at least 1 week. At the indicated time points, the cells were harvested for mRNA and protein analysis as well as for other assays.

| Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted using Trizol reagent (Invitrogen). Reverse transcription reaction kit (Takara, Japan) was used for the synthesis of the cDNA. The operation is carried out according to the manufacturer's instructions. The real-time fluorescent quantitative polymerase chain reaction was performed by ABI 7500 systems.

| Western blot analysis
RIPA lysate (containing PMSF and phosphatase inhibitors) was used for the extraction of total cell protein (Beyotime Ltd, China).

| Cell proliferation assay
Cell Counting Kit-8 (Dojindo, Japan) was used to detect cell proliferation. The experiment was carried out according to the instructions.
In a 96-well plate, 5 × 10 3 cells were added to each well. The next day, 10μl CCK-8 reagents were added to each well and incubated with cells at 37°C for 2 hours. Use microplate reader (Bio-Rad, USA) to measure 450nm absorption. Test once a day for 5 days. The experiment was repeated three times.

| EdU
The operation is carried out according to the instructions of the celllight TM EdU Apollo 567 in vitro kit (Ribobio Ltd, China). In 96-well plates, 5 × 10 3 cells were added to each well. 100μl culture medium (0.1%EdU) was added to each well for 2 hours before fixation in 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100, 100 μL 1×Apollo-staining reaction liquid was added to cells at 37°C for 30 minutes, the cells were counterstained with 4′,6-diamidino-2-phenylindole(DAPI) and imaged using a fluorescence microscope (Olympus, Tokyo, Japan). Proliferating cells was fluoresce red. Nuclei were counterstained with blue-fluorescent DNA stain DAPI.

| Cell clone formation test
In the 24-well plate, 1000 cells were added to each well. After   Repeat this infection process every other day 5 times. Three months later, the mice were sacrificed and gastric mucosae were retained.

| Animal experiments
The experimental operation was examined and approved by the Ethics Committee of Shandong University.

| Statistical analysis
All in vitro experiments were repeated more than three times, and three parallel groups were set up each time. The analysis data were expressed as (mean±SD). Use group t-test for two independent samples. The relationship between DEC1 expression and pathological parameters was examined by Fisher's exact test. p < 0.05 means the difference has statistical significance.

| DEC1 expression is positively correlated with H.pylori infection status and the progression of GC
In our previous study, we demonstrated that the DEC1 was upregulated in GC tissues compared to adjacent normal tissues. To dissect the potential relationship between DEC1 expression and infection of H. pylori in GC development, we firstly analysed the relative expression of DEC1 in public microarray data from the GEO database.
We downloaded two publicly available mRNA chips about interaction between H. pylori and gastric cells ( Figure 1A). The strains Kx1 and Kx2 used in GSE10262 chip came from a same patient. Kx1 was isolated from the gastric tissue of patients with chronic atrophic gastritis four years ago, and Kx2 was isolated from patients with gastric adenocarcinoma four years later. Differential gene expression

| DEC1 is an important mediator for H. pylori to perform tumorigenic activities
Our previous study has shown the DEC1 expression was high in MKN-45, but low in MGC-803. 19 We downregulated DEC1 ex-

| DEC1 regulates Akt/NF-κB pathway to support H. pylori-induced proliferation in GC cells
To gain insights into the mechanisms by which DEC1 are linked to the progression of H. pylori-positive GC, GSEA between low and high DEC1 expression was conducted based on the RNA-seq data from GSE62254. 34 H. pylori-positive GC patients with higher DEC1 expression showed enrichment of genes associated with the epithelial cell signalling in Helicobacter pylori infecting, apoptosis and pathway in cancer ( Figure 4A). Those pathways highlighted the association between DEC1 and H. pylori-positive GC progression. Notably, Akt and NF-κB were also significantly enriched in these pathways. The Akt/NF-kB signalling pathway plays a role in the proliferation of tumour cells. We next tested whether H. pylori infection could activate Akt/NF-kB pathway via enhancing DEC1 expression. As predicted, the Akt/NF-κB pathway was activated in GC cells by H. pylori infection ( Figure 4B). p-Akt and NF-κB expressions were inhibited by DEC1-shRNA while increased by DEC1 overexpression ( Figure 4C). In addition, ipatasertib, a specific inhibitor of Akt, was used in the present study to block the Akt/

| DISCUSS ION
In this study, we determined the pivotal role of DEC1 in uncontrolled proliferation and GC carcinogenesis induced by H. pylori infection. We observed that elevated DEC1 expression was directly correlated with GC H. pylori-infected status and progression. We also identified the novel mechanistic link between the DEC1 ex-  is plausible that activation of multiple cell proliferation pathways helps H. pylori to regulate the functions of multiple tumour suppressor genes and oncogenes, resulting in a higher proliferative capacity, more extensive spread and more rapid progression of H. pylori-positive GC.
In conclusion, we define the important role of the DEC1 expression mediated by H. pylori infection in GC. We also found that, when induced by H. pylori, DEC1 overexpression targeted Akt/NF-κB pathway, thereby leading to the promotion of cell proliferation.
These results provide novel insights into the molecular mechanism of GC, with DEC1 serving as a potential therapeutic target for GC linked to H. pylori.

ACK N OWLED G EM ENTS
Thanks to Dr. Jihui Jia (Department of medical microbiology, Shandong University, Shandong, China) for providing H. pylori standard strain NCTC 11637.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.