The Function And Molecular Mechanism Of DEC1 In H.pylori-associated GC

[Background]Gastric cancer(GC)is a kind of malignant tumor which originated from gastric epithelial cells.According to the latest relevant reports,GC's morbidity and mortality rates ranked among the world's top five cancer in 2018.Because the early symptoms of GC are not obvious,and the prevalence of early GC screening in routine physical examination in China is not enough,the incidence of GC in China accounts for about 7%of the total incidence of cancer,and the five-year survival rate is only 36%which is much lower than other countries in East Asia.Therefore,exploring the mechanism of the occurrence and development of GC is of great significance for finding the therapeutic molecular target and improving the curative effect of GC.Helicobacter pylori(H.pylori)is a Gram-negative microbial bacterium and it is the only bacterium that are now known which can survive in the human stomach.More than 50%of the world's population has been infected with H.pylori in the digestive tract since their childhood.H.pylori persistent infection could cause a series of gastric lesions,such as chronic gastritis and gastric ulcer,further leading to atrophic gastritis,intestinal metaplasia and dysplasia,and ultimately develop into GC.The International Agency for Research on Cancer(IARC)listed H.pylori infection as the first carcinogen in 1994.Therefore,exploring the mechanism of H.pylori in GC is of great significance for the early detection,diagnosis and treatment of H.pylori-related GC.DEC1(Differentiated Embryo-chondrocyte Expressed Gene 1)belongs to the basic helix-loop-helix(bHLH)transcription factor family.DEC1 is widely expressed in most of the human tissues and plays an important role in cell differentiation,proliferation cycle and circadian rhythm regulation.Previous studies have shown that DEC1 is involved in the occurrence and development of many diseases,and its expression has increased in many kinds of tumors.In GC related researches,DEC1 can be strongly induced in hypoxic environment,and has anti-apoptotic function.Meanwhile,the prognosis of GC patients with high DEC1 expression is poor.However,after curcumin treatment,the expression of DEC1 was down-regulated in mice.In conclusion,previous studies have shown that DEC1 plays an important role in the differentiation and progression of GC.Down-regulation of DEC1 related pathway could reduce the malignancy of GC.As one of the major carcinogens of GC,H.pylori's role in the occurrence and development of GC has not been fully elucidated.To elucidate the role and molecular mechanism of DEC1 and its signal pathway in H.pylori-related GC may provide a basis for new molecular targets and strategies for the treatment of H.pylori-related GC.[Objective]Although H.pylori has been listed as type I carcinogen of GC,and many studies have shown that DEC1 plays a carcinogenic role in GC,the role of DEC1 molecule in the malignant progression of H.pylori-related GC and related molecular pathways still needs to be further explored.In order to reveal the clinical value of DEC1 in the diagnosis and treatment of H.pylori-positive GC,we analyzed whether the expression of DEC1 in gastric diseases was related to H.pylori infection,and the relationship between clinical pathological parameters and the expression of DEC1 in H.pylori-positive GC patients.GC cells overexpressing or interfering with DEC1 were co-cultured with H.pylori standard strain NCTC 11637.The changes of DEC1 protein and mRNA levels and DEC1 effects on cell proliferation and apoptosis were determined.Then the related experimental results were validated by constructing an animal model of H.pylori infection in C57BL/6J mice.Finally,the downstream genes and molecular pathways were explored to further elucidate the pathogenesis of H.pylori-related GC,and to provide a preliminary theoretical basis for the treatment of H.pylori-related GC.[Material and methods]1.Collect gastric tissues from patients with gastritis or GC,determine the status of H.pylori infection by methylene blue staining,and analyze the relationship between DEC1 expression and H.pylori infection by immunohistochemical staining.The relationship between DEC1 expression and clinical pathological parameters was judged according to the corresponding clinical data.2.Use H.pylori and gastric cell interaction gene expression chip in GEO.Use Bioconductor Limma package in R 3.3.3 software to analyze whether DEC1 belongs to differentially expressed gene.3.The standard H.pylori strain NCTC 11637 was cultured on Colombian blood agar medium at 35 Cand 95%humidity under microaerophilic condition.Genomic DNA was extracted for sequencing and identification.The purity of the strain was tested by Gram staining and related biochemical reactions before used in the experiments.4.In GES-1,MKN-45 and MGC-803,Western blot and RT-PCR were used to detect the expression of DEC1 after co-cultured with H.pylori.Verify whether the regulation of DEC 1 expression requires active bacteria.5.The effects of H.pylori infection on the proliferation of GC cells were detected by CCK-8 method,EdU method and cell clone formation method.Western blot was used to detect the changes of apoptosis-related proteins Bcl2,Bax and Survivin.6.DEC]expression levels of MKN-45 and MGC-803 GC cells were detected.DEC1 interference was carried out on MKN-45 which has relatively high endogenous level,and DEC1 overexpression was carried out on MGC-803 which has relatively low endogenous level.CCK-8,EdU,cell cloning assay,flow cytometry and Western blot were used to detect the effects of H.pylori on DEC 1-mediated proliferation and apoptosis of GC cells.7.According to the previous literature,the feasibility and related methods of H.pylori standard strain NCTC 11637 infected C57BL/6J mice were preliminarily determined,and the H.pylori positive C57BL/6J mice model was constructed.Methylene blue staining and HE staining were used to evaluate the success of mouse model construction and the degree of gastritis.Immunohistochemical staining and total gastric protein were used to verify the molecular mechanism.8.By adding Akt inhibitors,we detected whether DEC1 regulated the proliferation and apoptosis of GC cells through Akt/NF-?B signaling pathway through cell function related experiments and Western blot.[Results]1.H.pylori promotes DEC1 expression in human gastric mucosal cells and analysis of the relationship between H.pylori infection and pathological parametersImmunohistochemical staining showed that the relative expression of DEC1 in H.pylori positive gastritis and GC was significantly higher than that in H.pylori negative gastritis and GC.Differential expression gene analysis of GSE10262,GSE70394 and GSE60661 microarrays showed that H.pylori infection resulted in up-regulation of DEC1 expression in npGECs or AGS cells.DEC1 staining of H.pylori positive GC mucosa showed that DEC1 expression was positively correlated with malignant degree.2.H.pylori culture and identificationWe first explored the cultivation conditions of H.pylori in our laboratory.The results showed that H.pylori grew well on Colombian blood agar solid medium under micro-aerobic gas environment,35 ?,90%humidity,and could recover after long-term freezing in 30%glycerol/Brucella Broth at-80 C.H.pylori can form needle-like transparent,wet,1-2 mm diameter colonies on the surface of culture medium.Gram-negative gull-like bacteria were seen under the microscope.H.pylori enters logarithmic growth period at 48-72h and it is sensitive to culture environment.Prolonged incubation time or destruction of micro-aerobic environment will lead to H.pylori spherical transformation.The whole genome DNA of the strain was extracted and sequenced,which confirmed that H.pylori cultured in our laboratory was the standard strain NCTC 11637.3.Activated H.pylori regulates DEC1 expression,GC cells proliferation and apoptosis levels in vitroStandard strain NCTC 11637 was co-cultured with immortalized gastric epithelial cells GES-1 and gastric adenocarcinoma cells MKN-45 and MGC-803.The number ratio of bacteria to cells was 100:1,and the time gradient was set to 4,8 and 12 hours.DEC1 mRNA level was increased after 4 h H.pylori infection.Compared with the blank control group,the expression of DEC1 was significantly increased in the experimental group after infected for 4,8 and 12 hours,and reached the highest level of DEC1 at 8 hours.Western blot results showed that the DEC1 expression levels increased at 4,8 and 12 hours after H.pylori infection,and DEC1 expression was the highest at 8 hours.Therefore,we chose MOI=100 and 8h infection time to carry out follow-up experiments.We placed the H.pylori in 100 C water bath for 10 minutes.DEC1 expression in GES-1,MKN-45 and MGC-803 cells co-cultured with inactivated bacterium did not change significantly.That is to say,the up-regulation of DEC1 in gastric cells by H.pylori depends on the interaction between cells and active bacteria.We used CCK-8,EdU and cell cloning formation test to detect the proliferation ability of GC cell lines MKN-45 and MGC-803 co-cultured with Helicobacter pylori for 8 hours.CCK-8 results were continuously detected and growth curves were drawn.The results showed that compared with the blank control group,the proliferation ability of H.pylori infected cells increased significantly.Simultaneous EdU experiments showed that the proportion of cells in the proliferative phase increased in the infected group.The results of cell colony formation experiment were consistent.The number and volume of cells in the infected group were significantly increased than those in the control group.Total cell protein was extracted and Western blot results showed that compared with the blank control group,the expression of DEC1,Bcl-2 and Survivin increased,while the expression of Bax decreased4.H.pylori regulates proliferation and apoptosis of GC cells through DEC1 molecule in vitroFirst,detected the background expression of DEC1 in two gastric adenocarcinoma cell lines.Western blot results showed that MKN-45 was an endogenous DEC1 high expressing cell line,while MGC-803 was endogenous DEC1 low expressing cell line.The DEC1 shRNA vector based on PKL-2099 plasmid was constructed and were transfected into MKN-45 cells.RT-PCR and Western blot results showed that the expression of DEC1 in MKN-45 cells after interference with DEC1 was significantly reduced.The overexpression vector DEC1 based on pGV plasmid was constructed and the results of RT-PCR and Western blot showed that the expression of DEC1 in MGC-803 cells transfected with DEC1 overexpression vector was significantly increased.We co-cultured GC cells overexpressing DEC1 or interfering with DEC1 with H.Pylori for 8 hours(MOI = 100).CCK-8 results showed that after interfering with DEC1 expression in MKN-45 cells,the proliferation ability of DEC1-sh cells was significantly inhibited,while co-culture of DEC1-sh cells with H.pylori could partially improve the inhibited proliferation ability.After overexpression of DEC1 in MGC-803 cells,DEC1 overexpression promoted cell proliferation,and co-culture of DEC1 overexpression cells with H.pylori could further enhance cell proliferation.EdU results showed that the proportion of MKN45/DEC1-sh+H.Pylori cells in the proliferative phase increased compared with the low proliferation rate of MKN45/DEC1-sh group.Compared with MGC-803/DEC1,the proportions of proliferative cells in H.pylori infection MGC-803/DEC1 cells increased significantly.The results of cell clone formation were consistent.PE-coupled Annexin-V flow cytometry and Western blot results showed that H.pylori co-culture partially counteracted the apoptotic effect of DEC1 interfering,or further increased the anti-apoptotic ability of DEC1 overexpressed GC cells.5.Relevant experiments of H.pylori infection mice modelIn order to further verify the action mechanism of related molecules in vivo,the standard strain of H.pylori NCTC 11637 was used to construct the gastritis model of mice infected with H.pylori colonization.Pathological observation showed that most of the mice in H.pylori gastric lavage group were chronic superficial gastritis with multiple foci,congestion and edema of mucosa.Methylene blue staining of paraffin sections of gastric tissues in mice showed that 20 of the 25 treated mice were infected and colonized by H.pylori.Hematoxylin-eosin staining showed that H.pylori-positive mouse had chronic inflammatory cell infiltration,gastric mucosal edema and epithelial necrosis and exfoliation.Immunohistochemical analysis showed that the expression of DEC1 protein in H.pylori positive gastritis group was significantly higher than that in control group.Western blot showed the same results after extracting total protein from mouse gastric mucosa.6.Effect of H.pylori infection on the expression of p-Aktl and NF-?B in gastric cellsAt the condition of MOI 100 and 8 h infection time,the expression levels of DEC1,p-Akt1 and NF-?B p65 in GC cells MKN-45 and MGC-903 co-cultured with H.pylori increased significantly.We validated the relationship between H.pylori and the expression of p-Aktl and NF-?B p65 in the mouse model of H.pylori gastritis.Western blot results showed that the expression of p-Aktl and NF-?B p65 in H.pylori positive gastritis mice were significantly up-regulated.Animal experiments confirmed the up-regulation of p-Aktl and NF-?B p65 by H.pylori,partly explaining the molecular mechanism of its carcinogenic effect.7.DEC1 promotes the expression of p-Aktl and NF-kB in GC cellsAfter overexpression of DEC1,the expression levels of p-Aktl and NF-?B p65 increased significantly,while the expression levels of p-Aktl and NF-?B p65 decreased significantly in MGC-803 cells after interference with DEC1.DEC1 promotes downstream p-Aktl and NF-?B p65 in GC cells.8.DEC1 regulates the expression of proliferation-related signal transduction pathway Akt/NF-KB in GC cellsWe chose Ipatasertib as an Akt inhibitor to further intervene in gastric cancer cells.MKN-45 cells were divided into NC-sh+DMSO group,NC-sh+Ipatasertib group,DEC1-sh+DMSO group and DEC1-sh+Ipatasertib group.Protein test results showed that Ipatasertib could further reduce the expression level of NF-?B p65 in MKN-45 after interfering with DEC1.MGC-803 was divided into NC+DMSO group,NC+Ipatasertib group,DEC1+DMSO group and DEC1+Ipatasertib group.Protein test results showed that Ipatasertib could partially offset the elevated expression of NF-?B p65 induced by the up-regulation of DEC 1.9.Akt/NF-?B signaling pathway is involved in DEC 1-mediated GC cells proliferationCCK-8 results showed that the addition of Ipatasertib could further inhibit the proliferation of MKN-45/DEC1-sh.In DEC1 over expression cell line MGC-803/DEC1,the addition of Ipatasertib could partially counteracted the increase of proliferation induced by DEC1.The results of EdU experiment and cell clone formation experiment were consistent with the CCK-8 results.[Conclusions]1.This study is the first to find that H.pylori infection can lead to high expression of DEC I in human gastric mucosa,and the expression level of DEC1 is positively correlated with the adverse prognosis of GC and GC patients.DEC1 expression was also up-regulated in H.pylori-positive mice.2.In vitro experiments showed that H.pylori infection could induce the high expression of DEC 1 in GC cells,which promotes the proliferation and anti-apoptotic ability of GC cells.3.H.pylori partially promotes the proliferation and anti-apoptotic ability of GC cells through DEC1.4.H.pylori promotes malignant transformation of GC through DEC1/Akt/NF-?B signaling pathway.