The Role Of JMJD2B In Invasion And Metastasis Of Gastric Cancer And The Regulation Of JMJD2B Expression By Helicobacter Pylori Infection In Gastric Cancer Cells

BackgroundGastric cancer (GC) is one of the most common malignancies worldwide and ranks second in terms of global cancer-related mortality. Although the overall incidence of gastric cancer has declined in United States, the incidence remains high in Asian countries. Invasion and metastasis is one of the main features of malignancy. Once into the stage of locally advanced or metastatic stage, surgery and combination chemotherapies play a minor role to improve the survival rate. Thus, a better understanding of the progression of gastric cancer is urgent. It is critical to identify the new diagnostic and prognostic markers and find novel targeted therapies. For this reason, it is very important to make further research of JMJD2B in invasion and metastasis of gastric cancer.Tumor invasion and metastasis is a multi-factor, multi-step complex biological process. Tumour invasion and metastasis is initiated by epithelial-mesenchymal transition (EMT). EMT, a developmental process in which epithelial cells show reduced intercellular adhesion and acquire migratory fibroblastoid properties, is considered to be critical for invasive and metastatic progression in cancer. The process of EMT is associated with the down-regulation of epithelial markers, abnormal translocation of β-catenin, and aberrant upregulation of mesenchymal markers. Repression of epithelial markers [e.g. E-cadherin (CDH1)], abnormal translocation of β-catenin and aberrant upregulation of mesenchymal markers [e.g. vimentin (VIM) and N-cadherin (CDH2)] are typical gene expression changes observed during EMT. Through EMT, epithelial cells with normal interaction with the basement membrane and polar, non-activities ability lost cell polarity and connection with the basement membrane of the epithelial phenotype, and then had the capacity of higher migration and invasion, anti-apoptotic and degradation of extracellular matrix in mesenchymal phenotype. Therefore, EMT is an important biological process of acquiring cells migration and invasion ability in epithelial cell-derived malignant tumors.JMJD2B, also known as KDM4B, is a jmjC domain-containing histone demethylase, which belongs to JMJD2family. JMJD2B primarily targets the tri-methylated lysine9of histone H3(H3-K9) and has been reported to play an important role in many biological processes such as heterochromatin formation, X-chromosome inactivation, homeotic gene silencing, and transcriptional regulation. Recently, increasing studies have revealed an epigenetic role of JMJD2B in stem cells differentiation, inflammation and tumorigenesis. In our previous study, we demonstrated that JMJD2B promoted proliferation and survival of gastric cancer cells. However, whether and how JMJD2B is involved in gastric cancer invasion and metastasis arouses our interest.The relationship between Helicobacter pylori(H. pylori) infection and gastric carcinogenesis is reported. Helicobacter pylori with persistent colonization in stomach result in hosts chronic atrophic gastritis, peptic ulcer, dysplasia and even gastric cancer. As a risk factor for gastric cancer, Helicobacter pylori has been considered as a class I carcinogenic factor by WHO. However, the molecular mechanism involved in Helicobacter pylori infection leads to gastric tumorigenesis and metastasis is not yet fully understood.Histone modification induced by pathogen promotes tumorigensis and cancer metastasis. The level of H3K9me decreases when cells are infected by H. pylori. JMJD2B primarily targets the tri-methylated lysine9of histone H3(H3-K9) and has been reported to play an important role in many biological processes. Therefore, the regulation of JMJD2B expression by H. pylori infection, not only reveal carcinogenic mechanisms of H. pylori infection but also reveals underlying molecular mechanisms of JMJD2B promoting oncogenesis.PurposeThis study investigated the role of histone demethylase JMJD2B in invasion and metastasis of gastric cancer and the regulation of JMJD2B expression by Helicobacter pylori infection in gastric cancer cells, and underlying molecular mechanisms in the tumorigenesis and progression of gastric cancer. Methods and ResultsPart I JMJD2B promotes epithelial-mesenchymal transition by cooperating with β-catenin and enhances gastric cancer metastasisOur study showed that, transformation from loosen cell-cell adhesion spindle-shaped mesenchymal-like phenotype to tight cell-cell contacts and cobblestone-like epithelial phenotype in cells was shown in JMJD2B knockdown HGC27and MGC803cells. To address whether JMJD2B affected the EMT program, we knocked down JMJD2B expression using JMJD2B specific siRNA in the gastric cell lines, JMJD2B repression resulted in downregulation of mesenchymal markers (vimentin and snail) and upregulation of epithelial markers (E-cadherin) by western blot and quantitative real-time PCR (QRT-PCR). The contrary results were observed in cells of ectopic expression of JMJD2B. Therefore, JMJD2B promote invasion and metastasis of gastric cancer by regulating EMT. To further reveal its underlying mechanisms, we performed a luciferase assay. Our results showed that VimPro-Luc luciferase activity was decreased in JMJD2B-depleted BGC823cells, while VimProMut-Luc luciferase activity (β-catenin/TCF binding sites inactivated mutant) failed to be repressed in JMJD2B-depleted BGC823cells. Opposite results were obtained in JMJD2B-overexpressed BGC823cells. Hence, we hypothesized a potential involvement of JMJD2B and β-catenin in vimentin transcription regulating EMT to enhance invasion and metastasis of GC. Furthermore, JMJD2B physically interacted with β-catenin and was recruited to the promoter of vimentin increasing vimentin transcription by co-immunoprecipitation and ChIP assay. Besides, JMJD2B inhibition attenuated TGF-β1-mediated β-catenin nuclear accumulation by immunofluorescence. And, JMJD2B inhibition attenuates migration and invasion of GC cells in vitro by scratch wound-healing assay and matrigel invasion assay. The biological effects of JMJD2B in invasion and metastasis in vivo were detected by injection JMJD2B siRNA-treated BGC823cells into immunodeficient nude mice through tail vein. The results showed that JMJD2B inhibition resulted in diminished invasion and metastasis in vivo. Finally,101cases of paraffin-embedded primary gastric cancer tissues were used for immunohistochemical staining. The expression of JMJD2B was positively correlated with tumor size (P=0.017), differentiation status (P=0.002), tumor invasion (P=0.045), lymph node metastasis (P=0.000), distant metastasis (P=0.024) and TNM stage (P=0.002) and vimentin expression (P=0.004) in101cases of gastric cancer patients.Part II The regulation of JMJD2B expression by Helicobacter pylori infection in gastric cancer cellsOur study showed that the level of JMJD2B and COX-2was upregulated induced by Helicobacter pylori infection0.5,1,1.5and2h in gastric cancer cells by western blot and quantitative real-time PCR. Gastric cancer cells were transfected with siJMJD2B, and then were infected by Helicobacter pylori, showing that JMJD2B and COX-2increased. And loss of JMJD2B in AGS and BGC823cells, caused the level of COX-2decreased. We detected JMJD2B and COX-2protein level, showing that JMJD2B and COX-2overexpressed in gastric cancer cells. This study has contributed to understanding the molecular mechanisms of epigenetic regulation of gastric tumorigensis caused by H. pylori infection in humans.ConclusionsThis study explores deeply the role of JMJD2B in invasion and metastasis of gastric cancer and its underlying mechanisms, and the regulation of JMJD2B expression by Helicobacter pylori infection. So JMJD2B inhibition is one of possible mechanisms of anti-tumor therapy, implicating that JMJD2B might be served as a new target for anticancer therapy. Wide applications of JMJD2B in the diagnosis and treatment of gastric cancer is promising.