| Background:malignant tumors are the serious diseases that pose great threaten to the health of people. It is shown that the incidence of tumors all over the world is still increasing and therefore it is important to reveal the regular pattern of their development and progression and to treat them effectively. It has become a key scientific problem during the investigation of gastric cancer to find the key molecule that is involved its development and progression to carry out early prevention and treatment targeting.The chronic inflammation of gastric epithelia induced by Helicobactor Pylori is closely associated with the development and progression of gastric cancer. Without being fully recovered, chronic inflammation can be turned into non-resolving inflammation and this will pave the way for the development of gastric cancer, providing the preferable microenvironment for the oncogenic process. The fact that chronic inflammation facilitates cancer development is explained in gastric carcinogenesis.People pay increasing attention to the role epigenetic regulation plays in the development and progression of cancer. To some extent, the development of cancer is the consequence of accumulation of abnormal gene expression and therefore the dysfunction of epigenetic regulation also can promote the development of cancer. Furthermore, the accumulation of this dysfunction will facilitate the malignant progression of tumors, endowing the ability of local invasion and distant metastasis of tumor cells.The malignant progression of cancer, that is local invasion and distant metastasis, is the main cause for the low survive rates, poor prognosis and recurrence. Epithelial-mesenchymal transition (EMT) plays an important role during this process. Therefore, revealing the law malignant progression of cancer, finding the key regulating molecule and developing the more effective targeting drugs have become the research focus of cancer.In a word, it is an important scientific problem to find the regular pattern that can link infection and inflammation, gastric carcinogenesis and malignant progression. For this, epigenetic regulation enlightens us much. We provide the evidence to prove that epigenetic regulation links chronic inflammation, gastric carcinogenesis and its malignant progression. This will be instrumental for us to understand more about the underlying mechanisms for cancer development and progression and to develop early prevention and targeting therapy.Aims:1 Investigate the mechanism through which RBP2 mediated the development of gastric cancer induced by Helicobactor Pylori, and find the effective early prevention.2 reveal the underlying mechanism through which RBP2 promoted malignant progression of gastric cancer, and provide the theoretical reference for effective tumor therapy.Methods and Results:1. RBP2 mediates malignant transformation of gastric epithelial cells through CagA-AKT-Sp1-RBP2-Cyclin D1 axis induced by Helicobactor Pylori (1) Helicobactor Pylori CagA induced the expression of RBP2. When transfected with CagA plasmid, RBP2 can be induced in gastric cancer cells. However, if we RBP2 was blocked, the pro-proliferation effect of CagA will be abrogated.(2) RBP2 induced by CagA is Spl dependent. Both RBP2 and Spl can be induced when CagA plasmid was transfected into gastric cancer cells. If Spl expression was blocked, RBP2 was also inhibited and the effect of CagA on RBP2 induction was abrogated. We also found the direct regulation of Spl on RBP2 promoter with the method of dual luciferase activity, EMS A and ChIP.(3) PI3K/AKT signaling pathway mediated the Spl dependent RBP2 induction by CagA. With the specific inhibitor LY294002 of PI3K/AKT to treat gastric cancer cells, the phosphorylated Spl was decreased, the induction of RBP2 by CagA was abrogated and at the same time, the pro-proliferation effect of CagA was also attenuated.(4) RBP2 induced by CagA targeted Cyclin D1 to promote the uncontrolled proliferation of gastric cancer cells. We used the methods of PCR and western blot to verify that Cyclin D1 was the dowmstream target of RBP2. Furthermore, we found the decreased Cyclin D1 expression in the tumors formed in the RBP2 knockdown group compared with that in the control group. Therefore this confirmed the regulation of Cyclin D1 by RBP2 in vivo.(5) The expression of RBP2 and Spl was positively related in the tissues from inflammation to cancer. We collected 36 cases of patients with chronic superficial inflammation,36 cases of patients with atrophic inflammation with metaplasia and 36 cases of patients with dysplasia. IHC staining was performed in these tissues and we found the increasing expression of RBP2 and Sp1 with the increasing severity of diseases. In addition, we observed the phenomenon of co-localization of RBP2 and Spl in the pathogenic tissues. RBP2 and Spl expression was positively related in these tissues.2. RBP2 promotes malignant progression of gastric cancer through TGF-β1-p-SMAD3-RBP2-E-cadherin-Smad3 feedback circuit.(1) RBP2 played an important role in the process of gastric cancer malignant progression and maintained the stem cell-like property of gastric cancer cells. The invasion and migration of gastric cancer cells obviously deceased with RBP2 knockdown. At the same time, the stem cell-like property, that is the ability of tumor cells to form sphere in the ultralow adherent plate, of tumor cells was also impaired with RBP2 inhibition. With the stable knockdown of RBP2 cancer cells to inject into mice via tail vein, distant metastasis was decreased compared that in the control group. In consistence, when RBP2 was overexpressed in gastric cancer cells, the ability to migrate increased significantly.(2) RBP2 can be induced by TGF-β dependent on Smad3 phosphorylation and is crucial for the EMT induced by TGF-β. TGF-β induced RBP2 expression in a time dependent manner. Phosphorylated Smad3 activated by TGF-P bound to the promoter of RBP2 to enhance its transcription. Inhibition of RBP2 abrogated the pro-invasive effect of TGF-β on gastric cancer cells. At the same time, the enhancement of stem cell-like property by TGF-β was also relieved by RBP2 knockdown. The effect of TGF-β on the expression of epithelial and mesenchymal cells was also reversed by RBP2 block.(3) RBP2 is involved in the regulation of gastric cancer malignant progression by directly inhibiting E-cadherin expression. E-cadherin expression was upregulated by RBP2 inhitition. RBP2 directly bound to the promoter of E-cadherin using ChIP assay to suppress its expression and this kind of suppression maybe was the histone demethylase dependent. When both RBP2 and E-cadherin expression was inhibited, the effect of RBP2 knockdown on cell invasion and migration as well as the stem cell-like property was reversed, which strongly indicated that RBP2 promoted gastric cancer progression via inhibiting E-cadherin expression.(4) RBP2 bound to p-Smad3 and was recruited to the promoter of E-cadherin to enhance suppression effect. Immunoprecipitation indicated that RBP2 bound to p-Smad3 and it was recruited by p-Smad3 to the promoter of E-cadherin to block its transcription.(5) E-cadherin feedback regulated RBP2 expression by inhibiting Smad3 phosphorylation. When E-cadherin was blocked in gastric cancer cells, the phosphorylation of Smad3 was increased, and then the expression of RBP2 was upregulated, contributing to the enhanced ability of tumor cells to migrate.(6) The co-localization of RBP2 and Snail1, and negative expression relationship between RBP2 and E-cadherin in primary gastric cancer specimen. We collected 130 cases of specimen from patients with primary gastric cancer and performed IHC staining for RBP2, Snail1 and E-cadherin. We found the co-localization of RBP2 and Snail1 in these tissues and the expression of RBP2 was negatively related with the expression E-cadherin, as the positive control Snail1 did.Conclusions:1. RBP2 mediated the malignant transformation of gastric epithelial cells induced by Helicobactor Pylori via CagA-AKT-Spl-RBP2-Cyclin D1 axis. This part of investigation revealed the new mechanism from chronic inflammation to cancer, that is RBP2 mediated the transformation from infection to tumor development. Therefore RBP2 can be considered as the target molecule for early prevention of gastric cancer.2. RBP2 promoted gastric cancer malignant progression by TGF-β1-p-SMAD3-RBP2-E-cadherin-Smad3 feedback circuit. This part of investigation elucidated the new epigenetic mechanism underlying gastric cancer progression and thus enriched our understanding of biological process of tumor metastasis. Therefore RBP2 may serve as the potential target molecule for gastric cancer treatment. |
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