The Relative Regulation Mechanism Between RBP2 And BECN1 In Gastric Carcinogenesis

Background and objective:Gastric cancer is a kind of common malignant tumors. Histone modification plays an important role as the epigenetic factors on carcinogenesis. Studies have demonstrated that RBP2 (RB Binding Protein 2) is a histone demethylase capable of H3K4 demethylation, whcih can regulate the promoters’activity of target genes and participate in the genesis and development of gastric cancer. Autophagy is a strict degradation pathway of long-lived proteins and damaged organelles catabolic. This process also may take part in gastric carcinogenesis. Autophagy-related protein BECN1 (Beclinl) is indispensable during the formation and maturation of autophagy. The promoter of BECN1 owns the sites where RBP2 can bind to specifically. Therefore, the role of RBP2 in BECN1-correlated gastric carcinogenesis and the relative regulation mechanism still need to be explored.Experimental methods:1.Human tissue speciesTo detect RBP2 and BECN1 expression level and their correlation in cancer tissues and the adjacent ones by immunohistochemical staining in 21 pairs of human gastric cancer tissues specimens.2.Cell levelIn gastric cancer cells (AGS, BGC-823 and GES-1) which were transfected with the RBP2 plasmid or RBP2 siRNA respectively, the regulatory relationship betweeen RBP2 and BECN1 in the levels of RNA and protein was examined by QRT-PCR and Western blotting. The PGL-3-BECN1-promoter plasmid was constructed by molecular cloning (including the region which contains CCGCCC sequence that RBP2 recognizes and binds to specifically). Then the plasmid was transfected into the gastric cancer cells. Dual luciferase assay was used to detect BECN1 promoter activity to check if it was regulated by RBP2 directly or indirectly. In gastric cancer cell line BGC-823 transfected with the RBP2 plasmid or RBP2 siRNA, the effect of the inhibition or the overexpression of RBP2 on the autophagy of gastric cancer cells was showed with immunofluorescence analysis.3.Animal modelRBP2 siRNA was transfected into gastric cancer BGC-823 cells with the help of lentiviral. Then the fluorescence intensity was observed to determine the suitable infection concentration of lentiviral by the inverted fluorescence microscope. Then the cells were trypsinized down to the cell culture bottles. With the selection of puromycin, we obtained the cell lines that could express RBP2 shRNA stably. These BGC-823 were injected into nude mice. The cells that could express the control shRNA was used as the negative one. When the tumors appeared, the tumor volume was measured to get the tumor growth curve. RBP2 and BECN1 expression levels were also detected by QRT-PCR and immunohistochemical staining method.Results:1.We carried out the immunohistochemical analysis of human tissue samples, which showed that the expression of BECN1 was increased in human gastric cancer species obviously, and it was positive correlated with RBP2 expression. At the same time, the expression of BECN1 had no relationship with the age, sex, size of the tumors and the differentiation of the tumors of the patients.2.Our findings suggested that the inhibition of RBP2 decreased the expression of BECN1 both at the RNA and the protein levels significantly in gastric cancer cell lines, which was determined by QRT-PCR and Western blotting. On the contrary, the overexpression of RBP2 could increase the expression of BECN1.3.The dual luciferase assay result showed that RBP2 could bind to the promoter of BECN1 and regulate its expression directly.4.Immunofluorescence analysis showed that RBP2 affected the autophagy in gastric cancer cells.5.In the nude mice models, the inhibition of RBP2 reduced the growth of the tumor which also showed the decrease of BECN1 expression at the same time.Conclusions:All the results revealed that RBP2 directly regulated the expression of BECN1 by affecting its promoter activity and took part in autophagy of gastric cancer cells. This showed a new regulatory mechanism of histone modification in carcinogenesis which might be a potential therapeutic way in gastric cancer.