| Background:Histone H3K4 demethylase RB-binding protein 2(RBP2)plays an important role in the carcinomas as an epigenetic regulator.Our research has confirmed that RBP2 plays a key role in the gastric cacinoma and participates in gastric cancer cells senescence,proliferation,invasion and metastasis.The significance of cellular metabolism in tumorigenesis and development has been paid close attention.However,the molecular mechanism and biological significance of RBP2 in gastric cancer cells by regulating key enzymes involved in glucose and lipid metabolism are still unclear.Peroxisome Proliferator-Activated Receptor γ(PPARγ),which is an important regulator of cell metabolism,has been shown to be involved in tumor proliferation and apoptosis.Studies have predicted that RBP2 might regulate PPARy expression.Therefore,it is the aim to study the effect of RBP2-regulated PPARy on the activity of key enzymes in glucose and lipid metabolism which leads to the abnonnal metabolism in gastric cancer cells.It is important to explore the molecular regulatory mechamism and biological effects of tumor cells metabolism at epigenetic level.Object:To study the molecular mechanism and biological effects of RBP2,a key epigenetic regulator of gastric cancer,involved in the metabolism of gastri2c cancer cells by regulating PPARy.Method:1.The level of human gastric cancer tissue specimenBioinformatics technology was used to analyze the expression of RBP2 and PPARy in human gastric cancer tissue specimen and the correlation between them,and the effect of PPARy on the prognosis of gastric cancer with the help of data bases.The expression of RBP2 and PPARy at the mRNA and protein levels were also detected by immunohistochemistry(IHC)and qRT-PCR in 16 pairs of gastric cancer tissues and corresponding adjacent normal tissues.2.The lever of molecular and cellsThe background expression of PPARy in four gastric mucosal epithelial cell lines GES-1,AGS,BGC-823 and SGC-7901 was verified by qRT-PCR and Western blotting.After transfection of RBP2 expression plasmid or specific small interfering RNA to overexpress or inhibit RBP2 expression respcetively,the expression of PPARy and downstream PKM2,PFKFB3,FASN and CPT1A which are the regulators in glucose and lipid metabolism were verified by qRT-PCR and Western blotting.Fluorescence and chemiluminescence were used to detect changes in cell ATP production and LDH activity in this process.At the same time,PPARy expression plasmid and specific small interfering RNA were transfected into the cells to overexpress or inhibit PPARy expression,which was used to detect their effects on the expression of downstream metabolic regulators and the corresponding biological effects respectively.The dual luciferase activity assay determined the direct regulation of RPP2 on the PPARy promoter.The recovery experiments confirmed that PPARy played an important role in the process of RBP2-regluated key moleculars expression in metabolism,ATP production and LDH activity.3.The lever of animal modelsBGC-823 cells with specific interference RNA of RBP2 were used for subcutaneous tumor formation in nude mice.The tumor formation was observed during this process.The expression and correlation of RBP2,PPARγ,PKM2,PFKFB3,FASN and CPT1A were detected in the tumors by qRT-PCR,Western blotting and IHC.It was further verified for the effect of RBP2 on the metabolism of tumor cells and the important role in the gastric cacinoma.Result:1.The level of human gastric cancer tissue specimensBioinformatics analysis,qRT-PCR and IHC results confirmed that RBP2 and PPARy were highly expressed in human gastric cancer tissue specimens and there was a positive correlation between them.PPARγ was negatively correlated with the prognosis of gastric cancer patients.2.The level of molecular and cellsPPARy was stably expressed in four gastric mucosal epithelial cell lines.Overexpression of RBP2 increased the expression of PPARy,which led to an increase in the expression of PKM2,PFKFB3 that are the key regulators of glycolysis and fatty acid synthase FASN.At the same time the expression of fatty acid oxidation rate-limiting enzyme CPT1A decreased which resulted in the relative increase of ATP production and LDH activity.On the contrary,after RBP2 expression was inhibited,PPARy expression decreased with the inhibition of glycolysis key regulators and FASN expression and activation of CPT1A expression.The cells ATP production and LDH activity relatively decreased.While directly overexpression or specific inhibition of PPARy in cells resulted in the consistent results.Dual luciferase activity assays confirmed that RBP2 could directly bind to the PPARγ promoter to activate its expression.Recovery experiments indicated that PPARγ was required for RBP2 to regulate key molecules of glucose and lipid metabolism,as well as cellular ATP production and LDH activity.3.The level of animal modelsAfter interfering with RBP2,the tumorigenic ability of gastric cancer cells in the subcutaneous of nude mice decreased,and growth was inhibited at the same time.The expression of PPARγ at mRNA and protein levels also decreased in the tumors,while the inhibition of glycolysis and fatty acid synthesis-related regulatory molecules was also involved in this process.The rise of the fatty acid oxidation regulator expression was also determined in the tumors.Conclusion:In human gastric cancer tissue samples,RBP2 and PPARγ were both highly expressed and positively correlated to each other.PPARγ expression was negatively correlated with gastric cancer prognosis.RBP2 could directly regulate PPARy promoter activity and thus promote its expression,which resulted in the regulation on the key molecules related to cell glucose and lipid metabolism.In this process,it could be detected the promotion of glycolysis and fatty acids synthesis while inhibition of fatty acids oxidation.The increase of cell ATP production and LDH activity was also determined at the same time.All these could promote the gastric carcinoma.In this study,it was investigated the important role of epigenetic molecule RBP2-regluated PPARγ in tumor metabolism and its molecular mechanism which could provide some theoretical basis for the early diagnosis and treatment of gastric cancer. |
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