ARRB1Mediates The Dysfunction Of Glucose Metabolism By Facilitating HIF1α-Dependent PKM2Expression

Obsjective:To investigate the molecular mechanism of beta-arrestinl in cancer metabolism, as well as its function in the development of gastric cancer, this will provide us a theoretical basis for prevention and treatment of gastric carcinoma and help us to develop a new drug for inhibiting the development of cancer.Methods:1. In the tissue level, we determined the expression of beta-arrestinl, HIF1A and PKM2in the gastric cancerous tissues and normal ones with immunohistochemical staining method and initially analyzed the relationship among them.2. In the molecular level, we transfected the CagA overexpression plasmid and its control plasmid into AGS, BGC-823and GES-1cell lines and determined the mRNA and protein expression of beta-arrestinl, HIF1A and PKM248hours later. We also analyzed the interaction of all of them after CagA overexpression plasmid transfected in these cells using immunofluorescence assay and luciferase reporter gene assay. The followings are the specific methods in this part:1) After CagA overexpression plasmid and its control plasmid transfection into AGS, BGC-823and GES-1cell lines for48hours, we determined the mRNA and protein expression of beta-arrestinl, HIF1A and PKM2by qRT-PCR and Western blotting.2) After beta-arrestinl siRNA and its NC siRNA transfection into AGS, BGC-823and GES-1cell lines for72hours, we determined the mRNA and protein expression of beta-arrestinl, HIF1A and PKM2by qRT-PCR and Western blotting.3) After CagA overexpression plasmid and its control plasmid transfection into AGS and BGC-823cell lines for12hours, we determined the location and interaction between beta-arrestinl and HIF1A using immunofluorescence assay.4) Luciferase reporter gene with HRE of PKM2and CagA overexpression plasmid co-transfected to see the directed binding between HIF1A and PKM2after CagA treatment using luciferase reporter gene assay. Besides, co-transfected luciferase reporter gene with HRE of PKM2and CagA overexpression plasmid after interference of beta-arrestinl, we determined the role of beta-arrestinl in the relationship between HIF1A and PKM2.3. In the organic level, using glucose and lactic acid test kit to detect the content of blood glucose and lactic acid, in order to determine the change of metabolism products level during the insistent infection of helicobacter pylori.Results:1. The expression of beta-arrestinl was stronger in gastric cancerous than non-cancerous tissues, as well as the expression of HIF1A and PKM2.2. After CagA overexpression plasmid transfection into AGS, BGC-823and GES-1cell lines for48hours, the expression of beta-arrestinl was significantly increased, the expression of HIFIA and PKM2were enhanced accordingly.Using immunofluorescence assay, we detected that beta-arrestinl transportation to the nucleus increased and the HIF1A regularly distributed after CagA transfection12hours.3. After beta-arrestinl siRNA transfection into AGS and BGC-823cell lines for72hours, the expression of beta-arrestinl almost none, accordingly, the expression of HIF1A and PKM2were decreased.4. The luciferase activity dramatically increased with CagA overexpression plasmid and PKM2-HRE co-transfection compared with their control group. Also, the activity decreased after beta-arrestinl interference.5. The detection of blood glucose and lactate, we found the glucose was consumed and the lactate was produced, which coincide with the Warburg effect.Conclusion:Beta-arrestinl participated in the carcinogenesis of gastric cancer through regulating PKM2expression, affecting the regulation of glucose metabolism.