Biological Function And Molecular Mechanism Of AAED1 And TCF7L1 In Gastric Cancer Cells

Part Ⅰ: Expression and biological function of AAED1 in gastric cancer cellsObjective: Ahp C/TSA Antioxidant Enzyme Domain Containing 1(AAED1)is located on chromosome 9.Its transcriptional protein is found in multiple regions,for example in brain tissue.It has been found to play a regulatory role in neurological diseases.AAED1 contains classic structure of antioxidant enzyme fragments,which suggests that it may play an important role in antioxidation reponse and may be critical in the regulation of malignant tumor metabolism.This part of study is based on this view,we assess the difference between AAED1 expression in tissu of gastric carcer and AAED1 expression in tissu in normal gastric mucosa.We also detect the biological function of gastric cancer cells in the condition of AAED1 knock-down.It is expected that this part of study can discover the expression and biological function of AAED1 in gastric cancer cells.Methods: Immunohistochemical staining is used to detect the difference between the expression of AAED1 in gastric cancer tissue and expression of AAED1 in normal tissue.Paraffin tissue specimens was collected from radical gastrectomy operation.By q RT-PCR and Western Blot assays,we established the stable transfected cell lines which were transfected with lentivirus containing plasmid of AAED1 knock-down.In the condition of AAED1 knock-down,AGS and MGC-803 cell lines were cultured and functional assessed.We use following assays:CCK8 assay to assess cell proliferation activity;scratch assay to assess cell migration;transwell assay to detect invasion ability;clone formation assay to assess clone formation ability.We also use in vivo assay,nude mice xenograft experiments to assess the ability of tumorigenesis in animals.Results: Immunohistochemical staining demonstrates that AAED1 expression in gastric cancer tissue is significantly higher than in normal tissue(p<0.01).CCK8 assay confirms that AAED1 knock-down inhibits proliferation ability of gastric cells,scratch assay confirms that AAED1 knock-down inhibits migration ability of gastric cells,transwell assay confirms that AAED1 knock-down inhibits invasion ability of gastric cells,and clone formation assay confirms that AAED1 knock-down inhibits clone formation ability of gastric cancer cells.Xenograft experiments demonstrates that AAED1 knock-down inhibits the tumorigenesis ability of gastric cancer cells in vivo.Conclusion: AAED1 is highly expressed in gastric carcinoma.AAED1 knockdown inhibits the gastric cancer cells proliferation ability,migration ability,invasion ability,clone-formation ability in vitro and xenograft tumorigenesis ability in vivo.Part Ⅱ: Expression and biological function of TCF7L1 in gastric cancer cellsObjective: TCF7L1 is one of the LEF/TCF(lymphoid enhancer factor/T cell factor)transcription factor family.The LEF/TCF transcription factor can bind to the β-catenin in the nucleus and transmit the Wnt signal to initiate the related target genes.Wnt/β-catenin signaling pathway has been reported in the regulation of gastric cancer.Functional enhancement of Wnt activators,such as Wnt-1,Wnt-2 and Wnt-2B,has been demonstrated to have high expression in gastric cancer.TCF7L1 was found to have high expression in poor survival cancer cases in several malignant tumors.This part of study is based on this view to detect the expression of TCF7L1 in gastric cancer and to determine whether TCF7L1 has a role of regulation in the progression of gastric cancer.Methods: We analyse the TCGA gastric cancer cases to observe the difference of TCF7L1 expression in gastric cancer tissues,and find if there is the relation in the malignant degree and prognosis of gastric cancer and TCF7L1 expression.In the cell assays,lentivirus infection technology is used to establish stable TCF7L1 knockdown AGS and MGC-803 gastric cancer cell lines.CCK8,clone formation,scratch and Transwell assays are used to observe the biological function of TCF7L1 knockdown in gastric cancer cells.Results: TCGA data analysis shows that TCF7L1 has high expression level in gastric cancer tissue,high TCF7L1 expression level predicts poorer prognosis and later T,N classification in gastric cancer.TCF7L1 knock-down inhibits the proliferation ability,clone formation ability,invasion ability and migration ability of gastric cancer cells in vitro.Conclusion: TCF7L1 expression is higher in gastric carcer cases.High expression of TCF7L1 is associated with the malignant degree and predicts poor prognosis of gastric cancer.Knockdown TCF7L1 inhibits the cell proliferation ability,clone formation ability,invasion ability and migration ability in gastric cancer cells.Part Ⅲ Mechanism of AAED1 and TCF7L1 regulating gastric cancer cells progressionObjective: AAED1 and TCF7L1 knock-down inhibits the progrssion of gastric cancer cells,but the mechanism of AAED1 and TCF7L1 in the regulation of gastric cancer is unknown.Both AAED1 and TCF7L1 are related to the redox cell metabolic pathway.In this part,we detect the regulation in glycolysis and redox pathway of AAED1 and TCF7L1 in gastric cancer cells.The purpose is to find the mechanism of AAED1 and TCF7L1 in the regulation of gastric cancer cells.Methods: RT-PCR and Western Blot are used to detect the glycolysis regulators and HIF1α expression in condition of AAED1 knock-down.Seahorse XF test is used to detect OCR and ECAR when AAED1 and TCF7L1 are knocked-down,and to confirm the effects of AAED1 and TCF7L1 in the glycolysis.DCFH-DA assay is used to detect ROS level change in in condition of TCF7L1 knock-down in AGS and MGC-803 cells.RT-PCR and Western Blot are used to detect the transcription of NRF2,Keap1 and their downstreams in the condition of TCF7L1 knock-down.Dual-luciferase assay is used to assess the regulation of TCF7L1 in Keap1 promoter activity.Results: AAED1 knock-down inhibits the phosphorylation of ERK1/2 and AKT1,and also inhibits their downstream HIF1α expression in AGS and MGC-803 gastric cancer cells.AAED1 knock-down inhibits glycolysis related genes expression,including GLUT1,HK2、PGK1,LDHA.TCF7L1 knock-down inhibits NRF2 protein expression and the transcriptional process in the downstream of NRF2(GCLC,GCLM,HMOX,ME1,TXNRD,NQO1).TCF7L1 knock-down enhances the expression of Keap1.TCF7L1 can regulate the promoter activity of Keap1.AAED1 regulates the HIF1α signaling pathway,TCF7L1 regulates the NRF2-Keap1 signaling pathway.AAED1 and TCF7L1 knock-down regulates the glycolysis and antioxidation response in gastric cancer cells.Conclusion: AAED1 regulates the HIF1α pathway,TCF7L1 regulates the NRF2-Keap1 pathway.AAED1 and TCF7L1 are regulators in the glycolysis and antioxidation response of gastric cancer cells.By the means of regulation in gastric cancer cell metabolism,AAED1 and TCF7L1 improve gastric cancer cells antioxidant capacity,and promote gastric cancer cells survive and proliferate.The present study uncovered AAED1 and TCF7L1 as novel markers of gastric cancer and provides the possible underlying molecular mechanism.