Analysis Of The Correlation Between DGKI And The Prognosis Of Gastric Cancer And Its Mechanism Of Regulating MAPK/ERK Pathway To Promote Gastric Cancer Progression

Research content:This study was divided into four chapters:1.Study on the relationship between DGKI and prognosis of gastric cancer based on TCGA database;2.Study on the relationship between DGKI and clinicopathological characteristics of gastric cancer;3.Effects of DGKI on the biological behavior of gastric cancer cells;4.Study on the mechanism of DGKI regulating MAPK/ERK pathway to promote gastric cancer progressionPart one:Study on the Relationship Between DGKI and Prognosis of Gastric Cancer Based on TCGA DatabaseObjective:Diacylglycerol kinase iota(DGKI)is overexpressed in a variety of cancers and is associated with poor prognosis in colon cancer.This study evaluated the prognostic value of DGKI in gastric cancer(GC)using data from The Cancer Genome Atlas(TCGA)Methods:RNA sequencing results and clinical data of gastric adenoma and adenocarcinoma samples were obtained from the TCGA database(https://portal.gdc.cancer.gov).Kaplan-Meier analysis was used to compare the survival time between high and low expression of DGKI in GC patients.Wilcoxon or Kruskal-Wallis test and logistic regression were used to analyze the relationship between DGKI and clinical pathological parameters of GC patients.Cox regression was used to analyze the relationship between the patient’s clinicopathological parameters and DGKI and overall survival,and to find out the high risk factors that affect the GC patient’s prognosis.Gene set enrichment analysis(GSEA)was performed using the TCGA datasetResults:DGKI was overexpressed in gastric tumors and was related to poor prognosis(P=0.003).Overexpression of DGKI in GC was significantly correlated with high grade(OR=1.71 for G3 vs.G2),stage(OR-2.08 for II vs.I)and T classification(OR=4.64 for T4 vs.T1;OR=3.99 for T3 vs.T1;OR=3.37 for T2 vs.T1)(all P<0.05).DGKI(OR=7.34;P=0.000)was an independent risk factor affecting the survival of GC patients.The MAPK signaling pathway was differentially enriched with DGKI overexpressionConclusion:DGKI overexpression may be a potential molecular marker for poor prognosis in GC.The MAPK signaling pathway may be one of the key pathways related to DGKI regulation in GCPart two:Study on the Relationship Between DGKI and Clinicopathological Characteristics of Gastric CancerObjective:To investigate the expression of DGKI in gastric cancer(GC)tissues and analyze its correlation with the clinicopathological characteristics of GC patients.Methods:Postoperative pathological data and paired specimens of cancer and paracancerous tissues were collected from 50 patients with D2 radical resection of gastric adenocarcinoma in our hospital from January 2017 to January 2018 Immunohistochemistry,western blotting and quantitative real-time fluorescence PCR(qRT-PCR)were used to detect the expression of DGKI protein and mRNA in GC and adjacent tissues,and to analyze the correlation between the expression level of DGKI mRNA in GC tissues and clinicopathological parameters and prognosis of GC patients.Results:The expression levels of DGKI mRNA and protein in GC tissues were higher than those in adjacent tissues(P<0.0001).The expression level of DGKI mRNA was related to vascular invasion(P=0.025),nerve invasion(P=0.048),histological grade(P=0.000),stage(P=0.036)and T classification(P=0.018)GC patients with high DGKI expression had a poor prognosis(P=0.003)Conclusion:The expression level of DGKI mRNA and protein in GC was markedly increased.The higher the DGKI expression was,the higher the possibility of tumor vascular invasion and nerve invasion was,the lower the degree of tissue differentiation was,the later the tumor stage was,and the deeper the invasion was and the worse the prognosis wasPart three:Effects of DGKI on the Biological Behavior of Gastric Cancer CellsObjective:To investigate the effects of DGKI silencing and overexpression on proliferation,migration,invasion in vitro and cell cycle of gastric cancer(GC)cellsMethods:GES-1,AGS,MKN-45,MGC-803 and HGC-27 cell lines were cultured.Using lentivirus-mediated gene silencing and overexpression technology,a stable transgenic cell line for DGKI silencing and overexpression were constructed The qRT-PCR and western blotting were used to detect the expression of DGKI protein in each cell line and the overexpression and silencing efficiency of DGKI The effects of DGKI silencing and over expression on the proliferation,migration and invasion of GC cells in vitro were determined by plate cloning,EdU proliferation,wound healing and Trans well assays.The effects of DGKI silencing and overexpression on cell cycle of GC cells were detected by flow cytometry.Results:The expression level of DGKI protein in GC cells was higher than that in normal gastric cells,MGC-803 expression was the lowest,and HGC-27 expression was the highest.The stable transformation of MGC-803 overexpressed by DGKI and HGC-27 silenced by DGKI were successfully constructed.The proliferation,migration and invasion ability of the GC cell line HGC-27 with DGKI silencing were decreased,and the proliferation,migration and invasion ability of the GC cell line MGC-803 with DGKI overexpression were increasedConclusion:DGKI silencing inhibits the proliferation,migration and invasion ability of GC cell lines in vitro;DGKI overexpression promotes the proliferation,migration and invasion ability of GC cell lines in vitroPart four:Study on the Mechanism of DGKI Regulating MAPK/ERK Pathway to Promote Gastric Cancer ProgressionObjective:To investigate the molecular mechanism of DGKI in promoting the proliferation,migration and invasion of gastric cancer(GC)cellsMethods:The total protein of GC cell line MGC-803 with DGKI overexpression and HGC-27 with DGKI silencing were extracted.Western blotting was used to detect the ERK signaling pathway related molecules(Raf,ERK1/2,p-ERK 1/2,Fos,and c-Myc),EMT-related molecules(E-cadherin,N-cadherin,β-catenin,and Vimentin)and MMPs-related molecules(MMP2 and MMP9)protein expression in GC cell line MGC-803 with DGKI overexpression and HGC-27 with DGKI silencing,and the expression of β-catenin was detected by cellular immunofluorescence experiments.Results:The protein expression of ERK signaling pathway-related molecules(Raf,ERK1/2,p-ERK1/2,Fos,and c-Myc),EMT-related molecules(N-cadherin,β-catenin,and Vimentin)and MMPs molecules(MMP2 and MMP9)was increased and that the protein expression of E-cadherin was decreased in the MGC-803 cell lines in which DGKI expression was upregulated(all P<0.05).In the HGC-27 cell lines in which DGKI expression was silenced,the results were the opposite(all P<0.05).DGKI overexpression enhanced the expression and nuclear accumulation ofβ-catenin,while DGKI silencing inhibited the expression and nuclear accumulation of β-catenin.Conclusion:DGKI may promote the proliferation,migration and invasion of GC cells by affecting EMT,upregulating MMPs and enhancing the MAPK/ERK pathway.