Study On The Induction Of HMGA2 Into EMT-like Gastric Cancer Stem Cells And Its Possible Molecular Mechanism

Part one: Expression of HMGA2 in gastric cancer and its clinical significanceObjective: To study the expression of HMGA2 in gastric cancer and its relationship with clinicopathological features and prognosisMethods:1?TCGA database,GTEx database and bioinformatics analysis tools(GEPIA,KM plotter)were used to analyze the expression of HMGA2 mRNA in gastric cancer and the survival time of patients.2?72 cases of gastric cancer tissue specimens treated by general surgery in southwest hospital from June 2018 to December 2018 were collected,and 72 cases of paracancer tissue specimens(more than 2cm away from the tumor tissue)were used as controls.Patients who had received chemoradiotherapy before surgery were not included in the study.Immunohistochemical staining was used to detect the expression of HMGA2 in gastric cancer and adjacent tissues,and the differences were compared.3?Using Ucsc xena website TCGA STAD(stomach)transcriptome sequencing data,screening HMGA2 transcriptome sequencing data at the same time matching patients' age,sex,T stage,M stage and related clinical data,delete the missing data,according to HMGA2 mRNA expression quantity is divided into a high and low expression group,analysis HMGA2 mRNA expression level clinicopathologic correlation.Result:1?Database analysis revealed that the expression of HMGA2 mRNA in gastric carcinoma was increased compared with that in normal gastric tissues(P<0.05),the overall survival and recurrence free survival of HMGA2 mRNA higher expression patients were shortened(P<0.05).2?The expression rate of HMGA2 in gastric cancer tissues was 69.4%(50/72),which was significantly higher than that in gastric cancer tissues 12.5%(9/72)(P<0.05).The difference was statistically significant.3?TCGA database was successfully used to screen HMGA2 sequencing data of 337 containing complete clinical information of patients.According to the expression level of HMGA2 mRNA,the first 168 cases were in the low expression group.The latter 169 cases were in the high-expression group,and the results showed that the expression level of HMGA2 mRNA was statistically significant in T stage(P<0.05).There were no statistical differences in gender,age,tumor differentiation,pathological stage,N stage and M stage(P>0.05).Conclusion:1?The expression level of HMGA2 in gastric cancer tissues is higher than that in paracancer normal tissues,and it is related to the prognosis of patients.2?The expression of HMGA2 mRNA was correlated with T stage,but not with gender,age,tumor differentiation,pathological stage,N stage and M stage.Part two: Isolation and identification of gastric cancer stem cell subsetsObjective: Stationary and invasive gastric cancer stem cells were isolated and identifiedMethods:1?Isolation and identification of gastric cancer stem cells.Based on the previous successful culture of gastric cancer stem cells by the research group,mgc-803 cell lines were selected for pellet culture,and the proliferation and invasion ability of pellet cells and normal adherent cells were compared by transwell experiment and plate cloning experiment.The expression of the markers OCT3,ALDH1A1 and Nanog in pellet cells and normal adherent cells was detected by western blot.2?Sorting and identification of stem cell subsets.Selection on the basis of related literature and research the experiment results CD26 gastric cancer stem cell markers and CXCR4 as EMT samples,through the flow separation for CD26 + CXCR4 + and CD26-CXCR4-two group of cells,and through the flat cloning experiments,transwell experiment comparing two subgroup cell proliferation ability,by western blot detection EMT related marker protein expression in both the group of cells.Result:1?Pellet culture was used to successfully isolate the integrated pellet cells.The results of plate cloning experiment and transwell experiment showed that the proliferation and invasion ability of pellet cells was higher than that of normal adherent cells.Western blot results showed that compared with normal adherent cells,the expression level of stem cell markers was higher in globular cells.2?The results of plate cloning experiment and transwell experiment showed that the proliferation and invasion ability of CD26+CXCR4+ cells was higher than that of CD26-CXCR4-cells.Western blot results showed that the expression of e-cadherin in CD26+CXCR4+ cells was significantly decreased and the expression of vimentin was significantly increased.Conclusion:1?The pellet-forming cells obtained by serum-free pellet-forming culture of gastric cancer cells have the characteristics of stem cells and are gastric cancer stem cells2?CD26 and CXCR4 were used as markers of invasive GCSCs and flow separation could separate stationary and invasive GCSCs3 ? The emt-like characteristics of invasive GCSCs may be due to the EMT transformation of stationary GCSCsPart three: Study on the induction of HMGA2 into emt-like gastric cancer stem cells and its mechanismObjective: In vitro studies were conducted to investigate the induction of HMGA2 into emt-like gastric cancer stem cells and its possible mechanismMethods:1?Cd26-cxcr4-cells were transfected with lentivirus for high expression of HMGA2.The changes in cell proliferation and invasion capacity before and after transfection were compared by plate cloning experiment and transwell experiment,and the expression changes of emt-related marker proteins were detected by western blot.2?By consulting relevant literature,the transcription factor Snail was selected as the research object after analysis.The expression of Snail was reduced in stationary gastric cancer stem cells with high expression of HMGA2,and the changes of cell invasion ability were detected by transwell assay,and the expression changes of emt-related marker proteins were detected by western blot.Result:1?Transwell assay results showed that after transfection with HMGA2,the proliferation and invasion ability of cd26-cxcr4-cells were enhanced.Western blot results showed that the expression of e-cadherin was decreased,while the expression of n-cadherin and vimentin were increased.2?After the expression of HMGA2 was overexpressed,the invasion ability of resting gastric cancer stem cells was significantly reduced(P<0.05),and the expression of e-cadherin was increased,while the expression of n-cadherin and Vimentin were decreased.Conclusion:1?HMGA2 can enhance the invasion ability of stationary gastric cancer stem cells and promote their transformation into emt-like gastric cancer stem cells.2?HMGA2 may promote the transformation of resting gastric cancer stem cells and emt-like gastric cancer stem cells by regulating the expression of Snail.