Effects Lumican On The Biological Behavior Of Gastric Cancer Cells And Its Mechanism

ObjectiveIn our previous experimental findings,we found and confirmed that the expression of Lumican in gastric cancer tissues and cells was higher than that in normal tissues and cells,and that Integrin Alpha 5（Integrin 5）had an important relationship with the occurrence and development of gastric cancer.The objective of this study was to further explore the effects of Lumican gene expression intervention on the biological behavior of AGS and HGC-27 gastric cancer cell lines and the mechanisms involved as well as the regulatory relationship of Integrin Alpha 5.MethodsGEPIA2 software was used to analyze the m RNA expression data of gastric cancer tissue samples and normal gastric tissue in the database of the Tumor Genome Atlas Project,to determine the expression difference of Lumican in cancer tissue and normal tissue,and meanwhile to analyze its generation curve.Then we carried out fluorescence quantitative PCR analysis on the collected 10 pairs of gastric cancer tissues and adjacent tissues.In the previous experiments,the most effective lumian-si RNA was screened out.Meanwhile,Lumian-overexpressed plasmid was selected to intervene in AGS and HGC-27of gastric cancer,and the intervention effect of Lumian-overexpressed plasmid was the best 24-48 hours after transfection.Therefore,this si RNA and overexpressed plasmid were selected to conduct experiments on AGS and HGC-27 cells of gastric cancer.Lumian-si RNA and Lumian-overexpressed plasmid were transiently transfected into AGS and HGC-27 cells using liposomes formed by Lipofectamine^TM3000.24h after transfection,cell proliferation was detected by CCK-8 assay and EDU with inhibition and enhancement of Lumican expression,respectively.Transwell and artificial matrix gel were used to detect the changes of cell migration and invasion ability.Annexin V-PE and 7-AAD double staining were used for flow cytometry detection to observe the changes of apoptosis of the two cell lines in different treatment groups.The expression sites of Lumican in the two cells were detected and confirmed by immunofluorescence.The expression of FAK,phosphorylated P-FAK and Integrin alpha 5 was detected by Western blot.Finally,we used Western blot and q PCR to detect the expression of Lumican in AGS and MKN-28 cells with stable gastric cancer transmutation after the intervention of Integrin Alpha 5 in the previous experiment.Results1.PCR verification results of database and 10 pairs of tissue samples:In408 gastric cancer tissue samples and 211 normal tissue samples from the Tumor Genome Atlas Project database,Lumican expression was significantly enriched in gastric cancer tissue（P<0.05）.In the survival analysis,the survival time of the low Lumican expression group was significantly prolonged in the high Lumican expression group（P<0.05）.PCR results of collected tissue samples indicated that the expression of Lumican in gastric cancer tissues was significantly higher than that in normal gastric tissues（P<0.05）.2.The results of cell proliferation detected by CCK-8:the proliferation rates of AGS cells and HGC-27 cells in the Lumian-si RNA transfection group were lower than those in the negative control group at all time points（24,48 and72 h）（P<0.05）;The proliferation rates of AGS and HGC-27 cells in Lumican overexpression group were significantly higher than those in negative control group at all time points（24,48 and 72 h）（P<0.05）.3.The results of EDU cell proliferation:48 hours after transfection,the proliferation rate of new cells in AGS cells and HGC-27 cells in the Lumian-si RNA transfection group was significantly lower than that in the negative control group（P<0.05）;The proliferation rate of new cells in the overexpression group was significantly higher than that in the negative control group（P<0.05）.4.Transwell test results of cell migration:48 h after transfection,the number of AGS cells and HGC-27 cells migrated in Lumian-si RNA group was significantly lower than that in the corresponding negative control group（P<0.05）.Compared with the negative control group,the migration inhibition rate of AGS cells was 53.90%and HGC-27 cells was 52.54%.In the overexpression group,the number of migrated cells of these two strains was significantly higher than that of the corresponding negative control group（P<0.05）.Compared with the corresponding negative control group,the inhibition rate of migration of AGS cells was-62%,and that of HGC-27 cells was-108%.5.Transwell test results of cell invasion:At 48 hours after transfection,compared with the negative control group,the number of cells invaded the stromal gel in AGS cells and HGC-27 cells in the Lumian-si RNA group was significantly decreased（P<0.05）.The invasion inhibition rate of AGS cells was49.60%,and that of HGC-27 cells was 44.70%.In the overexpression group,the number of cells invaded by AGS cells and HGC-27 cells increased significantly（P<0.05）,and the invasion rates of AGS cells and HGC-27 cells were-115.31%and-132.33%,respectively.6.Flow cytometry detection of cell apoptosis:48 h after transfection of AGS cells and HGC-27 cells,there was no significant difference in the apoptosis rate of Lumian-si RNA group compared with the negative control group（P>0.05）;Similarly,there was no significant difference in apoptosis rate between the two overexpression groups and the negative control group（P>0.05）.7.Immunofluorescence results:DAPI staining indicated that AGS cells and HGC-27 cells of gastric cancer were in good condition,meeting the requirements of the experiment,and Lumi CAN was widely expressed on the cell membrane.8.Western blot results:Compared with the control groups,the expression of FAK protein in the lumican-si RNA group in gastric cancer AGS cells and HGC-27 cells did not change significantly,and the expression of p-FAK protein was significantly reduced（P<0.05）,while Integrin There was no significant difference in the expression of alpha 5 protein;the expression of FAK protein in the overexpression lumican group in gastric cancer AGS and HGC-27 cells did not change significantly,the expression of p-FAK protein was significantly increased（P<0.05）,while the expression of Integrin alpha 5 protein was not Significant differences.In gastric cancer AGS and MKN-28 cells that inhibit the expression of Integrin alpha 5 protein,lumican expression was also significantly reduced（P<0.05）;in gastric cancer AGS and MKN-28 cells that enhanced Integrin alpha 5 protein expression,lumican expression was significantly increased（P<0.05）.9.Fluorescence quantitative PCR results:Lumican m RNA expression did not significantly change after Integrin Alpha 5 expression in AGS and HGC-27cells compared to the control group（P>0.05）.Conclusions1.Lumican is highly expressed in gastric cancer tissues,and the level of its expression is related to the survival rate of gastric cancer patients.2.Interference with Lumican expression can correspondingly change the proliferation,migration and invasion ability of gastric cancer cells;However,it was not related to the apoptosis of gastric cancer cells.3.Lumican performs its biological function through the FAK/p-FAK pathway,and Integrin Alpha 5 May perform its biological function by affecting the protein expression of the membrane protein Lumican.