LincRNA Z38’s Effect On Gastric Cancer Cell Proliferation,Migration And Tumor Microenvironment

Objective:As the"messenger"between cells,exosomes play an important role in promoting the tumor microenvironment and tumor biological process by mediating intercellular communication and transmitting intercellular macromolecules.The long non-coding RNA（Long non-coding RNA（linc RNA）plays an important role in the microenvironment of tumor occurrence,development and tumor metastasis.In order to further explore its function and biological mechanism,this study extracts exosomes from peripheral serum and cell culture medium to detect the expression of Z38 gene in exosomes;and through interference and overexpression of this gene,to study the effect of this gene on gastric cancer The biological effects of SGC7901 cells,and the expression of STK15,MMP-9 m RN and protein after silencing and over-expression of Z38 gene,lay the foundation for the later biological treatment of gastric cancer and liver metastasis of gastric cancer.Methods:The exosomes from human gastric mucosal epithelial GES-1 cells and gastric cancer SGC7901 cell culture medium,and peripheral sera of patients with normal stomach and gastric cancer liver metastases were extracted,and the expression of Z38 gene was detected by RT-PCR.Construct Z38 overexpression plasmid,design si RNA,use cationic polymer polyethyleneimine（PEI）to transfect logarithmic gastric cancer SGC7901 cells,divide the cells into blank group（no treatment）,negative control group（transform irrelevant interference）,silence group（transfected with Z38 interference fragment）,overexpression group（transfected with Z38 overexpression plasmid）,48h after transfection,observe the cell transfection efficiency under a laser confocal microscope,and extract total cell RNA and total protein.Real-time fluorescent quantitative PCR and Western-blot method to detect the expression of STK15 and MMP-9 m RNA and protein in SGC7901 cells;Plant SGC7901 cells in a six-well plate,and use CCK8 and stranswell assay to detect changes in the proliferation activity and migration ability of gastric cancer SGC7901 cells after silencing and overexpression of the Z38 gene;cell flow cytometry was used to detect the expression and silencing The cell cycle and apoptosis of each group after Z38 gene.Results:（1）Successfully extract exosomes from peripheral serum and culture medium,and verify that Z38 gene is highly expressed in peripheral serum exosomes of patients with gastric cancer SGC7901 cell culture and liver metastases from gastric cancer;（2）48 hours after PEI transfection of SGC7901 cells,red fluorescence was seen in the silence group and green fluorescence in the overexpression group SGC7901.The number of cell deaths in bright field was small,and the m RNA level verified that the Z38 gene can be successfully interfered and overexpressed;（3）After si RNA silencing,the m RNA down-regulation of Z38 gene was si RNA-1:38.0%,si RNA-2:64.0%,and si RNA-3:14.1%.The transfection efficiency of si RNA-2 group was significantly higher than that of si RNA-3 and si RNA-In group 1,the silencing effect of si RNA-2 was the most significant（P<0.01）;SGC7901 cells in the overexpression group showed obvious green fluorescence under the microscope after 48 hours of transfection.Combined with the results of real-time fluorescent quantitative PCR,it was compared with the negative control group and the overexpression group.The m RNA expression level of Z38 was significantly increased,with significant differences（P<0.01）.Western-blot results showed that STK15 protein in the silence group was significantly lower than that in the negative control group（p<0.05）,while the expression of STK15 protein in the overexpression group was significantly increased（p<0.01）;the expression of MMP-9protein in the silence group was significantly reduced（p<0.05）0.01）,the expression of MMP-9 protein in the overexpression group was significantly increased（p<0.01）;（4）The transwell assay results showed that the migration ability of the cells in the silent group was inhibited compared with the negative control group（p<0.05）,while the migration ability of the cells in the overexpression group was significantly stronger than that of the negative control group（p<0.05）;（5）The CCK8 experiment results showed that compared with the negative control group,the cell viability of SGC7901 in the silent group was significantly reduced（p<0.05）,while the cell viability of the overexpression group was significantly enhanced（p<0.05）;（6）The cell flow cytometry results showed that compared with the negative control,the number of cells in the S phase and G₂/M phase of the silence group was significantly reduced（p<0.05）,indicating that the silence group cells were mainly inhibited during the S-G₂transition,blocking cells from the S phase Progress to G₂/M phase;the apoptosis rate of the silent group was significantly up-regulated compared with the negative control group,and the average apoptotic rate of the overexpression group was down-regulated,and the difference was statistically significant（p<0.01）.Conclusion:（1）Transfection of Z38overexpression plasmid significantly increased the expression of Z38 m RNA,transfection of si RNA reduced the expression of Z38 m RNA,combined with the green fluorescence and red fluorescence observed under the microscope,indicating that p LVX-EFIa-IRES-GFP-Z38 was successfully transfected Plasmids and si RNA successfully enabled the overexpression and silencing of the Z38 gene in human gastric cancer SGC7901 cells.（2）The transwell assay,CCK8 and cell flow cytometry results prove that Z38 as an oncogene can promote the migration of SGC7901 cells,enhance proliferation activity,and inhibit gastric cancer cell apoptosis;conversely,silencing the expression of Z38 gene can inhibit SGC7901 cell activity,inhibit migration,and promote gastric cancer cells Apoptosis.（3）The cycle results showed that silencing the Z38 gene blocked the transition of cells from S phase to G2/M phase,thereby blocking the mitosis of gastric cancer cells.（4）Silencing SGC7901 gastric cancer cell Z38 gene expression,STK15,MMP-9 m RNA levels and protein levels are down-regulated,while over-expression of Z38 gene expression,STK15,MMP-9 m RNA levels and protein levels are up-regulated,indicating that silencing Z38 can inhibit STK15,MMP-9expression,linc RNA Z38 may achieve anti-tumor effect by regulating the expression of STK15 and MMP-9,and inhibit the metastasis of gastric cancer.