| Objective:To change the expression of KAI1 gene in different gastric cancer cell lines by transfecting lentiviral vectors,and to explore the effects of KAI1 gene on the biological behaviors of gastric cancer cells in vitro growth and migration ability.Methods:1.Group human low-differentiated gastric cancer cells BGC823 into experimental group(BGC823-OE group)transfected with KAI1 gene overexpression lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-KAI1-3Flag,negative control group(BGC823-NC group)transfected with empty virus vector pLenti-EF1a-EGFP-F2APuro-CMV-MCS,and Blank control group(BGC823-Ctrl group);at the same time,human highly differentiated gastric cancer cells MKN28 were grouped into experimental groups(MKN28-siRNA group)transfected with KAI1 gene lentiviral RNA interference vector pLKD-CMV-EGFP-2A-Puro-U6-shRNA(KAI1),and negative controls transfected with empty virus vector pLKD-CMV-EGFP-Puro-U6-shRNA Group(MKN28-NC group)and blank control group(MKN28-Ctrl group),72 hours after virus infection,observe the expression of green fluorescent protein EGFP in cells of each group under a fluorescence microscope,and establish stable transfected cells by drug screening method Strain.2.Western Blot and RT-PCR were used to detect the expression of KAI1 protein and mRNA in each group of cell lines.3.Tetrazole salt colorimetry(MTT)was used to detect the growth and proliferation of cell lines in each group.4.Cell cycle changes were detected by flow cytometry.5.Transwell assay was used to detect the migration of cell lines in each group.Results: 1.After 72 hours of lentiviral infection in each group of cells,the expression of a large amount of fluorescent protein EGFP was observed in two types of gastric cancer cells test group and NC group under fluorescence microscope,but no green fluorescence expression was observed in Ctrl group.The method of drug screening successfully expanded and constructed stable transfected cell lines of OE group and NC group.2.The relative expression of KAI1 protein and mRNA in BGC823-OE group was significantly higher than that of BGC823-NC group and BGC823-Ctrl group(P<0.05);while the relative expression of KAI1 protein and mRNA in MKN28-siRNA group was significant Compared with the MKN28-NC group and the MKN28-Ctrl group(P<0.05);there was no statistically significant difference in the relative expression of KAI1 protein and mRNA in the NC group and the Ctrl group of the two cells(P>0.05).3.The relative proliferation rates of the BGC823-OE group at 24 h,48h and 72 h were significantly lower than the relative proliferation rates of the BGC823-NC group and the BGC823-Ctrl group at the same period,and the differences were significant(P<0.05);while MKN28 The relative proliferation rates of the siRNA group at 24 h,48h and 72 h were significantly higher than the relative proliferation rates of the MKN28-NC group and the MKN28-Ctrl group at the same period,and the difference was also significant(P<0.05);the NC group of the two cells There was no statistically significant difference in the relative proliferation rates between 24 h,48h,and 72 h with the Ctrl group(P>0.05).4.The proportion of S-phase in BGC823-OE group was significantly less than that in BGC823-NC and BGC823-Ctrl groups,and the difference was significant(P<0.05).The proportion of S-phase in cell cycle in MKN28-siRNAgroup was significantly more than The difference between the MKN28-NC group and the MKN28-Ctrl group was also significant(P<0.05);there was no statistically significant difference in the proportion of the S phase between the NC group and the Ctrl group of the two cells(P>0.05).5.The migration number of BGC823-OE group was significantly less than that of BGC823-NC group and BGC823-Ctrl group,the difference was significant(P<0.05);the number of cell migration of MKN28-siRNA group was more than that of MKN28-NC group and MKN28-Ctrl group,the difference was significant(P<0.05);there was no significant difference in cell migration between the two types of NC group and Ctrl group(P>0.05).Conclusion: 1.The human gastric cancer poorly differentiated cell line BGC823,which stably and highly expresses the KAI1 gene,and the human gastric cancer highly differentiated cell line MKN28,which has stable and low expression of the KAI1 gene,have been successfully constructed.2.Up-regulating the expression of KAI1 gene can inhibit the proliferation and migration of BGC823 cell line in vitro;down-regulating the expression of KAI1 gene can promote the proliferation and migration of MKN28 cell line in vitro.3.KAI1 gene can block the growth cycle of gastric cancer cells in G0~G1 phase.4.KAI1 gene negatively regulates the growth and migration ability of different gastric cancer cell lines in vitro. |
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