Establishment Of Human C1GALT1 Stable Gastric Cancer Cell Line Mediated By Lentivirus-mediated ShRNA And Its Biological Characteristics

Objective : To construct a specific RNA interference(RNAi)lentiviral vector targeting human C1GALT1 gene,and establish a stable gastric cancer MGC803 cell line with C1GALT1 gene.The biological characteristics of lentivirus-mediated shRNA targeted interference with C1GALT1 gastric cancer MGC803 stable cell lines were preliminary observed,and the effects of silencing C1GALT1 gene expression on MGC803 cell proliferation,migration,invasion,cycle,and apoptosis were examined.Methods:1.Design specific shRNA target of C1GALT1 gene,construct pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO-h-C1GALT1 interference lentiviral packaging vector,package lentivirus and infect MGC803 cells,construct C1GALT1 gene stable and silent Gastric cancer MGC803 cell line.Stably transfected cells transfected with empty vector were used as negative controls.QPCR method was used to detect the change of C1GALT1 mRNA level in MGC803 cells,and Western Blot method was used to detect the expression of C1GALT1 protein level to identify the effect of C1GALT1 gene silencing.2.Using CCK8 proliferation test,cell scratch test,Transwell test,flow cytometry to detect the effect of C1GALT1 gene silencing on the proliferation,migration,invasion,cycle,and apoptosis of gastric cancer MGC803 cells.Results : 1.Successfully constructed a RNA interference(RNAinterference,RNAi)lentiviral vector specifically targeting the human C1GALT1 gene,and established a stable gastric cancer MGC803 cell line with C1GALT1 gene.After expanding the culture,the three cells of MGC803-C1GALT1-shRNA-1-1,MGC803-C1GALT1-shRNA-1-2,MGC803-C1GALT1-shRNA-1-3 were finally picked up by the limited dilution method for QPCR and Western blot detection,The identification showed that the mRNA and protein expression levels of the C1GALT1 gene silencing group were lower than that of the negative control group(p <0.05),which had an interference effect,of which MGC803-C1GALT1-shRNA-1-3 had the best interference downregulation effect in MGC-803 cells,Named C1GALT1-shRNA.C1GALT1-shRNA and stable transfected cells(Negative Control,NC)transfected with empty vector were selected for subsequent experiments.2.The results of CCK8 proliferation experiments showed that there was no significant change in the absorbance of C1GALT1-silenced MGC-803 stabilized cells compared with the empty vector group at various time points.The scratch test results showed that at 0h and 24 h after streaking,C1GALT1 silenced stable cells showed no significant difference in scratch area compared to the empty vector group,and after 48 h,the degree of scratch healing of C1GALT1 silenced stable cells Significantly lower than the empty vector group(p <0.05).Transwell invasion experiment results showed that the number of C1GALT1-silenced MGC-803 stable transfected cells passing Matrigel was significantly lower than that of the empty vector group(p <0.001).Flow cytometry cell cycle results showed that among the C1GALT1 silenced and stable cells,the proportion of cells in G0 / G1 phase was smaller than that of empty vector group,while the proportion of cells in G2 / M phase and S phase was higher than that of empty vector group.Flow cytometry detection of apoptosis showed that in C1GALT1-shRNA stable cells,0.53% of early apoptotic cells were detected,0.11% of late apoptotic cells,and no obvious early apoptosis was detected in empty vector cells Cells and late apoptotic cells.Conclusions:Silencing C1GALT1 gene expression can reduce the migration and invasion ability of MGC-803 cells and promote apoptosis.