| Objective:The aim of this study was to investigate the effect of over-expression of c1galt1 gene on the proliferation,migration and invasion of human gastric cancer cell line MGC-803.Method:Human gastric cancer MGC-803 cells were transfected b y lentivirus packaging vector of pHBLV-CMV-EF1-Zsgreen1-T2A-pu ro-h-C1GALT1,to establish a recombinant cell line for stable over-e xpression of C1GALT1.The successful constructed cell line were be s creened out by Puromycin.The expression of C1GALT1 mRNA and protein were examined by realtime-polymerase chain reaction(Realti me PCR)and Western blot.And then,MGC-803 cells transfected with empty vector were used as controls.The effect of over-expressi on of C1GALT1 on the proliferation of gastric cancer cell line MG C-803 were examined by CCK8.The effect of over-expression of C1GALT1 on migration and invasion of human gastric carcinoma cells were detected by wound healing assay and Transwell assay,respectively.The change of cell cycle was detected by flow cytometry.Results:1?After expanded culture,3 cell lines were finally selected by limited dilution method,there were MGC803-C1GALT1-1?MGC803-C1GALT1-2 and MGC803-C1GALT1-3.2?Realtime PCR showed that the target gene h-C1GALT1 had over-expressed in MGC-803 Cells.The over-expression of h-C1GALT1-1,h-C1GALT1-2 and h-C1GALT1-3 in MGC-803 cells was 6 times,18 times and 17 times,respectively.3?Western blot analysis showed that the target gene C1GALT1 was over-expressed in MGC803 Cells,among which the over-expression of MGC803-h-C1GALT1-2 group was the highest,named C1GALT1-OE,and the stable cells transfected with empty vector were named Control.C1GALT1-OE and Control were selected as follow-up experimental cell lines.4?CCK8 test showed that the absorbance of OD450 in the transfection group and empty vector group did not change significantly at 12 h and 24 h,but at 36 h and 48 h,the absorbance of cells in the over-expression group of C1GALT1 was significantly higher than that in the empty vector group(P < 0.01).At 36 h and 48 h,the absorbance of cells in the transfection group was(1.35±0.19)and(1.33±0.02),and that in the empty vector group was(0.89±0.09)and(1.10±0.02),respectively.The results showed that after 36 hours,the proliferation ability of MGC-803 gastric cancer cells could be significantly improved by increasing the expression of C1GALT1.5?Wound healing assay showed that there was no significant difference in scratch area between the transfected group and the control group at 0 h and 24 h after scribing,but after 48 h,the scratch healing degree of the transfected group was significantly higher than that of the control group(P < 0.05).At 48 h and 72 h,the healing rates of the transfected group were(20.02±1.08)%and(26.86±1.35)%,,and that of the empty vector group were(15.25±1.42)%and(22.45±1.46)%,respectively.The results showed that after 48 hours,the migration ability of MGC-803 cells could be improved by increasing the expression of C1GALT1.6?Transwell experiment showed that,compared with the empty vector group,the number of transfected cells passed through the Matrigel was significantly higher than that of the control group(P < 0.05).The number of cells passing through the Matrigel in the transfection group and the empty vector group were(73.50±5.4)and(45.67±4.92),respectively.7 ? Flow cytometry analysis showed that compared with the control group,the proportion of cells in S phase in the transfection group increased significantly(P < 0.05),but there was no significant difference between the two groups in G2 / G1 phase cell ratio(G2 / G1).Conclusion:The over-expression of c1galt1 can significantly improve the proliferation,migration and invasion of MGC-803 cells.The increased expression of C1GALT1 can accelerate the cell cycle of MGC-803 and promote the cell transformation to S phase. |
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