Effect Of RNF20 On DNA Damage Repair In UV-induced Gastric Cancer Cells

Objective:To explore the effect of knockdown Ring Finger Protein 20（RNF20）on DNA damage repair in gastric cancer cells under ultraviolet exposure.Methods:1.Construction of MGC803 gastric cancer cell line with stable low expression of RNF20Design short hairpin RNA sequences against RNF20 gene;（1）Design of short hairpin RNA sequences against RNF20 gene;（2）Construct p Lent-U6-GFP-Puro-sh RNF20,p Lent-U6-GFP-Puro-sh Ctrl lentiviral vectors;（3）Packaging lentivirus,infecting human gastric cancer cells MGC803,divided into Ctrl control group and RNF20 knockdown group,observing the green fluorescent protein expression in both groups of cells by the fluorescence microscopy,and screening both groups of cell lines by puromycin;（4）The expression of RNF20 protein in the cells was identified by western blotting.2.Effect of knockdown of RNF20 on proliferation of gastric cancer cellsThe effect of knockdown of RNF20 on the proliferation of gastric cancer cells was detected by CCK-8 method.3.Effect of knockdown RNF20 on DNA damage repair in gastric cancer cellsIrradiation of gastric cancer cells using UV light at a total irradiation dose of20J·m^-2;the expression ofγ-H2AX and RAD51 protein was detected by immunofluorescence staining,the expression ofγ-H2AX and p21 protein was detected by western blotting.Results:1.Establishment of MGC803 gastric cancer cell line with stable low expression of RNF20（1）Fluorescence microscopy observed that the infection efficiency of the stably transduced cells were higher than 85%;（2）The results of western blotting showed that the expression of RNF20 protein in gastric cancer cells in the knockdown group was reduced compared to the control group.2.Effect of knockdown of RNF20 on the proliferation of gastric cancer cellsThe results of CCK-8 assay showed that the proliferation ability of MGC803 cells with knockdown of RNF20 was enhanced compared with the control group.3.Effect of knockdown of RNF20 on DNA damage repair in gastric cancer cells（1）The results of immunofluorescence staining and Western blotting showed that the disappearance ofγ-H2AX was more delayed in MGC803 cells with knockdown of RNF20 than in the control cells;（2）The results of immunofluorescence staining showed that the recruitment of RAD51 protein was delayed in MGC803 cells with knockdown of RNF20 compared with control cells;（3）The results of Western blotting showed that the trend of p21 protein decreased more slowly in cells with knockdown RNF20 than in control cells.Conclusion:In this study,we constructed RNF20 knockdown gastric cancer cell lines by lentiviral vectors and found that knockdown of RNF20 promoted the proliferation of gastric cancer cells,and knockdown of RNF20 resulted in more severe DNA double-strand break damage and defective DNA damage repair in gastric cancer cells.