Effect Of Trim28(ser473)phosphorylation On Viability Of HCC Cells During DNA Damage

Objective: To observe effect of Trim28(ser473)phosphorylation on viability of HCC cells during DNA damage.Methods:Hep G2 cells were divided into SB203580+UV group,In the UV group,Hep G2 cells were irradiated with ultraviolet light(UV)to establish a DNA damage model of Hep G2 cells.In the SB203580+UV group,Trim28 phosphorylation inhibitor(SB203580)was used to treat cells on the basis of ultraviolet light,while the control group was not treated.After UV irradiation,the SB203580+UV group was treated with Trim28 phosphorylation inhibitor 4-(4-fluorophenyl)-2-(4-methyl-sulfon3-acyl phenyl)-5-4(-pyridinyl)-1h-imidazole(SB203580).Westernblot assay was used to detect the expression of Trim28,phosphorylated Trim28 ser473(P-Trim28 ser473)was used to observe the expression and intracellular distribution of ?-h2 ax under different treatment conditions.The viability of Hep G2 cells treated above was observed by MTT assay..Result:The expression levels of p-Trim28 ser473 at 2h,12 h and 24 h after ultraviolet irradiation of Hep G2 cells were(0.66±0.13,0.27±0.25,0.12±0.27).The control group was(0.12±0.462).The difference between 2h group and other groups was statistically significant(t=5.246 P < 0.05,t=7.143 P < 0.05,t=6.920 P < 0.05);After 2 hours of UV irradiation,there was no significant difference in Trim28 expression between SB203580+UV(0.96±0.16)group,the UV group(0.94±0.13)and the control group(1.03±0.12)(t=1.596 t=1.346 t=-0.190,P > 0.05);After 2 hours of UV irradiation,p-Trim28 in SB203580+UV group(0.32±0.04)was significantly lower than that in the UV group(0.72±0.05),and the difference was statistically significant(t=8.500,P < 0.05);After 2 hours of UV irradiation on Hep G2 cells,the expression of ?-h2 ax in SB203580+UV group(2.61±0.27)and UV group(2.51±0.09)was higher than that in the control group(0.89±0.05),but there was no significant difference between the two groups(t=-0.593,P >0.05).Immunofluorescence experiments: 2h after UV irradiation Hep G2 cells,?-H2 AX of UV groups and SB203580 + UV groups towards the nucleus gathered,strong fluorescence positive cells proportion respectively(82% ± 14%,84% ±16%),there was no significant difference(t = 0.254,P > 0.05),the 24 h after UV irradiation,strong fluorescent positive cell proportion respectively in SB203580+UV group and UV group(15%±15%,62% ±12%),statistically significant difference(t =4.745,P<0.05).Thiazole blue(MTT)experiment: There was no significant difference in OD values(0.34±0.04,0.43±0.04,0.59±0.06)between the SB203580 group(treated with SB203580 only)and the control group(0.33±0.04,0.41±0.04,0.64±0.03)(t=-0.410 P > 0.05,t=-0.373 P > 0.05,t=1.520 P > 0.05);the cell survival rate of UV group at 24,48 and 72 hours after UV irradiation(0.95±0.07,0.62±0.10,0.35±0.06)was significantly higher than that of SB203580+UV group(0.85±0.07,0.37±0.04,0.20±0.03),and the difference was statistically significant(t=-2.326,-5.212,-4.577,P<0.05)..Conclusion: Phosphorylation of Trim28 ser473 is induced when the DNA of HCC cell S Hep G2 is damaged.Inhibition of phosphorylation of tri-domain protein ser473(Trim28 ser473)may slow the process for DNA reparation and decreased the survival rate of HCC cells Hep G2.