范可尼贫血互补群B对丝裂霉素C诱导的293T细胞DNA损伤修复的研究

Study on the mechanism of Fanconi anemia complementary group B molecule in repair of mitomycin C induced DNA damage in 293T cells

摘要:

目的 研究丝裂霉素C诱导的DNA损伤的浓度和时间对293T细胞中范可尼贫血互补群B(FANCB)，DNA损伤因子gammaH2AX，修复因子RAD51表达量的影响，探索FANCB在DNA损伤中的作用机理。方法 利用人293T细胞系作为研究载体，通过Western blot和qRT-PCR技术定量分析FANCB，gammaH2AX，RAD51在25 μM丝裂霉素C浓度作用下，在1、3、5 h不同作用时间点评估FANCB，gammaH2AX，RAD51蛋白和基因表达水平差异;分析细胞在25、35、45、60 μM 丝裂霉素C浓度下作用3 h后gammaH2AX蛋白表达水平;siRNA敲低FANCB表达后FANCB、gammaH2AX和RAD51蛋白和基因表达检测。结果 相比对照组，在蛋白水平，gammaH2AX表达在1、3 h对比0 h差异具有统计学意义(P均<0.05)，而5 h对比0 h差异无统计学意义(P=0.223);FANCB表达在1、3、5 h对比0 h差异均具有统计学意义(P均<0.05); RAD51表达在1、3、5 h对比0 h差异均具有统计学意义(P均<0.05)。在基因水平，FANCB在1、3、5 h和RAD51在1、3、5 h相比对照组均具有统计学意义(P均<0.05);gammaH2AX蛋白表达水平与MMC浓度无明显关系。相比对照组，siRNA敲低组FANCB蛋白和基因表达下降，gammaH2AX蛋白表达上升，RAD51蛋白和基因表达下降。结论 在丝裂霉素C造成的293T DNA损伤模型中，FANCB在同源重组DNA修复途径中对DNA损伤具有潜在的调控功能。

Abstract:

Objective To study the mechanism of Fanconi anemia complementary group B (FANCB) in repair of DNA damage via detecting the expressions of Fanconi anemia complementary group B (FANCB), gammaH2AX and RAD51 in 293T cells with mitomycin C (MMC)-induced DNA damage.Methods Human 293T cell line was used as the study object. Compared with normal control group (NC), FANCB, gammaH2AX and RAD51 were detected by western blot and qRT-PCR, with 25 μM MMC treated for 1, 3, 5 h. Expressions of gammaH2AX protein in 293T treated with 25 μM, 35 μM, 45 μM and 60 μM MMC for 3 h were analyzed. After knockdown with siRNA, FANCB, gammaH2AX and RAD51 protein and gene expressions were detected.Results Compared with NC, the expression of gammaH2AX protein was increased in 1h and 3h (both P<0.05), and was insignificant different in 5 h (P=0.223). The expressions of FANCB and RAD51 at both protein and gene levels got increased in 1, 3, 5 h (all P<0.05). The expression level of GammaH2AX protein had no significant relationship with the concentration of MMC. Compared with NC, the expressions of FANCB and RAD51 were significantly decreased and those of gammaH2AX got increased at protein and gene levels (all P<0.05) in the siRNA knockdown group.Conclusion MMC can cause DNA damage, and FANCB may potentially regulate homologous recombination in 293T cells.