摘要
目的探讨Rad9错配修复功能与结直肠癌发生的关系。方法收集100例结直肠癌患者肿瘤样本，采用逆转录聚合酶链反应（RT—PCR）检测hRad9基因突变，采用快速点突变方法构建hRad9L101M点突变质粒并转染Rad9基因敲除的小鼠细胞。采用Westernblot方法检测Rad9表达．采用错配修复报告质粒及流式细胞仪检测活细胞错配修复能力。结果100例结直肠癌患者中，有7例发现了hRad9基因101位氨基酸由亮氨酸突变为甲硫氨酸。mRad9-＋L101M细胞（表达hRad9．L101M突变体的Rad9基因敲除小鼠细胞）和mRad9＋hRad9细胞（表达hRad9的Rad9基因敲除小鼠细胞）的错配修复效率分别为（34．0±5．6）％和（48．0±7．5）％，差异有统计学意义（P〈0．05）。用N-甲基亚硝脲（MNU）处理细胞后，mRad9-＋L101M细胞和mRad9-＋hRad9细胞的存活率分别为（33．7±5．9）％和（21.3±4．7）％，差异有统计学意义（P〈0．05）。hRad9基因101位氨基酸突变导致细胞错配修复效率下降及对MNU的抗性增加。hRad9基因多处翻译后修饰位点突变导致细胞错配修复能力下降。结论hRad9错配修复功能缺陷可能与结直肠癌发生有关。
Objective The aim of this study was to investigate the effects of Rad9 mutants with impaired DNA mismatch repair （MMR） function on the tumorigenesis of colorectal cancer. Methods The eoloreetal cancer tumor samples were collected from 100 patients. The mutation profiles of human Rad9 （hRadg） gene in these samples were detected by reverse transeriptase-polymerase chain reaction （RT-PCR） and sequencing. The plasmid of pFLAG-hRad9 （L101M） was constructed following the QuickChangemutagenesis procedure and transfected into mRad9-deleted mouse cells （mRad9-z- cells）. The expression of hRad9 protein was measured by western blot analysis. The MMR activity in live cells was detected by flow cytometry using the reporter plasmid for MMR function. Results Mutation from Leu to Met at the residue 101 （L101M） of hRad9 gene was detected in 7 of the 100 samples. The mismatch repair efficiency of mRad9-/- L 101 M cells （ mRad9-deleted mouse cells with ectopic expression of LIO 1M hRad9 gene ） was （34.0±5.6） %, which was significantly lower than that in the mRad9-/- ± hRad9 cells [ mRad9-deleted mouse cells with ectopic expression of hRad9 gene, （48.0±7.5）%, P〈0.05 ]. After N-nitroso-N-methylurea （MNU） treatment, the survival rate of mRad9-/-±LlO1M cells was （33.7±5.9）%, which was significantly higher than that in the mRad9-/- ± hRad9 cells [ （ 21.3 ±4.7 ） %, P 〈 0. 05 ]. Thus, ectopic expression of L101M hRad9 gene resulted in significantly reduced MMR activity and increased resistance to MNU. Furthermore, ectopic expression of brad9 gene with mutation at the target residues of post-translational modification in mRad9-/- cells also led to a reduced MMR activity. Conclusion Rad9 mutants with impaired DNA mismatch repair function may promote tumorigenesis of colorectal cancer.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2016年第5期351-356,共6页
Chinese Journal of Oncology
基金
国家自然科学基金（11179040、31400041）
