Objective:To explore the relationship between T85C mutation in dihydropyrimidine dehydrogenase gene (DPYD) and sensitivity of breast cancer cell to 5-fluorouracil(5-FU).Methods:Genomic mRNA was isolated from human liver tissue.DPYD coding sequence was amplified as wild type DPYD.The T was transformed into C by site-directed mutagenesis technique at nucleotide position 85 in DPYD.Then the sequences of GFP,wild type and mutation DPYD were inserted into PCDH-CMV-MCS-EF1-Puro vector respectively.The recombinant plasmids were transfected into MDA-MB-231 to obtain stable cells.After 24 hours of exposure to 5-FU at different concentrations (0,1,5,10,50 μg/mL),the cell viability was tested using the MTT colorimetric method.Results:The GFP,wild type and mutation DPYD were observed by restricting double enzyme digestion.The nucleotide of mutation DPYD transformed from T to C at 85 in DPYD coding sequence.Compared with control group,the mutation group cell survival rate was significantly increased (P ＜0.01),and the mutation group cell survival rate was higher than that of wild type group (P ＜0.05).Conclusion:The mutation of DPYD influences the sensitivity of breast cancer cell to 5-FU,and the sensitivity of T85C mutation phenotype cell to 5-FU could be significantly reduced.%目的:探讨二氢嘧啶脱氢酶基因(DPYD)T85C位点突变对乳腺癌细胞5-氟尿嘧啶(5-FU)敏感性的影响.方法:从肝脏组织中提取mRNA,扩增得到野生型DPYD编码序列.利用定点突变技术,获得T85C定点突变的DPYD编码序列.将GFP、野生型和突变型DPYD序列,连入PCDH-CMV-MCS-EF1-Puro载体.转染上述3种质粒到MDA-MB-231细胞中,获得稳定表达的细胞株.MTT法检测不同浓度5-FU(0、1、5、10、50 μg/mL)处理24h后,细胞存活情况.结果:酶切验证3种重组质粒,观察到目的序列.测序验证定点突变质粒,突变位点发生T到C的改变.相比对照组,突变型细胞的存活率显著增高(P＜0.01),且突变型细胞存活率高于野生型(P＜0.05).结论:DPYD基因突变影响乳腺癌细胞对5-FU敏感性,T85C突变型DPYD细胞对5-FU的敏感性显著降低.
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