缺失错配修复基因MLH1的结直肠癌HCT-116细胞对氟尿嘧啶耐药机制的研究

The mechanism of 5-Fu-based drug resistance in DNA mismatch repair deficient colorectal cancer HCT-116 cells

目的研究缺失错配修复基因MLH1(MutL homolog 1)的结直肠癌细胞HCT-116对氟尿嘧啶(5-Fu)耐药机制。方法通过构建MLH1缺失的结直肠癌细胞HCT-116稳定表达MLH1细胞株,CCK-8试剂检测细胞恢复MLH1表达后对化疗药物5-Fu耐药性的影响,并通过流式细胞仪检测细胞表面干细胞标志CD133和分化标志CK20以及CK8的表达变化。结果 HCT-116稳定表达MLH1分子后,其对5-Fu作用的化疗耐受性降低,5-Fu处理后细胞的活率显著降低(P<0.01);流式细胞仪检测结果显示CD133表达显著降低,并伴随细胞分化标志CK8和CK20表达上调。结论结直肠癌细胞缺失错配修复基因MLH1引起5-Fu耐药性可能与其促进肿瘤干细胞样特性密切相关。

Objective To study the mechanisms of the drug resistance of DNA mismatch repair （MMR） deficient colorectal cancer （CRC） HCT-116 to 5 -fluorouracil （5 Fu）. Methods MLH1 deficiency HCT-116 cells were transfeeted with pcD- NA3.1-MLH1 Vector. The expression of MLH1 was detected by Western blot. The change of resistance against 5-Fu was examined by detecting the cell viability with CCK-8 kits. The expression of CD133 （cancer stem cell marker） and CK8 ＆ CK20 （cell differentiation marker） were detected by flow cytometry. Results Comparing to HCT-116 control group,the viability of HCT-116 cells was markedly decreased （P〈0.01） after stable expressing MLH1 ,accompanied by the down-regulated expression of CD133 on the cell surface. Moreover, the up-regulation of cell differentiation marker CK8 and CK20 was observed in HCT-116 cells with stable expressing MLH1. Conclusion Our data indicated that the expression of MLH1 was associated with down-regulated CD133＋ stem-like cells in colorectal cancer HCT-116 with MLH1 deficiency. Therefore,CD133＋ stem-like cells may related to the drug resistance of MMR deficiency tumor. This study provides a possible theory to explain the 5-FU resistance in the colorectal cancer patients with MMR deficiency.