RUNX3基因对人乳腺癌细胞BCAP-37药物敏感性作用的实验研究

Effects of RUNX3 on drug sensitivity of BCAP-37 cells

目的:探讨RUNX3基因对人乳腺癌细胞BCAP-37的药物敏感性的调节作用。方法:构建RUNX3基因的小干扰RNA载体并将其转导入BCAP-37细胞,获得稳定转染的阳性克隆后,应用RT-PCR和Western blot进行鉴定;MTT法检测细胞转染前后药物敏感性的变化;流式细胞仪检测细胞转染前后对多柔比星的蓄积浓度的变化;DNALadder法和流式细胞仪检测细胞转染前后对DDP诱导的凋亡的变化;RT-PCR和Western blot检测耐药相关蛋白P-gp和MRP,凋亡相关蛋白Bcl-2和Bax的表达变化。结果:成功构建了RUNX3的小干扰RNA载体;成功建立了稳定的RUNX3低表达的乳腺癌细胞模型;下调RUNX3的表达能够显著降低BCAP-37细胞对长春新碱(P=0.011)、多柔比星(P=0.0004)5-FU(P=0.005)和DDP(P=0.0002)的敏感性,减少细胞内多柔比星的蓄积;转染RUNX3小干扰RNA后的细胞的抗凋亡能力明显增强,且高表达P-gp和Bcl-2,而Bax和MRP基因表达未见明显变化。结论:下调Runx3基因能够降低乳腺癌细胞对化疗药物的敏感性,提示RUNX3在乳腺癌的发生和发展中可能扮演重要角色。

OBJECTIVE： To investigate the effects of RUNX3 siRNA on the drug sensitivity of human breast cancer cell line BCAP-37. METHODS： The small interfering RNA eukaryotic expression vector specific to human RUNX3 gene was constructed by gene recombination, then transfected into BCAP-37 cells. Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blot analysis. MTT assay was used to investigate the effects of RUNX3 on the drug sensitivity of cancer cells. The flow cytometry assay was used to identify the effects of RUNX3 on the drug accumulation in cell models. The apoptosis after cisplatin treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses. The expressions of Bcl-2 ,Bax, P-gp and MRP were analyzed by RT-PCR and Western blot. RESULTS： mU6-RUNX3 siRNA was successfully constructed and transfected into BCAP-37 cells. The stable clones with decreased RUNX3 expression were successfully established. As detected by MTT and flow cytometry, down-regulation of RUNX3 was found significantly to decrease the drug sensitivity of BCAP-37 cells towards VCR （P=0. 011）, ADR （P=0. 000 4）, 5-FU （P=0. 005） and DDP （P=0. 000 2）, and inhibit the ADR accumulation in BCAP-37-siRNA. Down-regulation of RUNX3 was found significantly to inhibit the sensitivity of BCAP-37 cells towards DDP-induced apoptosis. The expressions of P-gp and Bcl-2 in transfected cells were up-regulated as compared with those of the control, while MRP and Bax not. CONCLUSION： Down-regulation of RUNX3 could significantly decrease the sensitivity of BCAP-37 cells to conventional chemotherapeutic agents, including VCR, ADR,DDP and 5-FU, suggesting RUNX3 might play important roles in the development of human breast cancer cells.