干扰Nrf2提高二氢青蒿素对结肠癌细胞增殖的抑制作用

Interfering RNA-mediated Nrf2 Gene Knockdown Enhances DHA Induced Cytotoxity in Colon Cancer Cells

目的探讨干扰沉默核转录因子(NF-E2-related factor 2,Nrf2)对二氢青蒿素(dihydroartemisinin,DHA)抑制人结肠癌HCT-116细胞增殖的影响.方法采用四甲基偶氮唑蓝比色法检测不同浓度DHA对细胞增殖抑制作用.流式细胞仪检测细胞内ROS的含量,Annexin V-FITC/PI双染检测细胞凋亡,Westernblot检测sh RNA干扰NRF2蛋白表达的效果.结果当DHA浓度为5μmol/L时sh NRF2组抑制率为24.05%,当DHA为10μmol/L浓度时sh NRF2组抑制率为31.32%,当DHA为20μmol/L时抑制率达46.18%,与Con组和sh Non组比较差异有统计学意义(P<0.05).应用流式细胞仪对细胞中的ROS进测定,结果发现干扰Nrf2后加入DHA时可提高HCT-116细胞的ROS含量,NAC能逆转Nrf2干扰和DHA引起的ROS升高.应用流式细胞仪对各组进行细胞凋亡检测,结果发现干扰Nrf2后加入DHA时可提高HCT-116细胞凋亡率,NAC能逆转Nrf2干扰和DHA引起的凋亡.结论 DHA能通过提高细胞内ROS抑制HCT-116细胞生增殖,沉默Nrf2后可提高细胞内的ROS含量,增强DHA的凋亡诱导作用.

Objective To investigate the effect of Nrf2 gene knockdown on DHA-induced cytotoxity in Compared with Con group and shNon group, there was significant difference human colon carcinoma HCT-116 cells. Methods The inhibition changes in the cell viability after treatment with different concentration of DHA were detected by MTT assay and the ROS was assayed by flow cytometry. AnnexinV-FITC apoptosis kit was used to detect the cell apoptosis. Nrf2 protein expression transfected with the shRNA targeting Nrf2 was analyzed with western blotting. Results The inhibitory rate of shNRF2 group was 24.05% when DHA concentration was 5 μmol / L, and 31.32% when DHA concentration reached 10 μmol/L. When DHA rose to 20 μmol/L, the inhibition rate climbed to 46.18%, compared with Con group and shNon group, there was significant difference. The ROS level in HCT-116 cells was found to increase by interfering with Nrf2. NAC reversed Nrf2 interference and DHA-induced ROS increased. It was detected by flow cytometry that the apoptosis rate of HCT-116 cells was induced by inhibiting Nrf2. Conclusion DHA can increase the proliferation of HCT-116 cells by increasing the ROS in cells, and can also increase the ROS content and enhance the apoptosis-inducing effect of DHA after knockdowning Nrf2.