Yin Jie, Tang Hui Fang, Xiang Qiong, Yu Jia, Yang Xiao Yang, Hu Nan, Lei Xiao Yong
Institute of Pharmacy and Pharmacology, The First Affiliated Hospital, University of South China Hengyang, PR China.
Pharmazie. 2011 Dec;66(12):975-81.
To investigate the changes in drug sensitivity of miR-122 transfected BEL-7402/5-FU cells. MiR-122 and negative miRNA expression vectors were constructed and stably transfected into BEL-7402/5-FU cells. Real-time RT-PCR was used to detect the level of miR-122, Bcl-XL, Bcl-2 and P53 mRNA. Western Blotting was used to detect Bcl-2, Bcl-XL and P53 protein expression. Drug sensitivity of the cells to 5-fluorouracil (5-FU) was analyzed with MTT and flow cytometry. Compared with negative miRNA transfectants or untreated cells, mRNA and protein expression level of Bcl-2, Bcl-XL in stable miR-122 transfectants were decreased. Accordingly, P53 protein expression showed a significant up-regulation; MTT results showed that after incubation with 5-FU, miR-122 transfectants had higher cell inhibitory rates than negative miRNA or untreated cells; flow cytometry results demonstrated that apoptosis rate increased in miR-122 transfected cells, compared with negative miRNA or untreated cells. After addition of 5-FU (10 and 100 micromol/I), miR-122 transfected cells showed higher apoptosis rate than negative miRNA or untreated cells. MiR-122 can specifically down-regulate the expression of Bcl-2 and Bcl-XL, and increase P53 activity in BEL-7402/5-FU cells, which increased cells spontaneous apoptosis and sensitize cells to 5-FU. Therefore, MiR-122 can be used as a potential therapy agent against human hepatoblastoma.
为研究转染miR-122的BEL-7402/5-FU细胞的药物敏感性变化。构建miR-122和阴性miRNA表达载体并稳定转染至BEL-7402/5-FU细胞。采用实时RT-PCR检测miR-122、Bcl-XL、Bcl-2和P53 mRNA水平。采用蛋白质免疫印迹法检测Bcl-2、Bcl-XL和P53蛋白表达。用MTT法和流式细胞术分析细胞对5-氟尿嘧啶（5-FU）的药物敏感性。与阴性miRNA转染细胞或未处理细胞相比，稳定转染miR-122的细胞中Bcl-2、Bcl-XL的mRNA和蛋白表达水平降低。相应地，P53蛋白表达显著上调；MTT结果显示，与5-FU孵育后，miR-122转染细胞的细胞抑制率高于阴性miRNA或未处理细胞；流式细胞术结果表明，与阴性miRNA或未处理细胞相比，miR-122转染细胞的凋亡率增加。加入5-FU（10和100μmol/L）后，miR-122转染细胞的凋亡率高于阴性miRNA或未处理细胞。MiR-122可特异性下调BEL-7402/5-FU细胞中Bcl-2和Bcl-XL的表达，并增加P53活性，从而增加细胞自发凋亡并使细胞对5-FU敏感。因此，MiR-122可作为治疗人类肝母细胞瘤的潜在治疗药物。
