MiR-122 increases sensitivity of drug-resistant BEL-7402/5-FU cells to 5-fluorouracil via down-regulation of bcl-2 family proteins.
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.
Institute of Pharmacy and Pharmacology, The First Affiliated Hospital, University of South China Hengyang, PR China.
Jie
J
Yin
Hui Fang
HF
Tang
Qiong
Q
Xiang
Jia
J
Yu
Xiao Yang
XY
Yang
Nan
N
Hu
Xiao Yong
XY
Lei
D000963:Antimetabolites / Q000494:pharmacology*; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004396:Coloring Agents; D015536:Down-Regulation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology*; D005822:Genetic Vectors; D006801:Humans; D035683:MicroRNAs / Q000494:pharmacology*; D046228:Microarray Analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D051020:bcl-X Protein / Q000096:biosynthesis
D000963:Antimetabolites; D004396:Coloring Agents; C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; C022616:thiazolyl blue; D005472:Fluorouracil