Lentivirus-mediated RNAi silencing targeting ABCC2 increasing the sensitivity of a human nasopharyngeal carcinoma cell line against cisplatin.
BACKGROUND
High resistance to drug is taken as a characteristic of human tumors, which is usually mediated by multidrug resistance-associated genes. ABCC2, an ATP-binding cassette multidrug resistance transporter, is found to be expressed in a variety of human cancers. In this study the effect of a RNAi construct targeting ABCC2 on the chemosensitivity of NPC cell line CNE2 against cisplatin was investigated.


METHODS
Lentiviral vectors were constructed to allow an efficient expression of anti-ABCC2 siRNA. The effective target sequence comprised nucleotides 1707-1727 of the human ABCC2 mRNA. The cell clones expressing the construct were picked and expanded, followed by identification using qRT-PCR and western blot method. As control, lentiviral vector containing invalid RNAi sequence was transfected to CNE2 cells. In vitro, cellular accumulation of cisplatin was detected by HPLC. The capacity of cellular growth and sensitivity of cells against cisplatin were detected by MTT assay. In vivo, the sensitivity of the tumor tissues against cisplatin were evaluated by transplanted CNE2 nude mice model.


RESULTS
Two CNE2 cell clones with reduced expression of targeted ABCC2 mRNA and protein for more than 70% by qRT-PCR and western blot were established, and no differences were shown in proliferation rates compared to control CNE2 cells by growth curves analysis. In vitro the accumulation of intracellular cisplatin in these CNE2 cell clones with reduced expression of ABCC2 increased markedly, accompanied by increased sensitivity against cisplatin. In vivo, the growth of CNE2 solid tumors with a stably transfected anti-ABCC2 siRNA construct was significantly inhibited by cisplatin in transplanted nude mice model.


CONCLUSIONS
Our investigation demonstrated that lentivirus-mediated RNAi silencing targeting ABCC2 might reverse the ABCC2-related drug resistance of NPC cell line CNE2 against cisplatin.
Cancer Research Institute, Key Lab for Transcriptomics and Proteomics of Human Fatal Diseases, Southern Medical University, Guangzhou City, Guangdong Province, PR China. xsming@sina.com
Si Ming
SM
Xie
Wei Yi
WY
Fang
Zhen
Z
Liu
Shuang Xi
SX
Wang
Xin
X
Li
Teng Fei
TF
Liu
Wei Bing
WB
Xie
Kai Tai
KT
Yao
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000172:deficiency* / Q000235:genetics; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000473:pathology*; D034622:RNA Interference / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D002945:Cisplatin; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000715087:ABCC2 protein, human