[Reversion of multidrug resistance (MDR) in human glioma cells by RNA interference (RNAi)].
OBJECTIVE
To explore whether the constructed vector of short haprin in vivo can induce human glioma cell line BT325 to produce RNAi duplexes and reverse the expression of MDR1 gene.


METHODS
Three 62nt oligonucleotide fragments (shRNA) were constructed according to GenBank MDR1 sequence and were cloned to the retrovirus-delivered vectors. After transfected these vectors directly into the human malignant glioma BT325 cells by lipofectamine 2000 with enhanced green fluorescence protein (EGFP) co-transfecting, the MDR1 gene silence effects were detected by the changing level of mRNA and P-glycoprotein including real time PCR (RT-PCR), Northern blot and Western blot analysis. To assess the multidrug resistance against adriamycin (ADR) and VCR, cell proliferation assays were performed by cell counting kit-8.


RESULTS
The RNAi plasmid vectors were constructed successfully. RT-PCR showed MDR1 mRNA was significantly reduced (P < 0.05). Northern blot analysis showed that the gene silence became most intense at 48 hours after transfection. Western blot analysis demonstrated that P-gp expression was reduced at different time to 12.9%, 30.3% and 4.8%, respectively. The chemosensitivity assays indicated that the transfected cells showed an enhanced sensitivity to ADR and VCR. Based on the value of IC(50), BT325 cells had significantly increased sensitivity to the drugs.


CONCLUSIONS
The sequence specific RNAi can inhibit MDR1 mRNA and P-gp expression in the glioma cell line. It may reverse multidrug resistance phenotype, therefore, may provide promising therapeutic modalities in the treatment of human glioma.
Zhao
Peng
P
Beijing Neurosurgical Institute, Beijing 100050, China.
Hu
Wei
W
Zhang
Ya-zhuo
YZ
Sun
Mei-zhen
MZ
He
Yue
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D014162:Transfection; D014750:Vincristine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014750:Vincristine; D004317:Doxorubicin