Antisense RNA of survivin gene inhibits the proliferation of leukemia cells and sensitizes leukemia cell line to taxol-induced apoptosis.
The effects of survivin antisense RNA on proliferation of leukemia cell line HL-60 and taxol-induced chemotherapy was explored. A cDNA fragment of survivin obtained by RT-PCR was inserted into a plamid vector named pcDNA3 in the reverse direction. The vector encoding antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was delivered into HL-60 cells by electroporation. Growth curves were plotted based on cell counting. Trypan blue dye exclusion assay and MTT assay were carried out after the cells were incubated with taxol. DNA gel electrophoresis and nuclear staining were performed for cell apoptosis assay. The correct construction of the recombinant plasmid has been identified by restriction enzyme digestion and DNA sequencing. A stable down-regulation has been achieved in HL-60 SVVas cells after G418 selection. Compared to HL-60 cells, the proliferation of HL-60 SVVas cells was significantly inhibited (P<0.05). Cytotoxicity assays indicated that IC(50) of HL-60 SVVas for taxol was relatively lower than controls (P<0.01). Apoptosis assays revealed that taxol-induced apoptosis was detected in HL-60 SVVas cells incubated with 50 ng/ml taxol for 12 h, while in HL-60 cells incubated with 100 ng/ml taxol for 72 h. It was suggested that Survivin antisense RNA could inhibit the proliferation of HL-60 cells and enhance taxol-induced apoptosis in HL-60 cells, which may lay an experimental foundation for further research on gene therapy in leukemia.
Li
Wenhan
W
Department of Immunology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. liwenhan@hotmail.com
Wang
Xiaojuan
X
Lei
Ping
P
Ye
Qing
Q
Zhu
Huifen
H
Zhang
Yue
Y
Shao
Jinfang
J
Yang
Jing
J
Shen
Guanxin
G
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D020128:Inhibitory Concentration 50; D007938:Leukemia / Q000188:drug therapy* / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis*; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D017239:Paclitaxel / Q000494:pharmacology*; D016372:RNA, Antisense / Q000737:chemistry*; D000077022:Survivin; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D000972:Antineoplastic Agents, Phytogenic; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016372:RNA, Antisense; D000077022:Survivin; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue; D017239:Paclitaxel