Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells.
OBJECTIVE
The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA (siRNA) on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells.


METHODS
Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.


RESULTS
One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the G0/G1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated.


CONCLUSIONS
The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.
Wang
Hao
H
Department of Dermatology, The Second Affliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an 710004, China.
Tan
Sheng-shun
SS
Wang
Xin-yang
XY
Liu
Dong-hua
DH
Yu
Chun-shui
CS
Bai
Zhuan-li
ZL
He
Da-lin
DL
Zhao
Jun
J
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D017931:DNA Primers; D005434:Flow Cytometry; D020868:Gene Silencing; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics*; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D009363:Neoplasm Proteins / Q000235:genetics*; D034741:RNA, Small Interfering
D048868:Adaptor Proteins, Signal Transducing; C419866:BIRC7 protein, human; D017931:DNA Primers; D051034:Inhibitor of Apoptosis Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering