Acta Pharmacologica Sinica 2007 December; 28 (12): 1968-1974; doi: 10.1111/j.1745-7254.2007.00724.x

Aim: 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.

Conclusion: The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.

Keywords: livin; malignant melanoma; siRNA; apopto-sis; cell cycle; proliferation