Effect Of RNAi-mediated Gene Silencing Of Livin On Apoptosis Of Human Breast Cancer Cell Line ZR-75-30

ObjectiveBreast cancer is the leading cause of cancer death in women worldwide.Despite advances in detection and therapy,many women with breast cancer continue to die of this malignancy.Apoptosis resistance of breast cancer cell may be one of many reasons, and has increasingly become one of the hot spot of research fields.Inhibitor of apoptosis protein family may play a key role in causing apoptosis resisitance,and have become the focus of research increasingly.Livin is a novel inhibitor of apoptosis protein(IAP)family member,It is selectively expressed in the most common human neoplasms,there is evidence that Livin gene high expression was typically detected in breast cancerous tissues,but not,or to substantially lower levels in the adjacent normal tissues.The aim of present study was to employ the sequence-specific siRNA silencing the Livin gene and investigate its effects on apoptosis of human breast cancer cell ZR-75-30.Methods1.Chemically synthesized double stranded RNA(dsRNA)targeting Livin was transfected into human breast cancer cell ZR-75-30 with lipofectamine^TM2000.2.The transfection efficiency was observed under a fluorescence confocal microscopy to optimize siRNA transfection conditions and evaluate the transfection efficiency.3.Twenty-four hours after transfection with siRNAs,the mRNA expression of Livin were detected by reverse transcription polymerase chain reaction(RT-PCR).4.Western blot assay and Immunohistochemical analyses were used to determine the protein expression of Livin. 5.The effects of siRNA-mediated gene silencing of Livin on apoptosis of ZR-75-30 cells were assessed by FCAS.Results1.Negative control FAM-siRNA was transfected into human breast cancer cell ZR-75-30.There was about 80%transfection efficiency with cell density at 4×10⁵/well and siRNA 250 pmol/well in six well play.2.when Livin was silenced by chemically synthesized siRNA 24h after transfection, Livin mRNA levels in ZR-75-30 cells transfected with Livin-siRNA was significantly inhibited(P＜0.01),the inhibition rate was 53.66%.3.Western blot analyses indicated the inhibition rate of Livin protein expression level was 58.32%;the results of Immunohistochemical analyses showed that transfection with Livin-siRNA markedly decreased Livin protein expression level compared to transfection with negative control siRNA or lipofectamine^TM2000.4.The apoptosis rate of cells with Livin-siRNA was elevated when compared with the negative control group,from(2.60±0.15)%to(8.33±0.20)%(P＜0.01).ConclusionsChemically synthesized short Livin-siRNA can effectively inhibit Livin over expression and remarkably induce apoptosis in human breast cell line ZR-75-30.Livin RNAi has a great potential value in the gene therapy of breast cancer.