脂质体介导的cFLIP反义寡核苷酸对肾细胞癌的凋亡作用

Liposome-mediated antisense oligonucleotides of cellular FLICE inhibitory protein induce human renal cancer cell to apoptosis

目的探讨cFLIP反义寡核苷酸(ASODN)对肾细胞癌的抑制作用。方法设计合成cFLIP的ASODN,按不同浓度在脂质体介导下转染786-0肾癌细胞株,设无义(NSODN)和空白对照组进行比较。观察细胞的生长状态,Western blot检测cFLIP蛋白的表达,MTT法检测细胞生长抑制率,应用流式细胞仪检测其凋亡率。结果与NSODN、空白对照组比较,ASODN组cFLIP蛋白表达显著降低(P<0.05),细胞抑制率(分别为10μmol/L组34.20%,20μmol/L组39.50%)显著增高,上述效应呈浓度依赖性;镜下观察ASODN转染细胞代谢衰退,当转染浓度至20μmol/L时,其凋亡率(56.11%)显著高于空白对照组(7.29%)和NSODN(4.69%)组。结论ASODN能特异性封闭肾癌细胞cFLIP基因的表达,并可抑制肾癌细胞的增殖,诱导其凋亡。

Objective To investigate the inhibitory effect of cFLIP antisense oligonucleotides on the growth of renal cancer cells. Methods ASODN of target cFLIP was synthesized and transfected to 786-0 renal cancer cells at different concentration. At the same time, positive control （nonsense oligonucleotides, NSODN） and normal control were set for comparison. Cell growth condition was observed under microscope; Western blot was used for the detection of cFLIP protein expression, mononuclear cell direct cytotoxicity assay （MTT） for cell growth inhibition ratio （IR）, and flow cytometry （FCM） for apoptosis. Results Comparing the transfection ASODN groups with NSODN group and normal control group, we found that the expression of cFLIP was apparently decreased （P〈0.05）, and cell growth IR （10 μmol/L group 34.20%, 20 μmol/L group 39.50%） obviously increased, which was in a dose-dependent manner. Cell growth in ASODN conditioned medium was obviously inhibited and apoptosis rate of 20 μmol/L group（38.40%） was significantly higher than that of normal control group （1.98%） and NSODN group （1.92%） （P〈0.01）. Conclusion ASODN can block the expression of cFLIP in 786-0 cell line specifically, inhibit cell proliferation and induce cell apoptosis.