利用RNA干扰效应阻抑鼻咽癌细胞bcl-xL基因表达和诱导癌细胞凋亡的研究

Knockdown of bcl-xL expression with RNA interference induces nasopharyngeal carcinoma cells apoptosis

目的研究RNA干扰(RNA interference)效应对人鼻咽癌低分化上皮细胞株CNE-2Z bcl-xL基因表达的抑制作用及其对CNE-2Z细胞增殖抑制和凋亡诱导的作用.方法使用美国Ambion公司提供的网上设计软件设计针对人bcl-xL基因的小干扰RNA(siRNA)序列,体外转录试剂盒合成siRNA;荧光素标记试剂盒标记siRNA;脂质体法将siRNA转入CNE-2Z细胞株.荧光显微镜下观察siRNA的转染效率;RT-PCR法半定量检测siRNA对bcl-xL基因表达的抑制作用;噻唑蓝法检测siRNA对细胞生长增殖抑制作用;流式细胞术检测siRNA诱导细胞凋亡作用.结果荧光显微镜下荧光素标记的siRNA转染组可见到细胞内清晰的绿色荧光,而在未转染siRNA对照组未见;各siRNA转染组bcl-xL mRNA表达水平有不同程度的下调,下调范围在10%～70%之间,而在未转染对照组内bcl-xL mRNA表达水平无明显改变;细胞生长增殖抑制率在一定范围内具有剂量依赖性(剂量增加,抑制率增高)和时间依赖性;各浓度siRNA4转染组可不同程度诱导CNE-2Z细胞凋亡.结论体外转录合成的siRNA能特异有效地下调bcl-xL基因的表达,不同序列特异性的siRNA下调bcl-xL基因表达的能力不同;瞬时转染bcl-xL siRNA4能有效抑制鼻咽癌细胞增殖并诱导其凋亡;siRNA不仅为基因组功能分析提供了强有力的工具,而且为抗鼻咽癌基因治疗提供了新的思路.

Objective To study the inhibition of the expression of bcl-xL gene induced by RNA interference in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction. Methods Small interfering RNAs targeting bcl-xL gene were synthesized by using web design software provided by Ambion and the silencer short interfering RNA (siRNA) construction kit; fluorescein-labeled siRNAs were done by FAM-silencer TM siRNA labeling kit; siRNAs were transfected into CNE-2Z cells by using lipofectamine TM 2000 reagent; siRNA transfection efficiencies were analyzed by fluorescent microscopy; down-regulation of bcl-xL was detected by RT-PCR; thiazolyl blue(MTT) assay was used to assess the cell growth; apoptosis of CNE-2Z cells was analyzed by flow cytometry. Results Green fluorescence in the cells was seen clearly in FAM-labeled siRNA transfected group under the fluorescent microscope while none in the untransfected group. Different down-regulations of bcl-xL mRNA expression were found in the transfected groups. The expression of bcl-xL mRNA decreased by 10%-70% in the siRNAs transfected CNE-2Z by RT-PCR scan analysis. The inhibitory rate of cell proliferation depended on time and concentrations to some extent. Different cell apoptosis could be induced by different concentrations of siRNA4. Conclusions The synthesized siRNAs in vitro were able to down-regulate the expression of bcl-xL. There were different capabilities of the specific siRNAs down-regulation. The transient transfected bcl-xL siRNA4 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for anti- nasopharyngeal carcinoma gene therapy.