Hua Yonghong, Hu Qiaoying, Piao Yongfeng, Tang Qiu, Feng Jiangguo
Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou 310022, China.
J Cancer Res Ther. 2015 Nov;11 Suppl:C239-43. doi: 10.4103/0973-1482.170529.
To observe the apoptosis of capilliposide against human nasopharyngeal cancer CNE-2 cells and to study its primary mechanisms.
Vectors pSilencer-PUMA-small interfering RNA (siRNA) were constructed to transcribe functional siRNA specially targeting PUMA. The interfering plasmids were used to transfect CNE-2 cells with lipofectamine 2000 transfection reagent. PUMA messenger RNA (mRNA) expression levels were analyzed by polymerase chain reaction. The proliferation of CNE-2 cells was detected using MTT colorimetry. Annexin V/propidium iodide double staining was applied to detect the apoptosis rate of CNE-2 cells. The protein levels of p53, PUMA, and Bax were detected using Western blot analysis.
Recombinant siRNA expression vector targeting PUMA was constructed. MTT assays showed capilliposide inhibited the proliferation of CNE-2 cells in a concentration-dependent manner. The inhibition was strengthened along with increased concentrations. Apoptosis detected by flow cytometry in control group, drug group, siRNA group, and drug combined siRNA group was 9.3 ± 2.3%, 31.4 ± 5.6%, 12.3 ± 4.1%, and 13.2 ± 3.7%, respectively. After pretreated by capilliposide, PUMA protein was upregulated, and BAX was distributed to mitochondria in CNE-2 cells using Western blot analysis, but this effect can be interrupted by PUMA-siRNA.
Capilliposide could induce the apoptosis of CNE-2 cells, which might be related with the increasing in PUMA-Bax pathway.
观察毛蕊花糖苷对人鼻咽癌CNE - 2细胞凋亡的影响并探讨其初步机制。
构建载体pSilencer - PUMA - 小干扰RNA（siRNA）以转录特异性靶向PUMA的功能性siRNA。使用脂质体2000转染试剂将干扰质粒转染至CNE - 2细胞。通过聚合酶链反应分析PUMA信使核糖核酸（mRNA）表达水平。采用MTT比色法检测CNE - 2细胞的增殖情况。应用膜联蛋白V/碘化丙啶双染法检测CNE - 2细胞的凋亡率。采用蛋白质免疫印迹分析检测p53、PUMA和Bax的蛋白水平。
构建了靶向PUMA的重组siRNA表达载体。MTT分析显示毛蕊花糖苷以浓度依赖性方式抑制CNE - 2细胞的增殖。随着浓度增加，抑制作用增强。流式细胞术检测对照组、药物组、siRNA组和药物联合siRNA组的凋亡率分别为9.3±2.3%、31.4±5.6%、12.3±4.1%和13.2±3.7%。蛋白质免疫印迹分析显示，经毛蕊花糖苷预处理后，CNE - 2细胞中PUMA蛋白上调，BAX分布至线粒体，但这种作用可被PUMA - siRNA阻断。
毛蕊花糖苷可诱导CNE - 2细胞凋亡，这可能与PUMA - Bax通路的激活有关。
