Wang Guojun, Hu Yanhua, Li Pengcheng
Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2006;26(5):614-7. doi: 10.1007/s11596-006-0536-1.
In order to construct a recombinant plasmid containing short hairpin RNA (shRNA) targeting survivin and to investigate its effect on survivin expression and cell apoptosis of human retinoblastoma cell line Hxo-rb44 in vitro, RNA interference plasmid pSIRENS that can express shRNA of survivin was designed, constructed, and transfected into human retinoblastoma cell line Hxo-rb44. Survivin and c-Myc expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Apoptosis of Hxo-rb44 cells was assayed by Honchest33258 staining and cell growth curve was drawn. The results showed that the oligonucleotide targeting survivin was identified in pSIRENS plasmid. After pSIRENS plasmid transfected, survivin and c-Myc expression in Hxo-rb44 cells was decreased significantly. Apoptotic rate of cells was up-regulated from (3.5+/-1.29) % to (36.1+/-19.66) %. The proliferation ability of Hxo-rb44 cells was inhibited. No significant effects on survivin expression and apoptosis of the cells were found when negative control plasmid was transfected. In conclusion, the plasmid containing shRNA targeting survivin was constructed successfully. It could inhibit efficiently the expression of survivin and c-Myc in human retinoblastoma cell Hxo-rb44 in vitro. The inhibition of the expression of c-Myc might be involved in the apoptosis of Hxo-rb44 cells.
为构建靶向生存素的短发夹RNA（shRNA）重组质粒，并研究其对人视网膜母细胞瘤细胞系Hxo-rb44体外生存素表达及细胞凋亡的影响，设计、构建了可表达生存素shRNA的RNA干扰质粒pSIRENS，并将其转染至人视网膜母细胞瘤细胞系Hxo-rb44。采用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测生存素和c-Myc的表达。用Ho33258染色法检测Hxo-rb44细胞凋亡情况，并绘制细胞生长曲线。结果显示，在pSIRENS质粒中鉴定出靶向生存素的寡核苷酸。转染pSIRENS质粒后，Hxo-rb44细胞中生存素和c-Myc的表达显著降低。细胞凋亡率从（3.5±1.29）%上调至（36.1±19.66）%。Hxo-rb44细胞的增殖能力受到抑制。转染阴性对照质粒时，对细胞生存素表达和凋亡无显著影响。综上所述，成功构建了靶向生存素的shRNA质粒。该质粒可有效抑制人视网膜母细胞瘤细胞Hxo-rb44体外生存素和c-Myc的表达。c-Myc表达的抑制可能参与了Hxo-rb44细胞的凋亡。
