Effects of shRNA targeting survivin on apoptosis of human retinoblastoma cell line Hxo-rb44 in vitro

Abstract

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 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.

Keywords

survivin / retinoblastoma / apoptosis / c-Myc

References

[1] MonzoM., RosellR., FelipE., et al.. A novel antiapoptosis gene expression of survivin messenger RNA as a prognosis marker in non small cell lung cancers. J Clin Oncol, 1999, 17(7): 2100-2104

[2] LingX., LiF.. Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology. Bio Techniques, 2004, 36: 450-460

[3] JiangB., ZhangY. L., ZhouD. Y.. General Experimental Methods of Molecular Biology (Chinese), 1996, Beijing, People’s Military Medical Press: 174-176

[4] BrummelkampT. R., BemadsR., AgamiR.. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296(5567): 550-553

[5] ZhouM., GuL., LiF., et al.. DNA damage induces a novel p53-Survivin signaling pathway regulating cell cycle and apoptosis in acute lymphoblastic leukemia cells. J Pharmacol Exp Ther, 2002, 303(1): 124-131

[6] ChangC. C., HellerJ. D., KuoJ., et al.. Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression. Proc Natl Acad Sci USA, 2004, 101(36): 13 239-13 244

[7] ChaoJ. I., KuoP. C., HsuT. S.. Down-regulation of survivin in nitric oxide-induced cell growth inhibition and apoptosis of the human lung carcinoma cells. J Biol Chem, 2004, 279(19): 20 267-20 276

[8] LuX., HuA. B., ZhangY., et al.. Effect of siRNA targeting survivin gene on biological behavior of hepatocellular carcinoma. World Chin J Digestol (Chinese), 2004, 12(7): 1534-1538

[9] WangY., ZhuH., QuanL., et al.. Downregulation of survivin by RNAi inhibits the growth of esophageal carcinoma cells. Cancer Biol Ther, 2005, 4(9): 974-978

[10] LohrK., MoritzC., ContenteA., et al.. p21/CDKN1 A mediates negative regulation of transcription by p53. J Biol Chem, 2003, 278(35): 32 507-32 516

[11] ZhuH. X., LiuS., ZhouC. Q., et al.. Anti-apoptosis gene survivin promotes cell growth and transformation. J Chin Med (Chinese), 2002, 82(5): 338-340

[12] EndohT., TsujiN., AsanumaK., et al.. Survivin enhances telomerase activity via up-regulation of specificity protein 1-and c-Myc-mediated human telomerase reverse transcriptase gene transcription. Exp Cell Res, 2005, 305(2): 300-311