Inhibition of siRNA on the expression of Cytoglobin gene in rat hippocampus cell in vitro
- ZHANG Xiao, SHI Xue-chuan
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Abstract
【Objective】 To investigate the inhibition of siRNA on the expression of Cytoglobin in hippocampus cell of rats in vitro. 【Methods】 Primary hippocampal neurons of newborn SD rats were cultured, and then identified them by Neu immunofluorescence antibody. siRNAs, which were designed against CYGB gene, were transfected into hippocampus cell with lipofectamine. The non-transfected cells and transfected with negtive-siRNA were taken as controls. The efficacy of transfection was detected by transfecting fluorescence-siRNA into cells, and the inhibitory effect of siRNA on mRNA level was detected by RT-PCR. 【Results】 The efficacy of transfection was about 70%. RT-PCR showed that the expression of CYGB was significantly decreased after transfecting siRNA-78 for 24 h(P＜0.01), 48 h(P＜0.001)and 60 h(P＜0.001), but it had no difference after 72 h. There was no difference in each time (24 h, 48 h, 60 h, 72 h, respectively) between siRNA-79/siRNA-80 and control group(P＞0.05). 【Conclusion】 The data of this study suggest that siRNA-78 designed against CYGB gene could significantly inhibit the expression of CYGB gene in hippocampus cell of rats in vitro after transfection for 24 h to 60 h.
Key words
siRNA / CYGB / gene / hippocampus / immunofluorescence
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References
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[2] Trent JT, Hargroves MS. A ubiquitously expressed human hexacoordinate hemoglobin[J]. Biol Chem,2002,277(22):19538-19545.
[3] Schmidt M, Gerlach F, Avivi A, et al. Cytoglobin is a respiratory protein in connective tissue and neuons, which is up-regulated by hypoxia[J]. Biol Chem,2004,279(9):8063-8069.
[4] Silva M, Chang K, Hannon GL, et al. RNA-Interference-based functional genomics in mammalian cells reverse genetics coming of age[J]. Oncogene,2004,23(51):8401-8409.
[5] 易浔飞,兰小鹏.siRNA介导人结肠癌细胞诱骗受体3基因表达抑制的研究[J].实用医学杂志,2009,25(7):1017-1019.
[6] 周毅彬,单玉喜.靶向survivin基因不同位点的siRNA对PC23细胞增殖和凋亡的影响[J].现代泌尿生殖肿瘤杂志,2009,1(6):355-359.
[7] Schubert S, Grunweller A, Erdmann VA, et al. Local RNA Target Structure Influences siRNA Efficacy: Systematic Analysis of Intentionally Designed Binding Regions[J]. J MolBiol,2005,348(4):883-893.
[8] Luo KQ, Chang DC. The gene-silencing effciency of siRNA is strongly dependent on the local structure of mRNA at the targeted region[J]. Biochemical and Biophysical Research Communications,2004,318(1):303-310.
[9] Overhoff M, Alken M, Far RK, et al. Local RNA Target Structure Influences siRNA Efficacy: A Systematic Global Analysis[J]. Mol Biol,2005,348(4):871-881.
[10] Fire A, Xu S, Montgomery MK, et al.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans[J]. Nature,1998,391(6669):806-811.
[11] Brummelkamp TR. A system for stable expression of short interfering RNAs in mammalian cells[J]. Science,2002,296(5567):550-553.
[12] Yu JA. RNA interference by expression of short-interfering RNAs and haipin RNAs in mammalian cells[J]. Proc Natl Acad Sci USA,2002,99(9):6047-6052.
[13] Dan LI, Xiao QC, Wu-Jian L. Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress[J]. Neurochem Res,2007,32:1375-1380 .
