title: The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells.
abstract: To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON(1)-MON(5)), MON(4) is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.
authors: Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China.
authors: Xue-Qin
authors: XQ
authors: Li
authors: Dong-Sheng
authors: DS
authors: Pei
authors: Guo-Wei
authors: GW
authors: Qian
authors: Xiao-Xing
authors: XX
authors: Yin
authors: Qian
authors: Q
authors: Cheng
authors: Lian-Tao
authors: LT
authors: Li
authors: Hui-Zhong
authors: HZ
authors: Li
authors: Jun-Nian
authors: JN
authors: Zheng
references: Urology. 2000 Apr;55(4):501-5
references: 10736491
references: J Cell Physiol. 2000 Mar;182(3):371-80
references: 10653604
references: Cancer Lett. 2004 Aug 30;212(2):211-23
references: 15279901
references: Life Sci. 2005 Mar 4;76(16):1873-81
references: 15698864
references: J Clin Invest. 2003 Jan;111(2):265-73
references: 12531883
references: Cancer Gene Ther. 2010 Jun;17(6):441-6
references: 20094073
references: Biochem Biophys Res Commun. 2010 Mar 19;393(4):592-7
references: 20152814
references: Cell. 1999 Nov 24;99(5):451-4
references: 10589672
references: Adv Genet. 2010;70:27-56
references: 20920744
references: Mol Cell Biol. 1997 Oct;17(10):5897-904
references: 9315647
references: Exp Cell Res. 2000 Jun 15;257(2):231-7
references: 10837136
references: Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3740-5
references: 11891299
references: Nat Rev Cancer. 2004 Feb;4(2):143-53
references: 14732866
references: Expert Rev Mol Diagn. 2009 Jan;9(1):75-83
references: 19099350
references: Urol Clin North Am. 2003 Nov;30(4):843-52
references: 14680319
references: J Immunol. 1984 Oct;133(4):1710-5
references: 6206131
references: Ann Surg. 2001 Jul;234(1):10-20
references: 11420478
references: Cancer Epidemiol Biomarkers Prev. 2009 Mar;18(3):894-900
references: 19240241
references: Int J Cancer. 2003 Jul 10;105(5):710-6
references: 12740923
references: BJU Int. 2005 Feb;95(3):416-20
references: 15679806
references: Nature. 2008 Mar 6;452(7183):45-50
references: 18322525
references: Cancer Res. 2008 Nov 15;68(22):9116-21
references: 19010880
references: Exp Cell Res. 1999 Oct 10;252(1):186-98
references: 10502411
references: PLoS Biol. 2008 Jan;6(1):e22
references: 18232738
references: Nat Rev Drug Discov. 2006 Jan;5(1):37-50
references: 16485345
references: Int J Cancer. 1983 Jan 15;31(1):13-20
references: 6339421
references: Life Sci. 2006 Jan 11;78(7):724-9
references: 16111722