Selection of optimal antisense accessible sites of uroplakin II mRNA for bladder urothelium

Abstract

The optimal antisense accessible sites (AAS) of uroplakin II (UP II) mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional cell carcinoma (TCC) of bladder. The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcripted total UP II cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UP II were selected. The RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODN) were synthesized. With the AS-ODN₀ designed only by RNADraw as a control, the AS-ODNs were transferred into UP II highly-expressing cell line RT₄. The cellular expression of UP II mRNA was detected by RT-PCR and Western blot. Twelve AAS of UP II mRNA were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 558–577, 552–571, 217–236 and 97–116 bp of UP II mRNA respectively. After transfection with the corresponding AS-ODN (AS-ODN₁, AS-ODN₂, AS-ODN₃ and AS-ODN₄) for 18 h, the UP II mRNA levels in RT₄ cells were reduced by 29.3%, 82.7%, 71.3% and 70.9%, while UP II protein levels were decreased by 20.2%, 78.5%, 65.2% and 64.4% respectively, which were significantly higher than those of AS-ODN₀ (14.3%, 12.1% respectively) (P<0.01). The AAS of UP II mRNA was effectively selected in vitro by random oligonucletide library/RNase H cleavage method in combination with computer software analysis, which had important reference values for further studying biological functions of UP II gene and targeted therapeutic strategy for TCC of bladder.

Keywords

bladder urothelium / uroplakin II gene / antisense accessible sites / random oligonucletide library

References

[1] WuX.R., LinJ.H., WalzT., et al. . Mammol/Lalian uroplakins. A group of highly conserved urothelial differentiation-related membrane proteins. J Biol Chem, 1994, 269(18): 13 716-13 724

[2] OlsburghJ., HarndenP., SouthgateJ., et al. . Uroplakin gene expression in normal human tissues and locally advanced bladder cancer. J Pathol, 2003, 199(1): 41-49

[3] HoS.P., BrittonD.H., StoneB.A., et al. . Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries. Nucleic Acids Res, 1996, 24(10): 1901-1907

[4] SambrookJ., RussellD.W.. Molecular cloning: A laboratory manual, 20023rd editionNew York, Cold Spring Harbor Laboratory Press

[5] LinJ.H., ZhaoH., SunT.T.. A tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice. Proc Natl Acad Sci USA, 1995, 92(3): 679-683

[6] WuR.L., OsmanI., WuX.R., et al. . Uroplakin gene is expressed in transitional cell carcinoma but not in bilharzial bladder squamous cell carcinoma:alternative pathways of bladder epithelial differentiation and tumor formation. Cancer Res, 1998, 58(6): 1291-1297

[7] LiS.M., ZhangZ.T., MclenanO., et al. . Detection of circulating uroplakin-positive cells in patients with transitional cell carcinoma of the bladder. J Urol, 1999, 162(3Pt1): 931-935

[8] YuasaT., YoshikiT., IsonoT., et al. . Expression of transitional cell-specific genes, uroplakin Ia and II, in bladder cancer: detection of circulating cancer cells in the peripheral blood of metastatic patients. Int J Urol, 1999, 6(6): 286-292

[9] XuX., SunT.T., GuptaP.K., et al. . Uroplakin as a marker for typing metastatic transitional cell carcinoma on fine-needle aspiration specimens. Cancer, 2001, 93(3): 216-221

[10] SohailM., SouthernE.M.. Selecting optimal antisense regents. Adv Drug Deliv Rev, 2000, 44(1): 23-34

[11] PatzelV., SteidlU., KronenwettR., et al. . A theoretical approach to select effective antisense oligodeoxynucleotide at high statistical probability. Nucleic Acids Res, 1999, 27(22): 4328-4334

[12] LloydB.H., GilesR.V., SpillerD.G., et al. . Determination of optimal sites of antisense oligonucleotide cleavage within TNFα mRNA. Nucleic Acids Res, 2001, 29(17): 3664-3673

[13] VickersT.A., KooS., BennettC.F., et al. . Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem, 2003, 278(9): 7108-7118

[14] MaZ., TaylorJ.S.. Nucleic acid-triggered catalytic drug release. Proc Natl Acad Sci USA, 2000, 97(21): 11159-11163