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