Selection of Optimal Antisense Accessible Sites of Uroplakin Ⅱ mRNA for Bladder Urothelium  

作　　者：郑丽端 童强松 陈方敏 曾甫清 汪良 董继华 

机构地区：[1]Department of Pathology,Union Hospital, Tongji Medical College, Huazhong University of Science and Technology [2]Department of Surgery,Union Hospital, Tongji Medical College, Huazhong University of Science and Technology [3]Department of Central Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

出　　处：《Journal of Huazhong University of Science and Technology(Medical Sciences)》2009年第3期344-349,共6页华中科技大学学报（医学英德文版）

基　　金：supported by grants from the National Natural Sciences Foundation of China (No. 30200284, No. 30600278, No. 30772359);Program for New Century Excellent Talents in University (NCET-06-0641);Scientific Research Foundation for the Returned Overseas Chinese Scholars (2008889)

摘　　要：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 UPⅡ cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UPⅡ were selected. The RNADraw soft- ware 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 UPⅡ mRNA was detected by RT-PCR and Western blot. Twelve AAS of UPⅡ 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 UPⅡ mRNA levels in RT4cells 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 UPⅡ 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.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 UPⅡ cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UPⅡ were selected. The RNADraw soft- ware 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 UPⅡ mRNA was detected by RT-PCR and Western blot. Twelve AAS of UPⅡ 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 UPⅡ mRNA levels in RT4cells 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 UPⅡ 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.

关 键 词：bladder urothelium uroplakin Ⅱ gene antisense accessible sites random oligonucletide library 

分 类 号：R737.14[医药卫生—肿瘤]