Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3...Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.展开更多
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 ce...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.展开更多
Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cel...Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results： The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion： The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy.展开更多
Objective To detect gene expressions of Uroplakin ( UP) family in interstitial cystitis ( IC) . Methods Gene expression of UP Ia,Ib,II,III,and III-c34 was quantitatively measured in bladder biopsy samples from patient...Objective To detect gene expressions of Uroplakin ( UP) family in interstitial cystitis ( IC) . Methods Gene expression of UP Ia,Ib,II,III,and III-c34 was quantitatively measured in bladder biopsy samples from patients with IC ( n = 29) and control subjects ( n = 16)展开更多
基金This project was supported by a grant from the National Natural Science Foundation of China (No. 30200284).
文摘Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.
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)
文摘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.
基金National Natural Science Foundation of China(30271301)
文摘Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results： The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion： The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy.
文摘Objective To detect gene expressions of Uroplakin ( UP) family in interstitial cystitis ( IC) . Methods Gene expression of UP Ia,Ib,II,III,and III-c34 was quantitatively measured in bladder biopsy samples from patients with IC ( n = 29) and control subjects ( n = 16)
