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ysis, which had important reference values for further studying biological functions of UP Ⅱ gene and targeted therapeutic strategy for TCC of bladder.
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Objective To investigate the feasibility of tissue-specific gene therapy for bladder cancer using human UroplakinⅡ(UPⅡ) promoter. Methods The recombinant adenoviruses,Ad-hUPⅡ-TNF carrying tumor necrosis factor (TNF-?) under control of the hUPⅡ,were generated.ELISA showed the production and secretion of TNF by bladder cancer cells infected with Ad-hUPⅡ-TNF.The level of TNF in urine was identified with ELISA. Results ELISA showed that production and secretion of TNF by bladder cancer cells infected with Ad-hUPⅡ-TNF was distinctly higher than by non-urothelium cells infected with Ad-hUPⅡ-TNF and MTT showed that proliferation of bladder cancer cells was obviously inhibited.Conditioned medium from bladder cancer cells apparently inhibited cells of L929 proliferation,compared with conditioned medium from non-bladder cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Intravesical inoculation of Ad-hUPⅡ-TNF also caused decreased tumor growth in orthotopic human bladder cancer model.The sustained high level of TNF in urine could be identified with ELISA. Conclusions TNF driven by hUPⅡ promoter has effective active in the inhibition of bladder cancer growth both in vivo and in vitro.These will undoubtedly yield a new approach of therapy for bladder cancer.
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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/ T3N0M0 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 Xba Ⅰ 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-UP Ⅱ 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.