Micro-ribonucleic acids (miRNAs) are endogenous single-stranded small non-coding RNAs. miRNAs bind to a com-plementary site in the 3' untranslated region of their target mRNAs through canonical base pairing, which can direct the degradation or translational repression of these transcripts. Thus, miRNAs can effectively silence the protein expression of target genes post-transcrip-tionally. miRNAs may also regulate the expression of oncogenes and tumor suppressor genes and could be involved in almost all known hallmarks of cancinogenesis. In this paper, we discuss the following in detail:(1) biogenesis and main functions of cellular miR-NAs, (2) stability and detectability of exosomal miRNAs in biological fluids;and (3) feasibility of miRNAs as a potential new class of biomarkers derived from urinary exosome in the malignancy of urinary system. Finally, we summarize studies on urinary exosomal miRNAs as potential biomarkers of prostate, bladder, and kidney cancers.

BACKGROUND:Cancer stem cell (CSC) hypothesis suggests that tumorous clones are maintained by a rare fraction of cells with stem cell proprieties. Several kinds of CSCs of solid tumor have been isolated in recent years. However, there have been fewer studies on the objective existence of bladder cancer stem cells (BCSCs) and on the methods to effectively isolate and identify BCSCs. OBJECTIVE:To investigate possibilities of BCSC existence and of epithelial membrane antigen (EMA) used as a surface marker of BCSC. DESIGN:A control observation experiment. SETTING:Tianjin Institute of Urinary Surgery & Second Hospital of Tianjin Medical University. MATERIALS:This study was performed at the Room for Tumor Immunity of Tianjin Institute of Urinary Surgery (key laboratory for State "211 Project") from March 2006 to July 2007. Nine specimens of human bladder were obtained from patients who received treatment in the Second Hospital of Tianjin Medical University. These specimens corresponded to the diagnostic criteria of low malignant potential papillary urothelial neoplasm and low-grade papillary urothelial carcinoma. Additionally, 40 samples of human low malignant bladder transitional cell carcinomas (BTCC) and 10 samples of normal urothelium that were used for immunohistochemistry were obtained from the patients who received treatment in the Department of Urinary Surgery, Second Hospital of Tianjin Medical University. Written informed consent for the specimen providing was obtained from the patients, and the protocol was approved by the hospital’s Ethics Committee. METHODS:The genes that were differentially expressed between normal urothelium and BTCC were identified through a DNA array assay to preliminarily determine the existence of BTCC. Overpressed stem cell related genes, Bmi-1 and EZH2, were verified by immunohistochemistry. A total of 27 potential surface markers of BCSCs were assayed to determine the location of positive cells. EMA- subsets were obtained through an immunomagnetic bead cell sorting system to analyze their abilities for colony forming, self-renewal and extensive proliferation. MAIN OUTCOME MEASURES:Normal urothelium and BTCC gene expression difference, Bmi-1 and EZH2 protein expression difference between low malignant BTCC and normal urothelium; and experimental results of colony forming. RESULTS:A total of 268 genes (including Bmi-1 and EZH2) that were differentially expressed between normal urothelium and low malignant BTCC were identified through a DNA array assay. The Bmi-1 and EZH2 had been found overexpressed in the low malignant BTCC. Except for EMA, above-mentioned 26 out of 27 potential surface markers were null for isolating BCSCs. EMA- subsets were located in the basal layer and suprabasal epithelial layers of both normal and tumorous urothelium (potential location of BCSCs). EMA- subsets possesed the abilities for colony forming, self-renewal and extensive proliferation. CONCLUSION:The experiment confirms the existence of BCSCs. EMA- might be a surface marker of BCSCs.

Objective: To investigate the molecular changes in bladder urothelial carcinoma via different pathways. Methods:Polymerase-chain reaction (PCR) or coamplification at low denaturation temperature-PCR and Sanger direct sequencing were per-formed to detect the status of fgfr3, p53, and h-ras gene mutations in 88 tissue samples of human bladder cancer and 10 normal control tissues. The relative mRNA expression levels of motility-related protein-1 (MRP-1)/CD9 and the relationship between genes and tumor recurrence were also determined. Logistic regression and relative analyses were conducted to compare the significance and interrelation of genes among tumor recurrences. Results:The mutation rate of p53 increased as pathological grades and stages increased. Recurrence rate was higher in patients with MT-p53 genotype than in patients with WT-p53 genotype. Conversely, the mutation rate of fgfr3 gene decreased as pathological grades and stages increased. Recurrence rate was also higher in patients with WT-fgfr3 genotype than in pa-tients with MT-fgfr3 genotype. In low-grade and early stage tumors, MT-fgfr3/WT-p53 was the most prevalent genotype;in high-grade and late stage tumors, WT-fgfr3/MT-p53 was the most prevalent genotype. The mutations of h-ras were mainly observed in low-grade tumors in early stages. Moreover, the relative mRNA levels of MRP-1/CD9 decreased as pathological grades and stages increased. The mRNA levels of MRP-1/CD9 were negatively correlated with p53 mutations and positively correlated with fgfr3 mutations. Logistic re-gression analysis results showed that patients with WT-fgfr3 genotypes exhibited 3.88 times higher relative risk of tumor recurrence than those with MT-fgfr3 genotypes;by contrast, patients with MT-p53 genotypes exhibited 4.53 times higher relative risk of tumor re-currence than those with WT-p53 genotypes. Conclusion:Fgfr3 and h-ras gene mutations may play important roles in tumorigenesis of low-grade and early stage bladder cancer. p53 gene mutation and mRNA levels of MRP-1/CD9 may be implicated in the tumorigenesis of high-grade tumors in late stage of bladder cancer. In general, the two variants of urothelial carcinoma exhibit distinct genetic defects. fgfr3 gene mutation revealed a pathway of favorable prognosis, and p53 gene mutation demonstrated a pathway associated with poor prognosis.

Tumor-derived heat shock protein-peptide complex 96 (HSPPC-96) containing tumor antigenic peptides can elicit po-tent tumor-specific and protective immunity. Autologous HSPPC-96 vaccine has been shown to effectively prolong recurrence-free sur-vival and increase the overall survival of many tumors, thereby suggesting extensive future applications. However, as an autologous tu-mor-derived individual vaccine, the development of HSPPC-96 vaccine is challenged by the lack of an adequate autologous tumor, lim-ited efficacy for advanced-stage cancer, etc. This paper summarized the progress, future perspectives, and challenges in the clinical de-velopment of HSPPC-96 vaccine immunotherapy.

Objective To screen the gene expression profiles of human bladder cancer cell line EJ,in which RNA interference was used to downregulate the EZH2 expression and to investigate the EZH2 molecular mechanism in cancer.Methods The vector expressing EZH2 shRNA was constructed and transfected into EJ cell line with Lipofectamine 2000.We used the gene chip to screen differential expression genes in EJ cells with transfection with EZH2 shRNA compared with untransfected EJ cells.The data were up-loaded to Passway miner serve((http://www.biorag.org/passway.htm))Results We found 436 differentiation genes,in which 257 genes upregulated and 179 genes downregulated.Bioinformation analysis showed that 115 genes locate in the known biological ways,including EGF,P53,proliferation,cell cycle,Notch and Wnt passway.EZH2 target genes such as WNT1,MYT1,CNR1,WT1 were upregulated,some of which were linked to tumor stem cell and cell differentiation.Conclusion The linkage between EZH2 expression and cell proliferation,which is induced by genes encoding cell differentiation.The research focusing on EZH2 regulating genes will explore its molecular mechanism in oncog-enesis.

Objective To express a mutated insulin gene in HepG-2 cell line to further research of insulin gene therapy. Methods Native human insulin cDNA was obtained from fetus pancreas with RT-PCR. Furin consensus cleavage sequence was introduced into proinsulin cDNA with site-directed mutagenesis (overlap extension PCR), and the new sequence was named as INS/furin. Subsequently, INS/furin was subcloned into the multiple clone sites of plasmid p(G1RE)3BP-1Luc. The new plasmid p(G1RE)3BP-11?furin was identified with the method of enzyme digestion by Hind Ⅲ and EcoR V. HepG-2 cells were transfected with the plasmid p(G1RE)3BP-11?furin by liposome-mediated method. The transfected HepG-2 cells were incubated for 48h in a glucose-containing medium (25mmol/L), and then the conditioned media were collected and HepG-2 cells were harvested respectively. The expression of INS/furin mRNA in transfected HepG-2 cells was examined by RT-PCR, the regained DNA was sequenced and insulin in conditioned media was investigated by radioimmunoassay. Results Two enzymes, Hind Ⅲ and EcoR V, digested p(G1RE)3BP-11?furin, and 2 fragments with length of 260 bp and 4 700bp, were obtained. The 260bp fragment was identified as insulin/furin, indicating that the target gene had been successfully inserted in specific sites. RT-PCR showed that insulin/furin mRNA was expressed in transfected HepG-2 cell, and the regained DNA was confirmed as insulin/furin by sequencing; while insulin was detected by radioimmunoassay in conditioned media. Conclusion The recombinant mammalian expression plasmid p(G1RE)3BP-11?furin has been successfully constructed, and transfected into HepG-2 cells, which therefore may efficiently secrete bioactive insulin.

Objective To prepare human Fab fragment antibodies against Interleukin-2 and identify their antigenic specificity and combining activities with antigens. Methods The specific Anti-interleukin-2 clones were screened from a natural human Fab fragment antibodies phage display library against the immobilized interleukin-2 antigens. Then the phagemid DNA from the specific clones was digested with Spe I and Nhe I to delete gⅢ (about 660bp). The digested 4.7kb DNA, which was purified after separation of bands from agarose gel using purification kit, was ligated with T4-DNA ligase and the ligated reaction mixture were transformed to the BL21 (DE3) pLysS. Positive clones on the LB agar plates were inoculated to liquid LB culture medium, and when the bacteria were grown to OD600≈0.5 at 37℃ with continuous shaking, IPTG was added to induce the expression of soluble Fab fragment antibodies at 30℃. The expressed products containing Fab fragment antibodies were determined by SDS-PAGE, Western blot and ELISA. Results The soluble products were identified as containing human Fab fragment antibodies against Interleukin-2 by Western blot and formed a Mr 47?103 band under non-reducing condition on SDS-PAGE. The band was then proved as anti-human Fab fragment antibodies by Western blotting. ELISA demonstrated that Fab fragments possessed good antigenic specificity as well as excellent combining activity with interleukin-2 antigens, and the fragments did not react with bovine serum albumin and IL-4 in ELISA. Conclusions The soluble human anti-interleukin-2 Fab fragment antibodies have been highly expressed and successfully identified, and an effective way has been searched out for constructing the engineering antibodies. All of the results may lay a potentially good foundation for engineering human Fab antibodies, and for the clinical application of the antibodies on the immunotherapy of tumor diseases.

Objective To study the biomarker panel of superficial bladder transitional cell carcinoma(SBTCC)and analyze the biological pathway in tumorigenesis by Shotgun proteomics strategy.Methods Normal urothelium cells and cancer cells were harvested by laser capture microdissection from clinical specimen and the proteomic expression profile was identified by two-dimensional liquid chromatography tandem mass spectrometry.The isoelectric point,molecular weight,grand average of hydropathicity,transmembrane helices were analyzed by using proteomics tools.Gene ontology was used to comment the identified proteins.The pathway analysis was performed by ArrayTrack software,and visualized by GenMAPP.Results There were 440 and 218 proteins expressed in cancer cells and normal cells respectively,among them 388 proteins were differerntially expressed.All the database about identified proteins was deposited in an accessible form to researchers at http://www.Proteome-SBTCC.org.cn and http://www.Proteome-NHTE.org.cn.There were 267(68.8%)differentially expressed proteins which had GO biological process comments.The biological pathwavs of these proteins included MAPK signaling pathway,focal adhesion,oxidative phosphorylation,ECMreceptor interaction,etc.Conclusion Shotgun strategy proteomies database of normal transitional epithelium and SBTCC is successfully constructed.And the basis for the understanding of cell biology and discovery of biomarker panel for SBTCC iS provided.

Objective To construct a naive human Fab fragment phage display library,from which the anti-gp96 antibodies may be panned by the gp96 purified from the tissue of urothelial carcinoma in the urinary bladder and provide a basis to new therapy for the malignant tumors.Methods Peripheral blood lymphocytes were isolated from 800 ml of blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lympbocytes were amplified by PCR with variable regions 5' and 3' primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E.coli XL1-Blue by electroperation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac Ⅰ,Xba Ⅰ,Xho Ⅰ and Spe Ⅰ, and both the phage antibody Fabs and phage-raids abstracted from amplified E.coil were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment.The gp96 purified from the urothelial carcinoma tissue of the bladder by affinity chromatog-raphy on eoncanavalin-A sephnrose and DEAE-sephnrose ion exchange chromatography were utilized as antigens to process three rounds of panning to the original Fab antibody library.Results The quantity of total RNA and cDNA were qualified.By combination of light chain and heavy chain genes, an antibody library containing 6.6×106 clones was obtained, and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.The gp96 protein was obtained from urothelial carcinoma tissue in the urinary bladder.After having been panned by gp96, the original antibody library gained enrichment by 68 times.Conclusion Utilizing the technology of phage surface display, specific antibody can be gained from the human naive Fab phage display library,which can be used for immunological therapy for tumors.

Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.