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.

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.

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.

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 construct a human Fab fragment phage display library and provide a platform for human antibody preparation.Methods:Peripheral blood lymphocytes were collected from healthy donor.The heavy chain Fd fragment and light chain of human immunoglobulin's genes were amplified by RT-PCR,and then cloned into phagemid pComb3XSS to generate human phage antibody library.Cutting with endonucleases such as SacⅠ,XbaⅠ,XhoⅠand SpeⅠto identify the insertion of the light chain or heavy chain Fd genes.IL-2 and digoxin as the antigen was used to scan the phage antibody library.Results:A phage antibody library of Fab had 8.4?107 members and it's recombinant rate was 70%.Through DNA sequencing of one positive clone,it was showed that its heavy chain belonged to IgG subvariety and its light chain to ? family.Conclusion:The success of constructing a nave human phage antibody library proves the useful of phage display system in human antibody preparation,and it can be used to select,purify and express of amylin Fab antibody.

Objective To explore the effects of specific T-cell immunity against prostate cancer(PC) cells induced by dendritic cells(DCs) derived from fetal organs.Methods Mononuclear cells(MNCs) were obtained from the fetal bone marrow and liver.Then MNCs were cultured in medium with induction of rhGM-CSF,rhIL-4 and rhTNF-?to get DCs.Lysates of DU145 containing HSP-peptide complex were prepared by 50%-70%(NH4)2SO4 saturation.T lymphocytes from fetal spleen were co-cultured with DC loading DU145 antigen for 72 h,whereby CTL was obtained.The cytotoxicity of CTL against DU145,PC3 and EJ was detected by MTT assay.Results Mature DCs were induced from fetal organs,which expressed CD1a,CD_(86),HLA-DR and CD_(83) at high levels.DC stimulated with tumor lysates transformed T cells to specific CD~+_8 CTL.Phenotype of CD~+_8 cell was(14.09?(2.46))% before transformation,and(62.76?2.64)% after transformation,respectively(P

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.