| Bladder cancer is the most common malignant tumor of the genitourinary system, and is also one of the ten most common malignant tumors, which is a serious threat to human health. The traditional therapy for bladder cancer is mainly based on operation, radiotherapy, chemotherapy and biological therapy, but with high recurrence rate and poor total survival rate. Therefore, based on the the latest research on molecular biology of bladder cancer, it is of great theoretical and practical significant to research and develop an effective treatment. Gene therapy is still the frontier in the treatment of tumor, and which suicide gene therapy is a hot research topic recent years. Herpes simplex virus thymidine kinase (HSV-TK)-mediated suicide gene therapy is one of the earliest, most complete and the most effective suicide gene therapis, which plays an important role in tumor gene therapy. Vector systems for cancer gene therapy mainly include viral vectors and non viral vectors (liposomes). Virus vector itself has many limitations, such as low virus titer, a limited host range, mutation, lack of security and so on, and as to non viral vectors (liposomes), low gene transfection efficiency, the lack of specific tumor targeting and so on. Bifidobacterium as an anaerobic bacterium with good tumor targeting overcomes poor targeting, safety and transduction efficiency.In our previous studies, a BI-TK/GCV treatment system was constructed and was proven to directly promote apoptosis and hence inhibit tumor growth in an in vivo bladder cancer rodent model. In this study, comparative proteomicswas used to obtain comprehensive differential proteins and thus to further identify novel molecular pathways and biomarkers of BI-TK/GCV treatment system. This article is composed of two parts.Part OnePROTEOMIC ANALYSIS OF BLADDER CANCER IN RAT MODEL AFTER BIFIDOBACTERIUM INFANTIS-MEDIATED HSV-TK/GCV SUICIDE GENE TREATMENTObjectives:To obtain comprehensive differential proteins after Bifidobacterium infantis-mediated HSV-TK/GCV suicide gene treatment by using iTRAQ technology, reveal the mechanisms of this treatment system on the overall level, and screen the key proteins playing critical roles.Methods:1. The rat models of bladder cancer were induced by intravesical N-methyl-N-nitrosourea (MNU), and the success of induction was confirmed by ultrasound and hematoxylin-eosin (HE) staining.2. Sixty tumor-bearing Sprague-Dawley rats were randomly divided into four groups (n=15 each):BI-pGEX-5X-1/GCV group (BI/PGEX-1 group), BI-pGEX-TK/GCV treatment group (BI-TK group), BI/GCV group (BI group), and normal saline/ganciclovir (GCV) control group (normal saline group). About 0.5 ml of the corresponding interventions was injected via the tail vein (bacterium count,4.4×109) once a week for 4 weeks. All groups received daily intraperitoneal injection of GCV (50 mg/kg) for 28 days.3. Part of the bladder cancer tissues from the four groups were preserved in paraffin for immunohistochemical analysis. Protein was extracted and digested by trypsin, and then labeled with the iTRAQ tags as follows:(i) normal saline group,114 tags; (ii) BI-TK group,115 tags; (iii) BI/PGEX-1 group,116 tags; (iv) BI group,117 tags. The peptide was identified by MALDI-TOF/TOF spectromety.4. Peptide identification and quantification were performed using ProteinPilot soft ware packages from Applied Biosystems. Each MS/MS spectrum was searched against the IPI rat protein database v3.49, and protein identification was accepted depending on ProteinPilot confidence scores. Gene Ontology (GO) analysis of the differencially expressed proteins were classified using the PANTHER classification system (www.pantherdb.org). The IPA program (www.ingenuity.com) was applied to analyze the pathway of differentially expressed proteins.5. Validation by Western blot and IHC:Western blot and IHC used to detect the protein level of Prx-I in each group.Results:1. During the the establishment of bladder cancer model by MNU, There are 7 rats died of overdose of anesthesia,,5 rats died of of unknown causes, and the success of induction was confirmed by ultrasound in the last remaining 63 rats. Three rats were randomly selected for hematoxylin-eosin (HE) staining. Finally,60 tumor-bearing rats were randomized into four groups.2. Mass Spectrometry Identify the Differentially Expressed Proteins:A strict cutoff value resulted in a final set of 402 differentially expressed proteins, of which 192 proteins were downregulated and 210 proteins were upregulated in the BI-TK group after treatment. Peroxiredoxin-I (Prx-I) was closely related to the NF-B signaling pathway by the bioinformatics analysis, and we found in the previous experiments that this treatment system can make the activated Caspase3 expression increased, which lead to the apoptosis increased in tumor cells significantly. Therefore, Prx-I could be involved in mechanisms of this treatment system.3. GO analysis of differentially expressed proteins:According to the bioinformatic function, the differentially expressed proteins are mainly involved in metabolic process (26.9%), cellular process (13.6%) and cell communication (8.3%). According to the molecular function, proteins are mainly involved in catalytic activity (36.8%), binding (28.4%) and structural molecule activity (15.1%). The protein classification was as follows:9.8% in oxidoreductase,8.9% in cytoskeletal protein,7.1% in transferase,6.8% in nucleic acid binding, and 6.2% in enzyme modulator.4. Prx-I expression was validated by Western blot:Compared with the normal saline group, expression of Prx-I is downregulated in the other three groups (especially in the BI-TK group), which is similar to the results obtained by iTRAQ (P<0.001).5. Prx-I expression in the tumor tissues was verified by IHC analysis: The Prx-I content in bladder cancer cells of BI-TK group was significantly lower than that in the other groups (P< 0.001), which is consistent with the results obtained by iTRAQ and Western blot.Conclusion:1. As a new technology of quantitative comparative proteomic, iTRAQ has high accuracy, repeatability and quantitative effect. It can be used to explore the molecular mechanisms of BI-TK/GCV treatment system and look for the key proteins and pathways.2. A strict cutoff value resulted in a final set of 402 differentially expressed proteins, of which 192 proteins were downregulated and 210 proteins were upregulated in the BI-TK group after treatment, and Prx-I closely associated with the NF-B signaling pathway. Together with the previous studies, we speculated that BI-TK/GCV treatment system exhibited a sustainable anti-tumor growth activity and induced apoptosis in bladder cancer tissues by inhibition of Prx-I through the NF-kB pathway.3. The results of Western Blot and immunohistochemical confirmed that the Prx-I expression was significantly down regulated in rat bladder cancer tissue after treatment, which verified the reliability of iTRAQ technology and provided key candidate targets for further research on BI-TK/GCV treatment system.Part TwoTHE EFFECTS AND MECHANISM OF PRX-I IN BLADDER CANCER CELLSObjectives:1. To investigate the effect on biological behaviors of bladder cancer cell line T24 by shRNA targeting Prx-I.2. To investigate the relationship between Prx-I and NF-κB signaling pathway.Methods:1. Construct three interference plasmids shRNA-Prx-I and transfect them into T24 to detect the inhibition efficiency of Prx-I gene by qPCR and Western blot, and then select out the most efficient recombinant plasmid shRNA.2. Examine the effect on proliferation by CCK-8 test after transferred the most efficient recombinant plasmid shRNA-Prx-I into T24.3. Examine the cell cycle and apoptosis by flow cytometry after transferred the most efficient recombinant plasmid shRNA-Prx-I into T24.4. Examine the expression of phospho-NF-xB p50 and p65 by Western blot after transferred the most efficient recombinant plasmid shRNA-Prx-I into T24.Results:1. The inhibition of Prx-I could suppress T24 cell growth in vitro, and the apoptosis rate in the shRNA-Prx-I group (21.99±1.10%) was significantly higher compared with the con shRNA group (4.51±0.73%) and parental group (4.96±0.46%) (P<0.05). Cell cycle analysis showed that G0/G1 phase ratio in the shRNA-Prx-I group (61.13±0.50%) was significantly higher compared with the con sh group (49.62±0.84%) and parental group (48.03±1.17%) (P<0.05), indicating that cell cycle arrested at the G0/G1 phase.2. A significant decrease (P<0.05) in the protein expression of both phospho-NFkB p50 and p65 was observed in shRNA-Prx-I group, compared with the con sh and parental groups, indicating that Prx-I knockdown inhibited activation of NF-κB P50 and P65 in T24 cells.Conclusion:1. After transfect by shRNA-Prx-I, the proliferation of bladder cancer cell line T24 was significantly inhibited, apoptosis cells increased and cell cycle arrested at G0/G1 phase.2. Prx-I knockdown inhibited activation of NF-κB P50 and P65 in T24 cells, which induced apoptosis in bladder cancer cells. |
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