Conditionally replicative adenovirus carrying shRNA targeting EZH2 inhibits prostate cancer growth and invasion.

The present study aimed to construct conditionally replicative adenovirus (CRAds) carrying small hairpin (sh)RNA targeting enhancer of zeste homolog 2 (EZH2), in order to study its effect on inhibiting prostate cancer (PCa) cell growth and invasion. Immunohistochemical analyses of EZH2 was performed in tumor tissue samples from PCa and benign prostate hyperplasia (BPH). The human telomerase reverse transcriptase (hTERT) promoter was chosen to transcriptionally control EZH2 gene expression to obtain adenoviral replication (Ad­hTERT­EZH2shRNA) in human PCa cell lines. The inhibitory effect of Ad­hTERT­EZH2shRNA on EZH2 expression was evaluated by reverse transcription­-quantitative polymerase chain reaction and western blot analyses. Cell Counting Kit­8 assays were used to examine the effects of the Ad­hTERT­EZH2shRNA on cell proliferation. Transwell Matrigel invasion assays were used to detected cell invasion. Immunohistochemistry showed that EZH2 staining was stronger in castration­resistant prostate cancer (CRPC) samples, compared with androgen­dependent prostate cancer (ADPC) samples, and was absent in BPH. Furthermore, EZH2 expression knockdown suppressed PCa cell proliferation and invasion. In addition, it was found that Ad­hTERT­EZH2shRNA selectively replicated and significantly reduced the expression of EZH2 in PCa cells lines. The growth ability and invasion of DU145 and PC3 cells in vitro was effectively inhibited by Ad­hTERT­EZH2shRNA. Silencing the expression of EZH2 led to decreased expression of CCND1 and Ki67 and increased expression of E­cadherin, as determined by western blot analysis. Thus, it was shown that CRAds armed with EZH2 shRNA exhibited significant antitumor effects in human PCa cells. Ad­hTERT­EZH2shRNA may be developed as a treatment for hormone­refractory PCa.

Mimic peptides bonding specifically with the first and second extracellular loops of the CC chemokine receptor 5 derived from a phage display peptide library are potent inhibitors of experimental autoimmune encephalomyelitis.

PURPOSE: To obtain mimic peptides that specifically bind with the first and second extracellular loops (ECL1, ECL2) of the CC chemokine receptor 5 (CCR5) and to study their treatment effects on experimental autoimmune encephalomyelitis (EAE) mice. METHODS: A phage display peptide library was applied to screen peptides that bond with ECL1 and ECL2. ELISA and DNA sequence analysis were used to identify positive clones. EAE mice were treated with synthesized peptides by intraperitoneal injection. RESULTS: Eighteen positive clones were obtained and four peptides with sequences STFTTTL, TPIPQLL, SLPLPKP and QTSSAAL were identified. These peptides could significantly protect against and reduce the severity of EAE. The infiltration of monocytes and lymphocytes into the spinal cord decreased significantly in treated mice, while abundant inflammatory cells and demyelination were observed in spinal cords of EAE mice. CONCLUSION: CCR5 mimic peptides provided a significant protective effect to EAE mice. These potent inhibitory mimic peptides could be useful in the clinical treatment of multiple sclerosis.

Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays.

AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipHO2 cDNA array. METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR. RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5, ccl16, GRObeta, GROgamma, IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level. RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings. CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1 mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GRObeta, GROgamma, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

Establishment of transgenic mice carrying gene encoding human zinc finger protein 191.

AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Kruppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo.

[Effect of recombinant prothymosin alpha on secretion of IFN-gamma, IFN-alpha and TNF-alpha in vitro].

AIM: To study the effect of prothymosin alpha (Pro T alpha) as a fusion protein on secretion of IFN-gamma, IFN-alpha and TNF-alpha in vitro. METHODS: The in vitro study was carried out on the culture of splenocytes, splenic and peritoneal macrophages isolated from Balb/c mice. Splenocytes were incubated with various concentrations of Pro T alpha (1 x 10(-7)-1 x 10(-10) mol.L-1) with or without Con A (5 micrograms.mL-1) for 72 h. Splenic and peritoneal macrophages were respectively treated with Pro T alpha (1 x 10(-7)-1 x 10(-10) mol.L-1) in the presence of LPS (10 micrograms.mL-1) for 24 h. Then IFN-gamma, IFN-alpha and TNF-alpha levels in the supernatant were detected by ELISA. RESULTS: Pro T alpha (1 x 10(-7) mol.L-1) was found to obviously increase IFN-gamma level (P < 0.05) in the supernatant of splenocytes compared with the control group. Moreover, Pro T alpha (1 x 10(-7) mol.L-1) significantly induced the secretion of IFN-alpha (P < 0.01) and TNF-alpha (P < 0.01) in splenic and peritoneal macrophages. CONCLUSION: In vitro, Pro T alpha could increase the secretion of IFN-gamma, IFN-alpha and TNF-alpha.

CCR5 gene expression in fulminant hepatitis and DTH in mice.

AIM:To isolate mouse CCR5 cDNA (muCCR5) and study its expression in vivo.METHODS: Marathon PCR was used to isolate muCCR5 cDNA and two animal models were designed to investigate the gene expression in vivo, one was mouse fulminant hepatitis induced by Propionibacterium acnes (P.acnes) and the other was that with delayed type hypersensitivity reaction (DTH). A specific GST-NH2-terminus of muCCR5 fusion protein antibody F(ab')(2) was prepared and clarified. RT-PCR and immunohistochemical analysis were used to observe the expression level of CCR5 gene in mice.RESULTS: A positive reaction of mouse macrophage was found in DTH but not expressed in P.acnes induced fulminant hepatitis by RT-PCR and immunohistochemical analysis.CONCLUSION: This muCCR5 expression may be involved in an allergic processmediated by cellular immunity but not acute inflammatory reaction induced by P.acnes.