Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.

Exosomes from cancer cells, which contain microRNA and reach metastasis loci prior to cancer cells, stimulate the formation of a metastatic microenvironment. Previous studies have shown that exosomal miR-141-3p is associated with metastatic prostate cancer (PCa). However, the role and regulatory mechanism of miR-141-3p in the microenvironment of bone metastases require further study. In this study, we performed a series of experiments in vivo and in vitro to determine whether exosomal miR-141-3p from MDA PCa 2b cells regulates osteoblast activity to promote osteoblastic metastasis. We demonstrate that extracts obtained from cell culture supernatants contained exosomes and that miR-141-3p levels were significantly higher in MDA PCa 2b cell exosomes. Via confocal imaging, numerous MDA PCa 2b exosomes were observed to enter osteoblasts, and miR-141-3p was transferred to osteoblasts through MDA PCa 2b exosomes in vitro. Exosomal miR-141-3p from MDA PCa 2b promoted osteoblast activity and increased osteoprotegerin OPG expression. miR-141-3p suppressed the protein levels of the target gene DLC1, indicating its functional significance in activating the p38MAPK pathway. In animal experiments, exosomal miR-141-3p had bone-target specificity and promoted osteoblast activity. Mice injected with miR-141-3p-mimics exosomes developed apparent osteoblastic bone metastasis. Exosomal miR-141-3p from MDA PCa 2b cells promoted osteoblast activity and regulated the microenvironment of bone metastases, which plays an important role in the formation of bone metastases and osteogenesis damage in PCa. Clarifying the specific mechanism of bone metastasis will help generate new possibilities for the treatment of PCa.

[Transcriptional activities of tumor-specific survivin promoter and PSMA promoter and enhancer in human prostate cancer: evaluation and comparison].

OBJECTIVE: To detect and compare the transcriptional activities of prostate-specific membrane antigen (PSMA) promoter and enhancer and survivin promoter in different human prostate cancer cell lines, and to search for some evidence for the targeting gene therapy of human prostate cancer. METHODS: The fragments of the PSMA promoter and enhancer and survivin promoter were amplified by PCR and inserted into pGL3-Basic. The recombinant plasmids were transiently transfected into human prostate cancer cell lines and normal Chang liver cells, and, their transcriptional activities in various cells were determined by measuring the expression of luciferase. RESULTS: The survivin promoter exhibited a higher transcriptional activity than PSMA promoter and enhancer in tumor cell lines, and the S2pro promoter showed the highest activity, reaching one third of that of the CMV promoter. CONCLUSION: The survivin promoter is highly activated in prostate cancer cell lines and may serve as a new tool for the transcriptional targeting gene therapy of prostate cancer.

[Effects of TFDP3 on regulating the autophagy and apoptosis of LNCaP cells].

AIM: To investigate the effect of TFDP3 on prostate cancer cell line LNCaP by transgenic method, and to explore the effect of TFDP3 on regulating the autophay and apoptosis by co-regulation with E2F1. METHODS: LNCaP cells were transfected with pcDNA3.1-TFDP3, pCMV-E2F1-HA or pcDNA3.1 empty vector.The expression of TFDP3, E2F1 and LC3B were detected by real-time PCR after transfection for 24 h. Western blotting was used to monitor the changes in autophagy-associated protein LC3B, Apoptosis of transfected cells were analyzed by flow cytometry. RESULTS: The results showed that activation of TFDP3 upregulates the expression of autophagy genes-microtubule-associated protein-1 light chain-3B (LC3B), and E2F1 antagonizes TFDP3-induced autophagy, and TFDP3 can inhibit E2F1-induced apoptosis. CONCLUSION: TFDP3 upregulates the expression of autophagy gene LC3B and inhibits E2F1-induced apoptosis, and may play an important role in prostate cancer.

Evaluation of a new rapid influenza A diagnostic test for detection of pandemic (H1N1) 2009 and seasonal influenza A virus.

BACKGROUND: Rapid influenza A diagnostic tests (RIDTs) play an important role in the clinical setting, especially in the influenza post-pandemic era with three influenza A viruses in circulation. OBJECTIVES: Determine the sensitivity and specificity of a new RIDT (FluA Dot) by comparison with BD Directigen EZ FluA+B and CDC rRT-PCR. STUDY DESIGN: Two sets of experiments were conducted to determine the performance of the new test. (1) Serial dilutions of eight pandemic (H1N1) 2009 (pH1N1) isolates, five seasonal H3N2 isolates, five seasonal H1N1 isolates and three recombinant nucleoproteins were tested by FluA Dot assay, Directigen EZ FluA+B test and CDC real-time RT-PCR. (2) Using CDC rRT-PCR as the gold standard, the clinical sensitivity and specificity of the FluA Dot and Directigen EZ FluA+B were evaluated in nasopharyngeal swab (NPS) specimens of 807 patients presenting with influenza-like illness. RESULTS: The average analytical sensitivity of FluA Dot (0.06 ng/mL for recombinant nucleoproteins and 2.16 ± 0.85 log 10 TCID(50) for viruses) was approximately 10-fold higher than Directigen EZ FluA+B (1-2 ng/mL for recombinant nucleoproteins and 3.54 ± 0.81 log 10 TCID(50) for viruses), and was approximately 10-fold lower than the CDC rRT-PCR (1.09 ± 0.69 log 10 TCID(50) for viruses). Among 807 NPS specimens tested, the sensitivities and specificities of FluA Dot were 91.1% (95%CI: 86.7-94.4%)/99.7% (95%CI: 98.7-99.9%), and the Directigen EZ FluA+B were 71.9% (95%CI: 65.7-77.6%)/99.8%(95%CI: 99.0-99.9%). CONCLUSION: The new test (FluA Dot) exhibit higher sensitivity than Directigen EZ FluA+B both in pH1N1 and seasonal influenza A detection. The promising RIDT can play important roles in influenza diagnosis and therapy.

Inhibition of prostate cancer by suicide gene targeting the FCY1 and HSV-TK genes.

Prostate cancer is one of the most prevalent tumors. The switch of androgen signal dependence makes therapy more complex. Although reports on introduction of a single suicide gene exist, double suicide gene therapy has not been reported yet. In the current study, two suicide genes were constructed in the pIRES plasmid driven by PSMA promoter. 5-FC and GCV combination in vitro led to a higher growth inhibition on prostate cancer compared to a single pro-drug. Retarded xenograft tumor growth was observed in castrated nude mice after double suicide gene activation. Furthermore, decreased metastasis was observed with double suicide gene treatment. These findings suggest that specific double suicide gene strategy could be a potential option for the therapy of prostate cancer.

[Immune response induced by Rpf domain from Micrococcus luteus in mice].

AIM: To investigate the immunobiology of Rpf domain from Micrococcus luteus. METHODS: BALB/c mice were immunized with Rpf domain three times at 2-week interval. ELISA was used to detect the title of the anti-Rpf domain antibody titer in the immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. The levels of secreted IFN-gamma, IL-10 and IL-12 upon specific antigen stimulation was detected by ELISA. The Rpf domain immunized BALB/c mice were intravenously infected with 10(5) CFU MTB H37Rv. The number of CFU in the spleens was determined four weeks after final injection. RESULTS: The titer of the specific antibody in sera of the immunized BALB/c mice was 1:128 000. The SI of Rpf domain immunized group (2.10+/-0.12) was significantly higher than that of saline immunized group (0.90+/-0.21). The lever of IFN-gamma, IL-10 and IL-12 levels in culture supernatant of spleen lymphocytes from the fusion protein immunized mice was (1 126+/-36) ng/L, (368+/-13) ng/L and (289+/-14) ng/L, respectively, which was markedly higher than that of saline immunized group (P<0.01). Compared with normal saline immunized mice (6.64+/-0.13) four weeks after final injection, dramatic reduction in MTB replication was observed in the spleen (5.03+/-0.11) from the BALB/c mice immunized with fusion proteins. CONCLUSION: Rpf domain can be used as a candidate for a new TB vaccine.

[Preparation and characterization of monoclonal antibodies against Micrococcus luteus Rpf domain].

AIM: To express Micrococcus luteus Rpf domain in prokaryotic cells and prepare monoclonal antibodies against Rpf domain. METHODS: The gene encoding Micrococcus luteus Rpf domain was amplified from genome of Micrococcus luteus by polymerase chain reaction(PCR), and inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf domain gene was subcloned into the expression vector pPro-EXHT and transfected into E.coli DH5alpha. After induced by IPTG, the bacteria controlled by T7 promoter expressed the fused Micrococcus luteus Rpf domain protein with a hexahistidine tail at its N-terminal and the target protein was purified under denaturing conditions. Using this protein as antigen to immunize the BALB/c mice and prepare monoclonal antibodies against Micrococcus luteus Rpf domain. Then specifities and relative affinities of mAbs were identified by ELISA. RESULTS: The fusion protein was purified by metal chelate affinity chromatography under denaturing condition. Three cloned mAbs were prepared from the mice immunized by Rpf domain. All of them could recognize Rpf domain. specifically. CONCLUSION: The prepared mAbs against Rpf domain have strong specificity with high titers, which provides useful tools for further study of the function of Rpf domain in TB prevention.

[Detection of transcriptional activities of tumor-specific survivin promoter in human prostatic carcinoma].

OBJECTIVE: To clone DNA sequence of the survivin promoter and study is transcriptional activities in human prostate cancer cells and normal Chang liver cells. METHODS: The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME vectors to reconstruct a recombinant plasmid named pPRIME-S1pro and pPRIME-S2pro. Then the reconstructed plasmid was transiently transfected into human prostate cancer cells lines LNCaP and normal Chang liver cells. The transcriptional activities of the survivin promoter in various cells was determined by measuring the expression of green fluorescent protein (GFP). RESULTS: The survivin promoter had transcriptional activities in LNCaP cells and the transcriptional activity of the S2pro was much higher that of the S1pro, reaching a level of 39% of the transcriptional activity of the CMV promoter. CONCLUSION: The survivin promoter cloned in the therapy for prostate cancer.

[Construction of the eukaryotic expression vector of bioactive fragment of human bactericidal/permeability-increasing protein and its expression in CHO cells].

AIM: To construct the eukaryotic expression vector of the cDNA sequence encoding bioactive N-terminal fragment of human bactericidal/permeability-increasing protein (BPI) and express it in CHO cells. METHODS: Total RNA was extracted from human polymorphonuclear leukocytes (PMN) and subjected to reverse transcription, then the human BPI cDNA gene was amplified by nested PCR. The PCR product was cloned into pUC19 plasmid and confirmed by restriction enzyme digestion and DNA sequencing. Then the specific BPI encoding fragment was subcloned into pcDNA3 plasmid to form pcDNA-BPI(N) eukaryotic expression vector. CHO cells were transfected with the recombinant plasmid and the stable clones were selected by G418. The expression of BPI was identified by immunofluorescent assay. RESULTS: Restriction enzyme digestion and DNA sequence analysis revealed that the sequence encoding signal peptide and bioactive N-terminal fragment of BPI had six nucleotide substitutions, compared with that of the established human BPI sequence. The expression products of the selected CHO positive cell clones were detected by anti-BPI monoclonal antibody. CONCLUSION: The construction of the eukaryotic expression vector of bioactive fragment of human BPI and its successful expression in CHO cells are helpful to further study of the role of BPI.

Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer.

BACKGROUND: The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-gamma-seminoprotein E(4)B(7) murine mAb by guided selection. METHODS: Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E(4)B(7) mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified gamma-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for gamma-seminoprotein were selected and further identified. RESULTS: First, using the E(4)B(7) Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against gamma-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E(4)B(7) mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-gamma-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. CONCLUSIONS: Through guided-selection, we successfully produced a human anti-gamma-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.

[Inhibition of survivin gene expression in PC-3 cells by RNAi].

OBJECTIVE: To construct eukaryotic expression vectors by using the pSilencer3. 1-H1 neo vector for inhibiting human survivin gene by RNA interference, and to detect the effect of the silenced survivin gene on PC-3 cells. METHODS: Three target gene segments were synthesized and cloned into the pSilencer3. 1-H1 neo vector respectively to construct three recombinant eukaryotic expression vectors: pSilencer3. 1-SVV1, pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3, which were identified by enzyme digestion analysis and DNA sequencing. Then the PC-3 cells were transfected with the recombinant vectors and the interference effect detected by RT-PCR, Western blot and immunohistochemical staining. The apoptosis index of the PC-3 cells was detected by flow cytometry and their proliferation detected by MTT method. RESULTS: Enzyme digestion analysis and DNA sequencing showed that three target segments were cloned into pSilencer3. 1-H1-neo vectors. The results of RT-PCR, Western blot and immunohistochemical staining indicated that pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors could knock down the transcription and expression of survivin gene. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased by 10% - 15% and their growth obviously slowly down. CONCLUSION: The transcription and expression of survivin gene were inhibited effectively by the recombinant eukaryotic expression vectors (pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3) in the prostate cancer cell line PC-3. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased and their growth inhibited.

[Screening of human anti-gamma-seminoprotein light chain guided with the murine Fd fragment].

AIM: To screen human anti-gamma-sm (gamma-seminoprotein) light chain (Lc) with guided selection of murine Fd fragment. METHODS: The human Lc repertorie genes were amplified by RT-PCR from PBMC in patients with prostate cancer, and cloned into the phagemid vector pComb3X with murine Fd gene against gamma-seminoprotein to construct the human-mouse hybrid Fab antibody library. The size of the library, antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified gamma-sm was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected by ELISA with pIII-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST. RESULTS: A 1.2x10(7) CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds' panning with gamma-sm, 5 positive clones were selected by ELISA and 2 clones with higher affinity were selected. Sequence analysis suggested these two positive clones contained the same light gene with high V(L) homology to human germline gene IGKV4-1*01. CONCLUSION: Human anti-gamma-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.

[Study on the nucleic acid of E. coli bacteriophage with broad host range and its sterilization effect to sewage samples from the environment].

OBJECTIVE: To study the change of nucleic acid sequence and the germicidal effect of an E. coli bacteriophage with broad host range isolated from hospital sewage as well as to study the mechanism of phage host specificity and the effect of killed bacteria by phage-disinfectant to the samples from sewage water. METHODS: To extract the nucleic acid from phage f(2) and phage with broad host range using anti-serum-carbamidine hydrochloride assay. Purity with agarose gel electrophoresis was then evaluated. Differences of nucleic acid sequence between phage f(2) and phage with broad host range with reverse transcription-polymerase chain reaction (RT-PCR) and random amplified polymorphic DNA (RAPD)-PCR were also comparing and analysed. Through observing the germicidal test of phage f(2) and phage with broad host range to samples from environment, different sterilization effects between the two phages were compared. RESULTS: Analystic test for nucleic acid revealed that the two phages both belonged to 6000 bp, single-stranded RNA bacteriophage. Significant differences in their specificity of RAPD-PCR and RT-PCR were found during the changed of host range; with 26 RAPD-cDNA differential fragments found that in two phages RAPD-PCR products. The RT-PCR product of phage f(2) was 450 bp cDNA fragment, but the phage with broad host range did not show PCR product. Treating the sewage water with phage under broad host range, the germicidal test showed that the cleaning rate of E. coli bacteria and phage f(2) in water samples from environment could reach 36.75% - 56.28%, 30.84% - 47.96%, 19.19% - 35.06% and 13.05% - 27.85%, respectively. CONCLUSION: The cleaning rates to E. coli and bacteria by phage with broad host range were obviously higher than phage f(2) (P = 0.000). Analytic test for nucleic acid indicated that host-specific lytic effect of phage with broad host range had been changed at genetic level.

[Detection of expression of endometriosis-related cytokine and their receptor genes by cDNA microarray technique].

AIM: To study the pathogenesis of endometriosis(EM) by investigating cytokine(CK) and CKR genes expression involved in the development of EM. METHODS: The CK and CKR gene expression pattern in samples of EM and normal endometrial tissues were analyzed by using cDNA microarrays. RESULTS: 119 genes were expressed differently between 3 cases EM tissues and 3 cases normal endometria tissues. Among them, 15 were CK and CKR genes, including IL-1, IL-2, IL-6, IL-8, VEGFR, TGF, EGF, FGF, and EPOR etc. CONCLUSION: These 15 differently expressed CK and CKR genes may be correlated with the development of EM.

[The diagnostic significance of Ig heavy chain rearrangement detected in B cell lymphoma patients' serum or plasma blood samples].

OBJECTIVE: To evaluate the diagnostic significance of detecting immunoglobulin (Ig) heavy chain (IgH) by using serum or plasma as blood samples. METHODS: First, collect serum and plasma blood samples of patients with B-NHL and extract tumor-derived DNAs. Then design the primer to amplify framework3 (Fr3) from the V segment regions to the J regions of the IgH complementary determining region III (CDR-III) gene. Detect the positive ratio of IgH rearrangement with PCR and evaluate its diagnostic significance. RESULTS: B lymphoma cell line Raji was used as the positive control. Of the 30 B-NHL cases diagnosed with morphologic study, 25 (83.3%) showed IgH rearrangement. DNAs extracted from healthy adults and chronic lymphadenitis patients showed negative result. There was no statistical pertinence between IgH gene rearrangement and clinical manifestation, clinical staging and tumor burden. CONCLUSION: Tumor-derived DNA can be detected in serum or plasma of the majority of patients with B-cell lymphoma. Testing of serum or plasma for tumor associated DNA may be a novel parameter for clinical diagnosis without the restriction of the position of enlarged lymph node.

[Construction of antisense recombinant adenoviral vector for c-myc and its antiproliferative effect on rat lymphocytes].

AIM: To observe the antiproliferative effect of antisense recombinant adenoviral vector for c-myc on rat thymus lymphocytes. METHODS: Antisense and sense bacterial plasmids for c-myc were constructed. Bacterial plasmids and El detected adenoviral plasmid were cotransfected into 293 cells. Recombinant adenoviral vectors were obtained after cotransfection. The antiproliferative effects were assayed by MTS. The expression of c-myc mRNA was detected by RT-PCR. RESULTS: The results showed that antisense recombinant adenoviral vector for c-myc could inhibit rat thymus lymphocytes proliferation. The expression of c-myc mRNA was decreased after antisense recombinant adenoviral vector for c-myc was transfected into cells. CONCLUSION: Recombinant antisense adenoviral vector for c-myc could inhibit rat thymus lymphocytes proliferation.

[Study on isoniazid-resistant Mycobacterium tuberculosis isolates by multiple-polymerase chain reaction-single strand conformation polymorphism].

OBJECTIVE: To develop a new multiple-polymerase chain reaction-single strand conformation polymorphism (multi-PCR-SSCP) system for detecting the aphC promoter, inhA, and katG gene mutations in isoniazid-resistant Mycobacterium tuberculosis isolates in the single reaction, and for the quick diagnosis of isoniazid-resistant Mycobacterium tuberculosis isolates. METHODS: Three pairs of oligonucleotide primers were designed according to the aphC promoter, inhA, and katG genes of Mycobacterium tuberculosis to examine isoniazid-resistance by multi-PCR-SSCP. RESULTS: Isoniazid-sensitivity and resistance were analyzed with general PCR and multi-PCR at the same time, and H(37) Rv was used as a control. These two protocols amplified the anticipated fragments, the rate of consistency being 100%. By single gene PCR-SSCP, the mutation rates of aphC promoter, inhA, and katG gene were 17%, 20%, and 66%, respectively. The mutation rate detected by multi-PCR-SSCP was 83%. CONCLUSIONS: Multi-PCR-SSCP is a sensitive and specific method for rapid detection of aphC promoter, inhA, and katG gene mutations in isoniazid-resistant Mycobacterium tuberculosis isolates. Drug-resistant gene detection may be clinically useful in the therapy of tuberculosis.