Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Analysis of differentially expressed genes in LNCaP prostate cancer progression model.

The LNCaP/C4-2 human prostate cancer progression model was established to mimic phenotypic and genotypic changes during prostate cancer development from androgen dependence to androgen independence, from nonmetastasis to metastasis. In this study, cDNA microarrays were performed using a microarray chip from Affymetrix to characterize and compare gene expression profiles in LNCaP and C4-2, which may provide novel insight into the molecular mechanism mediating prostate cancer progression. Three hundred eighteen genes consistently exhibited differential expression in LNCaP and C4-2 in 2-time microarray data. Based on their function, the differentially expressed genes can be grouped into several subcategories, including growth factors and signal transducers, oncogenes and tumor suppressors, tumor-specific antigens, transcriptional factors, transporters, and factors involved in invasion, metastasis, and metabolism. Some genes are novel and unexplored in prostate cancer progression and are of potential interest for follow-up investigation. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to corroborate the microarray results, and 76 differentially expressed genes were validated out of 104 candidates. Expression pattern analyses were performed in these 76 differentially expressed genes, and a series of genes was found to be positively or negatively correlated to prostate cancer progression in the LNCaP prostate cancer progression model and to possess predominant prostate cell specificity. ELF5/ESE-2b and long-chain acyl coenzyme A dehydrogenase (ACADL) expressions were found to be positively associated with malignant progression in LNCaP, C4-2, and C4-2B, and predominantly expressed in prostate cancer cells. Functional evaluation revealed that ELF5/ESE-2b and ACADL expressions contributed to the malignant phenotypes of prostate cancer cells. Accordingly, our microarray data may provide clues for finding novel genes involved in prostate cancer progression to androgen independent and metastasis, and shed light on finding new targets for diagnosis and therapy of prostate cancer.

[Molecular mechanisms of regulation of estrogen receptor a expression level in breast cancer].

Estrogen receptor alpha (ERalpha) plays an important role in breast cancer development and progression and thus becomes a useful molecular target for breast cancer therapy. ERalpha is differentially expressed in breast cancer patients. Moreover, ERalpha expression levels may change at different stages of breast cancer even for the same patient. ERalpha expression is closely associated with the effect of endocrine therapy and prognosis. The mechanisms underlying ERalpha expression are complicated, because ERalpha expression is regulated at different levels, including chromatin, transcriptional, post- transcriptional, translational, and post-translational levels. Many proteins modulate the transcription of ERalpha gene at the chromatin and transcriptional levels through direct or indirect interaction with the ERa promoter. Some microRNAs decrease ERalpha levels possibly by induction of the degradation of ERalpha mRNA and/or repression of the mRNA translation. At the post-translational level, many proteins regulate ERalpha protein levels through ubiquitin-proteosome pathway. This review focuses on molecular mechanisms of regulation of ERalpha expression at different levels.

Ubiquitous mitochondrial creatine kinase is overexpressed in the conditioned medium and the extract of LNCaP lineaged androgen independent cell lines and facilitates prostate cancer progression.

BACKGROUND: Androgen independent prostate cancer (AIPC) is not responsive to androgen ablation therapy. The biomarkers of AIPC are lack. Numerous proteomics studies have focused on finding new markers of AIPC and exploring their possible functions, but little is known about the difference between conditioned medium (CM) from AIPC and androgen dependent prostate cancer (ADPC) cells. METHODS: We performed a proteome analysis of CM from LNCaP, C4-2, and C4-2B cells by a two dimensional electrophoresis based technology. Western blots and immunohistochemical studies were employed to explore the expression pattern of the identified protein in prostate cancer cell lines and clinical specimens, respectively. Then we examined the possible roles and mechanisms of the ubiquitous mitochondrial creatine kinase (uMtCK) in vitro. RESULTS: Besides prostate specific antigen (PSA) and insulin-like growth factor binding protein-2 (IGFBP2), uMtCK was identified in the CM of AIPC cells. uMtCK was up-regulated in AIPC cells and in human prostate cancer tissues at WHO grade III. Stably transfected exogenous uMtCK showed a growth promoting effect rather than mock vector in LNCaP cells, with or without bicalutamide in culture medium. Further assays showed that higher degrees of ROS generation and Akt signaling pathway activation in LNCaP-uMtCK than in LNCaP-neo cells. CONCLUSIONS: We showed that uMtCK could be easily detected in CM of LNCaP lineaged AIPC cells. Exogenous uMtCK in LNCaP cells surprisingly contributed to overproduction of ROS, activation of Akt signaling pathway and more aggressive phenotypes including androgen independence development.

Silencing PinX1 compromises telomere length maintenance as well as tumorigenicity in telomerase-positive human cancer cells.

The nucleolar protein PinX1 has been proposed to be a putative tumor suppressor due to its binding to and inhibition of the catalytic activity of telomerase, an enzyme that is highly expressed in most human cancers in which it counteracts telomere shortening-induced senescence to confer cancer cell immortalization. However, the role of PinX1 in telomere regulation, as well as in cancer, is still poorly understood. In this study, we showed that the PinX1 protein is constitutively expressed in various human cells regardless of their telomerase activity and malignant status. Most interestingly, we found that silencing PinX1 expression by a potent short hairpin RNA construct led to a robust telomere length shortening and growth inhibition in telomerase-positive but not in telomerase-negative human cancer cells. We further showed that silencing PinX1 significantly reduced the endogenous association of telomerase with the Pot1-containing telomeric protein complex, and therefore, could account for the phenotypic telomere shortening in the affected telomerase-positive cancer cells. Our results thus reveal a novel positive role for PinX1 in telomerase/telomere regulations and suggest that the constitutive expression of PinX1 attributes to telomere maintenance by telomerase and tumorigenicity in cancer cells.

High mobility group box-1 protein acts as a coactivator of nuclear factor of activated T cells-2 in promoting interleukin-2 transcription.

High mobility group box-1 protein, an abundant and conserved constituent of vertebrate nuclei, has recently been reported to be an endogenous immune signal [Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Reports 2004;5:825-30]. High mobility group box-1 protein can trigger the release of interleukin-2 and interleukin-12 from lymphocytes. However, at present the underlying mechanism remains unknown. It has been clarified that nuclear factor of activated T cells-2 transduces most immunological signals in T cells and modulates the production of interleukin-2. So it is natural that we asked whether high mobility group box-1 protein could promote production of interleukin-2 in a nuclear factor of activated T cells-2-dependent way. Our experiments firstly showed that high mobility group box-1 protein could bind to nuclear factor of activated T cells-2 in vivo and in vitro. High mobility group box-1 protein cotransfection markedly upregulated the transcription activity of nuclear factor of activated T cells-2 in promoting interleukin-2 reporter gene transcription, which was demonstrated to be dose-dependent. Cotransfection of high mobility group box-1 protein and nuclear factor of activated T cells-2 induced an 18.4-time increase of interleukin-2 activity in 293T cells and a 117.7-time increase in Hela cells. Moreover, inhibition of either high mobility group box-1 protein or nuclear factor of activated T cells -2 expression by sRNAi led to significant decrease of transcription activity of interleukin-2 reporter gene, suggesting that high mobility group box-1 protein and nuclear factor of activated T cells-2 both take important roles in facilitating interleukin-2 transcription, and high mobility group box-1 protein could act as a coactivator for nuclear factor of activated T cells-2 in enhancing transcription of interleukin-2. This discovery has not been reported elsewhere, and helps to understand the newly highlighted immunological role of high mobility group box-1 protein.

[Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1].

OBJECTIVES: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1). METHODS: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo. RESULTS: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1. CONCLUSIONS: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

Identification and characterization of the novel human prostate cancer-specific PC-1 gene promoter.

Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5'-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5' deletion analysis identified the -1579 bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; -4939 bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6 kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between -345 and -359 bp of the PC-1 5'-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy.

[Stress proteins of Bifidobacterium longum NCC2705].

Stress proteins of Bifidobacterium longum strain NCC2705 were identified and characterized during stationary phase. According to the proteomic map of L. lactics IL1403 and theoretical Mr/pl of stress proteins in B. longum NCC2705 genome annotation, spots of stress proteins in gels were localized, and proteins were identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or ESI-MS/MS mass spectrometry. For protein identification by peptide mass fingerprinting, peptide masses were searched against database of B. longum NCC2705 by Mascot licensed in-house. 44 spots representing 8 protein entries have been identified, these proteins were hydrophilic proteins and predicted acid proteins. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Except for DnaJ, the stress proteins were encoded by genes with CAI value above 0.5, and represented a large proportion of the most abundant proteins. Moreover, the results of scavenging effects on free radicals in vitro showed that B. longum NCC2705 can inhibit fatty acid oxidation and scavenge DPPH, but they scavenge weakly active oxygen free radicals. We identified a key protein that can reverse oxidative damage to proteins and lipids: alkyl hydroperoxide reductase (ahpC, BL0615)synthesized under our experimental conditions.

[An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli].

OBJECTIVE: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli. METHODS: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography. RESULTS: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded. CONCLUSION: This study may provide a basis for further study on the biological function of HBx at the protein level.

[Effects of exogenous ER beta expression on the cell growth properties of MCF-7 breast cancer cell line].

OBJECTIVE: To study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment. METHODS: An eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed. RESULTS: A stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed. CONCLUSION: Exogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.

Cancer immunotherapy targeting the telomerase reverse transcriptase.

The human telomerase reverse transcriptase (hTERT) is expressed in more than 85% of tumor cells but is usually not found in normal cells, which makes hTERT as an ideal tumor-associate antigen (TAA) to develop potential vaccine specifically destroying cancers without impairing normal tissues in human cancer immunotherapy. Here are reviewed the fundamental advances of studies on immunogenicity of hTERT or its peptides and the early clinical trials using the hTERT vaccine approach in the last decades.

[Construction of recombinant plasmid using Neo/E Technology].

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

[Effect of PC-1 gene expression on migration ability of prostate cancer cells].

BACKGROUND & OBJECTIVE: PC-1 gene is highly expressed in C4-2 cell line, an androgen-independent and aggressive prostate cancer cell line, but lowly expressed in LNCaP cell line, an androgen-dependent prostate cancer cell line. This study was to assess the contribution of PC-1 gene to migration of prostate cancer cells. METHODS: LNCaP cell line with stable expression of PC-1 gene and C4-2 cell line with antisense nucleotide-down-regulated PC-1 were established. The migration abilities of LNCaP and C4-2 cells were tested using an in vitro transwell invasion assay. RESULTS: Migration cell count of LNCaP cells with up-regulation of PC-1 gene was similar to that of control LNCaP cells(P>0.05); migration cell count was significantly lower in C4-2 cells with down-regulation of endogenous PC-1 gene than in control C4-2 cells (P<0.05). CONCLUSION: PC-1 may be involved in the invasion of prostate cancer cells.

[SMAD3 inhibits the expression of MMP-2 in mouse embryonic fibroblasts].

SMAD3 is one of the receptor-activated SMADs which are important in TGF-beta signal transduction. Smad3-null mice show accelerated cutaneous wound healing compared with wild-type mice. In this work, we investigated the functions and the mechanism of Smad3-mediated TGF-beta signal on matrix metaloproteinase-2 (MMP-2) in mouse fibroblast. We found that MMP-2 at wound bed was expressed earlier in Smad3-null mice than that in wild type and heterozygotes. In the sera of wounding mice, the activity of MMP-2 was also remarkably higher in Smad3-null mice than that in the other two. The embryonic fibroblasts were separated from Smad3 knockout mice to test the function of Smad3 in modulating the expression of MMP-2. The results showed that the expression and activity of MMP-2 in Smad3-null fibroblasts were higher than those in wild type cells. TGF-beta1 could increase the MMP-2 activities in both Smad3 mutant and wild type fibroblasts. The expression and activity of MMP-2 were inhibited by over expression of SMAD3 in Smad3-null fibroblasts, while the expression and activity of MMP-2 were increased by over expression of anti-sense Smad3 in wild type cells. All these results showed that SMAD3 inhibited the expression of MMP-2 in mouse embryonic fibroblasts.

[Development of a new recombineering system by gap repair].

Using lambda phage Red recombinase mediated in vivo homologous recombination system, a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique, and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria. To confirm the recombination functions of pBR322-Red, a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T-->G mutation in galK gene on the bacterial chromosome. The result demonstrated that a new lambda Red-mediated recombineering system based on pBR322-Red was successfully established.

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli.

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

[Screen in vivo induced gene of Mycobacterium tuberculosis by IVIAT].

In order to search new candidates of pharmaceutical target, in vivo induced antigen technology (IVIAT) was used to screen in vivo induced (ivi) genes of Mycobacterium tuberculosis (M. TB). Genomic expression library of M. TB was first constructed with an inducible plasmid pKK223-8; the titer of the library was 1.02 x 10(5) CFU. Sera from ten tuberculosis patients were pooled and absorbed against in vitro-grown M. TB and Escherichia coli, and used to probe the genomic expression library. 16 positive clones were identified by immunological screen, including 22 ORF: two encoding lipid metabolism proteins, five information pathways proteins, two PE/PPE proteins, six intermediary metabolism and respiration proteins, one cell wall and cell processes protein, four conserved hypothetical proteins and two conserved hypothetical proteins with an orthologue in Mycobacterium bovis. Parts of these genes can be used as candidates of pharmaceutical target because they may be relate with virulence.

[Malignant transformation of NIH3T3 cells induced by ectopic expression of PC-1 gene].

OBJECTIVE: To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. METHODS: Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression. RESULTS: NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice. CONCLUSION: Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T.

AIM: To screen the immunogenic membrane proteins of Shigella flexneri 2a 2457T. METHODS: The routine two-dimensional polyacrylamide gel electrophoresis (2-DE) and Western blotting were combined to screen immunogenic proteins of S. flexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was performed to determine the molecular weight of peptides. Electrospray ionization (ESI-MS/MS) was performed to determine the sequences of the interesting peptides. RESULTS: A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein (YaeT) was detected, which might be a candidate target of vaccine. CONCLUSION: Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.