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1.
Biomed Res Int ; 2017: 7587395, 2017.
Article in English | MEDLINE | ID: mdl-28828388

ABSTRACT

Yeast MET18, a subunit of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery which is responsible for the maturation of Fe/S proteins, has been reported to participate in the oxidative stress response. However, the underlying molecular mechanisms remain unclear. In this study, we constructed a MET18/met18Δ heterozygous mutant yeast strain and found that MET18 deficiency in yeast cells impaired oxidative stress resistance as evidenced by increased sensitivity to hydrogen peroxide (H2O2) and cumene hydroperoxide (CHP). Mechanistically, the mRNA levels of catalase A (CTA1) and catalase T (CTT1) as well as the total catalase activity were significantly reduced in MET18-deficient cells. In contrast, overexpression of CTT1 or CTA1 in MET18-deficient cells significantly increased the intracellular catalase activity and enhanced the resistance ability against H2O2 and CHP. In addition, MET18 deficiency diminished the replicative capacity of yeast cells as evidenced by the shortened replicative lifespan, which can be restored by CTT1 overexpression, but not by CTA1, in the MET18-deficient cells. These results suggest that MET18, in a catalase-dependent manner, plays an essential role in enhancing the resistance of yeast cells to oxidative stress and increasing the replicative capacity of yeast cells.


Subject(s)
Aging/genetics , Catalase/genetics , Oxidative Stress/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Aging/physiology , Benzene Derivatives/toxicity , Cytosol/metabolism , Hydrogen Peroxide/toxicity , Oxidation-Reduction , Oxidative Stress/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Transcription Factors/deficiency
2.
Mol Med Rep ; 16(4): 4113-4118, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28765958

ABSTRACT

Interleukin-12 (IL-12) serves an important role in immune responses and antitumor activity. The study of the association between autophagy and cancer cells remains controversial. The present study aimed to investigate the effect of IL­12 on autophagy in the human breast cancer cell lines MDA­MB­231 and MCF­7, and the possible molecular mechanism. Breast cancer cells were treated with different concentrations of recombinant IL­12. The expression of the autophagy-associated protein microtubule­associated protein light chain 3 (LC3) was determined using western blot analysis, fluorescein isothiocyanate­labeled LC3 was detected using fluorescence microscopy and autophagosomes were examined using transmission electron microscopy. Alterations in the phosphatidylinositol 3­kinase/Rac­α serine/threonine protein kinase (PI3K/Akt) and 5'­AMP­activated protein kinase subunit ß­1 (AMPK) pathways, in addition to pathway­associated proteins, were detected using western blotting, following treatment with IL­12 and pretreatment with the PI3K/Akt activator insulin­like growth factor or the AMPK inhibitor compound C. It was observed that IL­12 was able to upregulate the expression of the autophagy­associated protein LC3 in a concentration­ and time­dependent manner, and induce the formation of autophagosomes in the two cell lines, and that the above effects involved the inhibition of the PI3K/Akt signaling pathway and the activation of the AMPK signaling pathway.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Autophagy/drug effects , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Interleukin-12/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Autophagosomes/drug effects , Autophagosomes/metabolism , Autophagosomes/ultrastructure , Breast Neoplasms/ultrastructure , Cell Line, Tumor , Fluorescent Antibody Technique , Humans , Microtubule-Associated Proteins/metabolism
3.
Saudi J Biol Sci ; 24(1): 95-102, 2017 Jan.
Article in English | MEDLINE | ID: mdl-28053577

ABSTRACT

To investigate the effect of over-expression of tissue factor pathway inhibitor-2 (TFPI-2) on the differentiation of hepatocellular carcinoma (HCC) cells (Hep3B and HepG2). The TFPI-2 recombinant adenovirus (pAd-TFPI-2) was constructed using the pAdeasy-1 vector system. Transfected by pAd-TFPI-2, the cell proliferation of HCC cells was evaluated by CCK-8 assay, flow cytometry was used to detect cell apoptosis and CD133 expression. Real-time PCR and Western blot were used to detect the expression levels of markers of hepatocellular cancer stem cells (CSC) and hepatocytes. The over-expression of TFPI-2 significantly suppressed cell proliferation, induced apoptosis, and dramatically decreased the percentage of CD133 cells, which was considered as CSC in HCC. Real-time PCR and Western blot showed that the expression of markers of CSC in Hep3B cells and HepG2 cells infected with pAd-TFPI-2 was markedly lower than those of the control group (P < 0.05), while the expression of markers of hepatocytes was significantly increased (P < 0.05). Hence, TFPI-2 could induce the differentiation of hepatocellular carcinoma cells into hepatocytes, and is expected to serve as a novel way for the treatment of HCC.

4.
Biosens Bioelectron ; 92: 342-348, 2017 Jun 15.
Article in English | MEDLINE | ID: mdl-27836609

ABSTRACT

Herein, a simple and novel fluorescence biosensing strategy has been developed for ultrasensitive determination of microRNA (miRNA) by combining target-triggered DNA nanoassembly on quantum dots (QDs) with DNAzyme-modulated double quenching of QDs. In presence of miRNA target, the target triggered catalytic hairpin assembly (CHA) amplification and powered highly efficient DNA nanoassembly on the surface of QDs, leading to exhibition of numerous G-quadruplexes close to the QDs. The G-quadruplex folded properly and bound hemin to form a stable G-quadruplex/hemin complex. Then the luminescence of QDs was quenched via photoinduced electron transfer by hemin associated with the particles and the electron acceptor of O2 which was in situ generated with the horseradish peroxidase-mimicked G-quadruplex/hemin DNAzymes toward H2O2. Based on this target-triggered highly efficient DNA nanoassembly and DNAzyme-modulated double quenching mechanism, the proposed biosensing strategy showed admirable signal amplification capability. Using miRNA-21 as model analyte, the designed nanosensor could detect miRNA down to 37 fM with a wide linear detection range of 1×10-13M to 1.0×10-8M, and exhibited good selectivity, acceptable reproducibility and low matrix effect. This proposed strategy presented a simple, powerful platform toward ultrasensitive miRNA detection and had great potential for bioanalysis and clinic diagnostic application.


Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/chemistry , MicroRNAs/analysis , Quantum Dots/chemistry , G-Quadruplexes , Hemin/chemistry , Humans , Hydrogen Peroxide/chemistry , Limit of Detection , MCF-7 Cells , Oxygen/chemistry , Reproducibility of Results , Spectrometry, Fluorescence/methods
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(7): 865-9, 2016 Jul.
Article in Chinese | MEDLINE | ID: mdl-27363262

ABSTRACT

Objective To investigate the effect of endogenous interferon ß (IFN-ß) on the polarization of M1 macrophages as well as the proliferation and invasion activities of hepatocellular carcinoma cells (HCCs) mediated by M1 macrophages. Methods U937-M1 macrophages derived from human monocytic tumor cells U937 was established and the cell phenotypes were identified by real-time quantitative PCR, ELISA and flow cytometry. After IFN-ß gene was knocked down with siRNA or IFN-ß was neutralized with IFN-ß monoantibody in U937-M1 macrophages, the change of M1/M2 phenotype was again analyzed by the above methods. The expressions of interferon regulatory factor 1 (IRF1) and IRF5 were detected by real-time quantitative PCR and Western blotting. The proliferation and invasion activities of HCCs, which were cultured with conditioned medium (CM) collected from different macrophage groups, were analyzed by CCK-8 assay and Transwell(TM) experiments, respectively. Results U937-M1 macrophages showed higher expressions of interleukin 12p35 (IL-12p35), interleukin 12p40 (IL-12p40), interleukin 12p70 (IL-12p70), interleukin 23p19 (IL-23p19), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and CD86 than U937-M0 did. But both U937-M0 macrophages and U937-M1 macrophages showed low expression of CD206. However, compared with the U937-M1 macrophages, the IFN-ß-blocked U937-M1 macrophages presented decreased expressions of the above M1 macrophages-associated markers, but increased expressions of M2 macrophages-associated markers IL-10 and CD206, as well as lower expressions of IRF1 and IRF5. The inhibited proliferation/invasion activities of HCCs mediated by U937-M1 macrophages were reversed by IFN-ß-blocked U937-M1 macrophages. Conclusion Blocking endogenous IFN-ß could inhibit the U937-M1 polarization status and U937-M1 macrophages-mediated anti-tumor activity of HCCs. IFN-ß might be involved in modulating the expressions of IRF1 and IRF5 as well as maintaining the M1 polarization status and its function.


Subject(s)
Cell Proliferation , Interferon-beta/metabolism , Macrophage Activation , Macrophages/metabolism , Blotting, Western , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Survival/drug effects , Culture Media, Conditioned/pharmacology , Flow Cytometry , Hep G2 Cells , Humans , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-1/metabolism , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Interferon-beta/genetics , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-12/genetics , Interleukin-12/metabolism , Interleukin-23/genetics , Interleukin-23/metabolism , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Liver Neoplasms/pathology , Macrophages/classification , Mannose Receptor , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/metabolism , Neoplasm Invasiveness , RNA Interference , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , U937 Cells
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(7): 870-5, 2016 Jul.
Article in Chinese | MEDLINE | ID: mdl-27363263

ABSTRACT

Objective To investigate the effect of IL-12 on autophagy and the relative possible mechanism in HepG2 and SMMC-7721 human hepatoma cells. Methods The hepatoma cells were treated with IL-12 (10 ng/mL) for 6 hours. Western blotting was applied to detect the expressions of microtubule-associated protein 1 light chain 3 (LC-3), Beclin 1 and the phosphorylated levels of protein kinase B (AKT), mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3); immunofluorescence assay (IFA) and transmission electron microscopy (TEM) were used to observe the formation of autophagosome. After STAT3 was inhibited by STATTIC or siSTAT3 and AKT was activated by insulin-like growth factor (IGF-1), Western blotting and IFA were performed again to analyze the change of IL-12-induced autophagy. After the cells were treated with IL-12 (10 ng/mL) for 1, 2, 3, 4, 5 days, CCK-8 assay was used to determine the growth ability. After the hepatoma cells were treated with IL-12 (10 ng/mL) for 48 hours, trypan blue staining was used to detect the death rate of the cells. After cell autophagy was inhibit by siBeclin 1, CCK-8 assay and trypan blue staining were performed again to study the effect of IL-12 on the proliferation and death of human hepatoma cells. Results IL-12 induced autophagy and inhibited cell growth in the hepatoma cells. Silencing Beclin 1 gene enhanced IL-12-mediated growth inhibition and cell death. Furthermore, IL-12 treatment also decreased the expressions of p-AKT, p-mTOR and p-STAT3. The pretreatment of siSTAT3 or STATTIC inhibited STAT3-enhanced IL-12-induced autophagy. Accordingly, activation of AKT with IGF-1 decreased IL-12-induced autophagy. Conclusion IL-12 could induce autophagy through AKT/mTOR/STAT3 signaling pathways and the induction of autophagy attenuates the growth-inhibitory effect of IL-12 on hepatoma cells.


Subject(s)
Autophagy/drug effects , Interleukin-12/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Autophagosomes/drug effects , Autophagosomes/metabolism , Autophagosomes/ultrastructure , Autophagy/genetics , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/genetics , RNA Interference , STAT3 Transcription Factor/genetics , Signal Transduction/genetics , TOR Serine-Threonine Kinases/genetics , Time Factors
7.
Int J Mol Med ; 38(1): 148-60, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27176664

ABSTRACT

Macrophages that differentiate from precursor monocytes can be polarized into a classically activated (M1) or alternatively activated (M2) status depending on different stimuli. Generally, interferon (IFN)-γ and lipopolysaccharide (LPS) are considered the classical stimuli with which to establish M1 polarization. IFN regulatory factor (IRF)1 and IFN-ß are two crucial molecules involved in IFN-γ- and LPS-initialed signaling. However, the association between IRF1 and IFN-ß in the context of the M1 polarization of macrophages is not yet fully understood. In this study, we demonstrate that U937-derived macrophages, in response to IFN-γ and LPS stimulation, readily acquire an M1 status, indicated by the increased expression of interleukin (IL)-12, IL-6, IL-23, tumor necrosis factor (TNF)-α and the M1-specific cell surface antigen, CD86, and the decreased expression of the M2-specific mannose receptor, CD206. However, the knockdown of IRF1 in U937-derived macrophages led to an impaired M1 status, as indicated by the decreased expression of the above-mentioned M1 markers, and the increased expression of the M2 markers, CD206 and IL-10. A similar phenomenon was observed in the M1 macrophages in which IFN-ß was inhibited. Furthermore, we demonstrated that IRF1 and IFN-ß may interact with each other in the IFN-γ- and LPS-initiated signaling pathway, and contribute to the IRF5 regulation of M1 macrophages. In addition, the conditioned medium collected from the M1 macrophages in which IRF1 or IFN-ß were inhibited, exerted pro-tumor effects on the HepG2 and SMMC-7721 cells, as indicated by an increase in proliferation, the inhibition of apoptosis and an enhanced invasion capability. The findings of our study suggest that the interactions of IRF1, IFN-ß and IRF5 are involved in the M1 polarization of macrophages and have antitumor functions. These data may provide a novel antitumor strategy for targeted cancer therapy.


Subject(s)
Antineoplastic Agents/metabolism , Cell Polarity , Interferon Regulatory Factor-1/metabolism , Interferon-beta/metabolism , Macrophages/cytology , Macrophages/metabolism , Apoptosis/drug effects , Biomarkers/metabolism , Culture Media, Conditioned/pharmacology , Hep G2 Cells , Humans , Interferon Regulatory Factors/metabolism , Interferon-gamma/metabolism , Lipopolysaccharides , Protein Binding/drug effects , U937 Cells , Up-Regulation/drug effects
8.
Mol Cell Biochem ; 415(1-2): 157-68, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27003285

ABSTRACT

Hepatocellular carcinoma is the third most common cause of cancer death worldwide. Novel early detection biomarkers and efficacious therapy strategies are needed. Macrophages recruited from circulation monocytes are the major component of solid cancer and play an important role in the carcinogenesis. Whether overexpression of L-12 in monocytes could induce the phenotype directional differentiation into tumoricidal M1 macrophages and inhibit HCC growth in tumor microenvironment was investigated in this study. For the establishment of the monocyte/IL-12 and polarization of M1-like macrophage, the IL-12 overexpressing recombinant monocyte/IL-12 cells were established by infecting with pAd5F35-CMV/IL-12 adenovirus and co-cultured with HCC SMMC-7721 and Hep3B cells. It was found that the phenotype of monocyte/IL-12 polarized to M1-like macrophages with CD197high IL-12high CD206low IL-10low, and decreased expression of TGF-ß, VEGF-A, and MMP-9. In order to explore the mechanism underlying the macrophages polarization, we detected the Stat-3 pathway and its downstream transcription factor c-myc, and found that the p-Stat-3 and c-myc were down-regulated. To evaluate the effects of monocyte/IL-12 on inhibiting HCC growth, various assays including CCK8, flow cytometry, colony-forming and Transwell assays in vitro, and xenograft mouse models and immunohistochemical analyses in vivo were used to detect the HCC growth and relative markers. Treated with IL-12 overexpressing monocytes, the xenograft tumor growth was significantly inhibited in vivo. These results have proven that IL-12-overexpressed monocytes could directionally differentiate to M1-like macrophages through downregulation of Stat-3 and result in the inhibition of HCC growth.


Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Polarity , Down-Regulation , Interleukin-12/physiology , Liver Neoplasms/pathology , Macrophages/pathology , STAT3 Transcription Factor/physiology , Cell Line, Tumor , Cell Proliferation , Coculture Techniques , Humans , Neoplasm Invasiveness
9.
Int J Clin Exp Pathol ; 8(8): 9196-201, 2015.
Article in English | MEDLINE | ID: mdl-26464666

ABSTRACT

This study aims to explore the relationship between Sirt3 expression and lipid accumulation in macrophages by inducing mitochondrial IDH2 deacetylation. In this study, Sirt3 interference and overexpression lentiviral vectors were constructed. Macrophages collected from C57BL/6J mice by peritoneal lavage were used to construct Sirt3 gene interference and overexpression models, and cultured in medium containing 1 mg/ml ox-LDL for 72 h to observe the enrichment of ox-LDL. Reverse transcription PCR was used to detect the expression of Sirt3 mRNA, western blot to detect Sirt3 and acetylated IDH2 proteins, and Nile Red staining and flow cytometry to detect intracellular lipids in macrophages. The results indicated that as compared to Sirt3 overexpressed and normal groups, the acetylation of IDH2 and accumulation of ox-LDL were significantly higher in the Sirt3 inhibited group. In conclusion, the expression of Sirt3 can inhibit lipid accumulation in macrophages by inducing mitochondrial IDH2 deacetylation.


Subject(s)
Isocitrate Dehydrogenase/metabolism , Lipid Metabolism/genetics , Macrophages/metabolism , Mitochondria/metabolism , Sirtuin 3/metabolism , Acetylation , Animals , Female , Isocitrate Dehydrogenase/genetics , Lipoproteins, LDL/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/genetics , Oxidative Stress/genetics , Sirtuin 3/genetics
10.
Int J Mol Med ; 36(4): 1028-34, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26311523

ABSTRACT

The classification of hepatitis C virus (HCV) genotypes is of clinical importance as it may help to predict drug therapy responses and estimate treatment duration. The classical method of HCV subgenotype classification is whole genome sequencing (WGS). However, the high cost and time-consuming nature of WGS limits its usage in clinical practice. A number of studies have been conducted to confirm whether specific regions of HCV could replace WGS in the classification of HCV subgenotypes. In the present study, we used the HCV database to select HCV sequences from different countries. The neighbor-joining method was used to construct phylogenetic trees based on different regions of HCV (core, E1, E2 and NS5B), to confirm which region could replace WGS in subgenotype classification. Our results indicated that the core, E1 and E2 regions could not be used to classify the HCV subgenotype correctly (core failed to recognize subgenotypes c and a, E1 failed to discriminate between subgenotypes a and b, and E2 failed to identify subgenotypes a and c). The NS5B region provided the correct subgenotype classification. The HCV samples (n=153) collected from patients in Sichuan province, (Southwest China) were sequenced and classified based on the NS5B region. The results indicated that the major subgenotype of HCV in patients from Sichuan was 1b (51.6%, n=79); other subgenotypes included 3b (30.1%, n=46), 3a (7.8%, n=12), 6a (8.5%, n=13), 2a (n=2) and 6n (n=1). The data from our analysis may prove to be helpful in future epidemiological investigations of HCV, and may aid in the prevention and clinical treatment of HCV.


Subject(s)
Genotype , Hepacivirus/genetics , Hepatitis C/genetics , Phylogeny , Viral Nonstructural Proteins/genetics , China/epidemiology , Female , Hepatitis C/epidemiology , Humans , Male
11.
J Microbiol Methods ; 117: 22-7, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26172085

ABSTRACT

To prevent epidemic and pandemic cholera disease, an indispensible approach is to develop cholera vaccines based on comprehensive epitope information of this pathogen. This study aimed to utilize our previously raised monoclonal antibody IXiao3G6, which can recognize an epitope in lipopolysaccharide (LPS) sites of Ogawa, to identify mimetic peptides, which may represent Ogawa LPS's epitope information. A phage display library screening using IXiao3G6 antibody resulted in identification of a mimic peptide (MP) with high avidity. A recombinant protein, containing one cholera toxin subunit B (CTB) and two MP repeats (CTB-(MP)2), was subsequently constructed and investigated for its immunological characteristics. The findings collectively demonstrated that the MP presenting phages and CTB-(MP)2 recombinant protein were both capable of inhibiting the interaction between IXiao3G6 and Ogawa/Ogawa LPS specifically in a dose-dependent manner.


Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Epitopes/immunology , Recombinant Proteins/immunology , Antibodies, Bacterial/chemistry , Antibodies, Bacterial/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Epitopes/chemistry , Epitopes/genetics , Escherichia coli/genetics , Peptide Library , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Vibrio cholerae O1/immunology
12.
Hepat Mon ; 15(1): e23729, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25741372

ABSTRACT

BACKGROUND: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) served as a vital role in the life cycle of the virus and persistent infection. However, specific and quantitative methods for cccDNA detection have not been available. OBJECTIVES: Our aim was to develop and primarily evaluate a quantitative method for HBV cccDNA based on magnetic capture hybridization and quantitative PCR technology. MATERIALS AND METHODS: The functionalized-nanoparticles specifically to capture HBV cccDNA, located on both sides of relaxed circle DNA (rcDNA) gap, were designed. Then, magnetic capture hybridization and quantitative PCR (MCH-qPCR) assay were developed and its performance was primarily evaluated with cccDNA standards and serum samples of patients with chronic hepatitis B. RESULTS: Specific nanoparticles of cccDNA capture were prepared and a magnetic capture hybridization and quantitative assay method for cccDNA was developed successfully. The limit of detection was 90 IU/mL, and a good linear relationship in the range of 10(2)-10(6) IU/mL was revealed (r(2) = 0.994) with the MCH-qPCR. Compared with directly real-time PCR, a high content of HBV DNA did not affect the detection of cccDNA for the MCH-qPCR method, and there was no cross-reactivity between cccDNA and rcDNA. CONCLUSIONS: The novel MCH-qPCR method has good sensitivity and specificity. It could meet the requirement of clinical routine detection.

13.
Mol Cell Biochem ; 402(1-2): 157-69, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25563480

ABSTRACT

Interleukin-12 (IL-12), a member of interleukin family, plays a critical role in immune responses and anti-tumor activity. In this study, the effects of IL-12 on monocytic tumor cell lines differentiation to macrophagocyte and its likely mechanism was investigated. We examined the differentiation markers, morphological and functional changes, and possible mechanism in IL-12-treated THP-1 and U937 cells. It was found that IL-12 could up-regulated macrophage surface marker CD68 and CD11b expression in a time-dependent manner. Morphologically, after IL-12 treatment, THP-1 and U937 cells became round or irregular shape, even stretched many cell membrane protuberances; some cell nuclei became fuzzy or completely disappeared, and the chromatin appeared dense and cordlike. Furthermore, IL-12-induced monocytic tumor cell differentiation was accompanied by the growth arrest with G1-phase accumulation and S-phase reduction; apoptosis increased with anti-apoptosis protein Bcl-2 down-expression and pro-apoptosis protein Fas up-regulation, and enhanced phagocytosis function. The IL-12-induced macrophage differentiation of THP-1 and U937 cells was associated with the up-regulation of c-fms expression and the CSF-1R Tyr 809 site phosphorylation. These findings have revealed that IL-12 could induce monocytic tumor cells directional differentiation into macrophage-like cells, and its mechanism is possible connected with the up-regulation of c-fms expression and the phosphorylation of CSF-1R Tyr-809 site.


Subject(s)
Cell Differentiation , Interleukin-12/physiology , Macrophages/physiology , Antigens, CD/metabolism , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cell Shape , Endocytosis , Humans , Phagocytosis , Phosphorylation , Protein Processing, Post-Translational , Receptor, Macrophage Colony-Stimulating Factor/metabolism
14.
Asian Pac J Cancer Prev ; 15(14): 5767-72, 2014.
Article in English | MEDLINE | ID: mdl-25081699

ABSTRACT

BACKGROUND AND AIM: B7-H1, a co-inhibitory molecule of the B7 family, is found aberrantly expressed in ovarian cancer cells and infiltrating macrophage/dendritic-like cells, and plays a critical role in immune evasion by ovarian cancer. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects on ovarian cancer. However, whether IL-12 regulates B7-H1 expression in human ovarian cancer associated-macrophages has not been clarified. Therefore, we investigated the effects of IL-12 on the expression of B7-H1 in ovarian cancer-associated macrophages and possible mechanisms. METHODS: PMA induced THP-1-derived macrophages or human monocyte-derived macrophages were treated with recombinant IL-12 (rIL-12) or infected with adenovirus carrying human IL-12 gene (Ad-IL-12-GFP) for 24 h, then cocultured with the SKOV3 ovarian cancer cell line for another 24 h. Macrophages were collected for real-time PCR and Western blot to detect the expression of B7-H1, and activation of the NF-κB signaling pathway. Moreover, supernatants were collected to assay for IL-12, IFN-γ and IL-10 by ELISA. In addition, monocyte-derived macrophages treated with IFN-γ were cocultured with SKOV3 and determined for the expression of B7-H1. Furthermore, the expression of B7-H1 in monocyte-derived macrophages was also evaluated after blocking NF-κB signaling. RESULTS: The expression of B7-H1 was significantly upregulated in monocyte-derived macrophages treated with rIL-12 or Ad-IL-12-GFP compared with the control groups (p<0.05), accompanied by a remarkable upregulation of IFN-γ (p<0.05), a marked downregulation of IL-10 (p<0.05) and activation of NF-κB signaling. However, the upregulation of B7- H1 was inhibited by blocking the NF-κB signaling pathway (p<0.05). Expression of B7-H1 was also increased (p<0.05) in monocyte-derived macrophages treated with IFN-γ and cocultured with SKOV3. By contrast, the expression of B7-H1 in THP-1-derived macrophages was significantly decreased when treated in the same way as monocyte-derived macrophages (p<0.05), and IL-10 was also significantly decreased but IFN-γ was almost absent. CONCLUSIONS: IL-12 upregulates the expression of B7-H1 in monocyte-derived macrophages, which is possible though inducing the secretion of IFN-γ and further activating the NF-κB signal pathway. However, IL-12 downregulates the expression of B7-H1 in THP-1-derived macrophages, associated with a lack of IFN-γ and inhibition of expression of IL-10.


Subject(s)
B7-H1 Antigen/biosynthesis , Interferon-gamma/pharmacology , Interleukin-12/pharmacology , Ovarian Neoplasms/immunology , Transcription Factor RelA/metabolism , B7-H1 Antigen/immunology , Cell Line, Tumor , Enzyme Activation/immunology , Female , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-10/biosynthesis , Interleukin-10/immunology , Interleukin-12/genetics , Interleukin-12/immunology , Macrophages/immunology , Macrophages/metabolism , Ovarian Neoplasms/pathology , Transcription Factor RelA/antagonists & inhibitors , Tumor Escape/immunology
15.
Int J Oncol ; 45(2): 619-28, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24819045

ABSTRACT

Long non-coding RNAs (lncRNAs) have been shown to play a critical role in cancer biology and are frequently aberrantly expressed. Despite their important role in pathology, little is known mechanistically regarding their role in gastric cancer (GC) pathogenesis. To characterize the role of lncRNAs in GC pathogenesis, 8 paired human GC tissue samples and matched adjacent normal tissue were examined. Large scale expression profiling of lncRNA and mRNA was performed utilizing microarray technology and validated by qPCR. Differentially expressed lncRNAs were subjected to bioinformatic analysis to predict target genes, followed by the integration of differentially expressed mRNA data and GO and network analysis to further characterize potential interactions. In our study, 2,621 lncRNAs and 3,121 mRNAs were identified to be differentially expressed (≥2.0-fold change) in GC samples relative to their matched counterparts. lncRNA target prediction revealed the presence of 221 potential lncRNA-mRNA target pairs for the 75 differentially expressed lncRNAs and 60 differentially expressed genes. KEGG pathway analysis showed that these target genes were significantly enriched in 7 different pathways, of which the p53 signaling pathway was the most significant and has been previously implicated in GC pathogenesis. Construction of a lncRNA-mRNA correlation network revealed 10 differentially expressed lncRNAs potentially regulating the p53 signaling pathway. Overall, this is the first study perform global expression profiling of lncRNAs and mRNAs relating to GC. These results may provide important information for further insights into the pathogenesis of GC and provide potential targets for future therapeutics.


Subject(s)
Adenocarcinoma/genetics , RNA, Long Noncoding/analysis , RNA, Messenger/analysis , Stomach Neoplasms/genetics , Transcriptome , Female , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction
16.
Int J Mol Med ; 32(5): 1115-25, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24071828

ABSTRACT

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and is associated with numerous deleterious phenotypes, including cognitive impairment, childhood leukemia and immune defects. Five Hsa21­derived microRNAs (i.e., hsa-miR-99a, let-7c, miR-125b-2, miR-155 and miR-802) are involved in variable phenotypes of DS. However, the changes involved in the genome-wide microRNA expression of DS fetuses under the influence of trisomy 21 have yet to be determined. To investigate the expression characteristic of microRNAs during the development of DS fetuses and identify whether another microRNA gene resides in the Hsa21, Illumina high-throughput sequencing technology was employed to comprehensively characterize the microRNA expression profiles of the DS and normal fetal cord blood mononuclear cells (CBMCs). In total, 149 of 395 identified microRNAs were significantly differentially expressed (fold change >2.0 and P<0.001) and 2 of 181 candidate novel microRNAs were identified as residing within the ̔DS critical region̓ of human chromosome 21 (chr21q22.2­22.3). Additionally, 7 of 14 Hsa21-derived microRNAs were detected, although not all seven were overexpressed in DS CBMCs compared with the control. Gene ontology enrichment analyses revealed that a set of abnormally expressed microRNAs were involved in the regulation of transcription, gene expression, cellular biosynthetic process and nucleic acid metabolic process. Significantly, most of the mRNA targets in these categories were associated with immune modulation (i.e., SOD1, MXD4, PBX1, BCLAF1 and FOXO1). Findings of the present study provided a considerable insight into understanding the expression characteristic of microRNAs in the DS fetal CBMCs. To the best of our knowledge, this is the first study to examine genome-wide microRNA expression profiles in the DS fetus. Differentially expressed microRNAs may be involved in hemopoietic abnormalities and the immune defects of DS fetuses and newborns.


Subject(s)
Down Syndrome/genetics , RNA, Messenger/genetics , Sequence Analysis, RNA/methods , Down Syndrome/diagnosis , Female , Fetus/metabolism , Fetus/pathology , Humans , Pregnancy
17.
Gene ; 530(2): 278-86, 2013 Nov 10.
Article in English | MEDLINE | ID: mdl-23933415

ABSTRACT

Given the important roles of miRNAs in post-transcriptional regulation and its implications for the development of immune tissues and cells, characterization of miRNAs promotes us to uncover the molecular mechanisms underlying the pathway of trisomic chromosome 21 that disrupts the disomic genes expression and immunological defects related to Down syndrome (DS). In the present study, we analyzed global changes and chromosome distribution characteristics of miRNAs expression in lymphocytes from children with trisomy 21 by means of the Illumina high-throughput sequencing technology. Two small libraries were constructed using pool RNA of normal and DS children. The results have been further validated by stem-loop quantitative RT-PCR. Comparison between DS and normal profiles revealed that most of identified miRNAs were expressed at similar levels. The chromosome 21 that contributes to the abundantly expressed miRNAs was small, and not all Hsa21-derived miRNAs were over-expressed with ratios significantly ≥ 1.5 in Down syndrome children lymphocytes. Based on the deep sequencing technology, 108 novel candidate miRNAs have been identified, and 2 of them were derived from human chromosome 21. For the 114 significantly differentially expressed miRNAs, function annotation of target genes indicated that a set of highly abundantly and significantly differentially expressed miRNAs were involved in hematopoietic or lymphoid organ development, thymus development, and T/B cell differentiation and activation. Our results indicated that these abnormally expressed miRNAs might be associated with the mechanisms that trisomy 21 results in dysregulation of disomic genes and involved in the immunological defects seen in DS.


Subject(s)
B-Lymphocytes/metabolism , Chromosomes, Human, Pair 21/genetics , Down Syndrome/genetics , MicroRNAs/genetics , T-Lymphocytes/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Base Sequence , Case-Control Studies , Cell Differentiation , Child , Child, Preschool , Chromosome Mapping , Chromosomes, Human, Pair 21/immunology , Down Syndrome/immunology , Down Syndrome/pathology , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , Immunity, Innate/genetics , MicroRNAs/immunology , Molecular Sequence Annotation , Molecular Sequence Data , T-Lymphocytes/immunology , T-Lymphocytes/pathology
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 29(6): 633-6, 2013 Jun.
Article in Chinese | MEDLINE | ID: mdl-23746249

ABSTRACT

OBJECTIVE: To observe the expression of Wnt2 in the breast cancer cells (MDA-MB-231, ZR-75-30 and MCF-7) and tissues, and analyze the correlation of Wnt2 expression with CA-153 in the sera of patients with breast cancer. METHODS: The expression of Wnt2 at both mRNA and protein levels was measured respectively by real-time fluorescent quantitative PCR and Western blotting in the three human breast cancer cell lines. In addition, the immunohistochemistry (IHC) was applied to detect the expression of Wnt2 in 5 corresponding tissues adjacent to cancer, 9 breast carcinoma in situ and 9 invasive breast cancer; ELISA was used to detect Wnt2 and electrochemiluminescence immunoassay was used to detect CA-153 in the sera of 15 normal women and 30 patients with breast cancer. Furthermore, we collected complete clinicopathological data from the participating subjects, and analyzed their relationships with serum Wnt2 levels. RESULTS: The mRNA and protein of Wnt2 were negative in the three human breast cancer cells, while Wnt2 in the epithelial and interstitial cells of breast cancer tissues was significantly higher than that in normal tissues (P<0.05). The expression levels of Wnt2 and CA-153 in the sera of patients with breast cancer were higher than those in normal women (P<0.05), and Wnt2 levels in the sera of breast cancer patients were positively correlated with serum CA-153 levels. CONCLUSION: Simultaneous tests of Wnt2 and CA-153 levels in the serum may help the diagnostic screening of breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Wnt2 Protein/metabolism , Breast/metabolism , Breast Neoplasms/blood , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression , Humans , Mucin-1/blood , RNA, Messenger/genetics , Wnt2 Protein/blood , Wnt2 Protein/genetics
19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 29(2): 113-7, 2013 Feb.
Article in Chinese | MEDLINE | ID: mdl-23388326

ABSTRACT

Objective To investigate the efficiencies of transfection and expression of human recombinant adenovirus Ad5F35-IL-12 in the different kinds of human mononuclear macrophages. Methods The human recombinant adenovirus Ad5F35-IL-12 was used to infect human peripheral blood monocytes, pleural fluid macrophages as well as THP-1, U937 monocyte cell lines and their phorbol myristate acetate (PMA)-induced macrophages. 48 h later, green fluorescence was observed under the fluorescence microscope to detect the transfection efficiency. The expressions of IL-12 double-subunits (p35, p40) mRNA were tested by RT-PCR and the level of IL-12p70 protein in the cell culture supernatant was detected with ELISA. Results The human recombinant adenovirus Ad5F35-IL-12 successfully infected the human peripheral blood monocytes, pleural fluid macrophages, THP-1 monocytes, U937 monocytes, and THP-1 and U937 macrophages induced with PMA. All above infected mononuclear macrophages effectively secreted IL-12p70 protein, and they were listed from high to low of IL-12p70 protein level as pleural fluid macrophages, U937 and THP-1 macrophages induced with PMA, U937 monocytes, human peripheral blood monocytes and THP-1 monocytes. Conclusion The human recombinant adenovirus Ad5F35-IL-12 could infect different kinds of mononuclear macrophages, and IL-12 p70 protein could be successfully expressed in cell supernatants.


Subject(s)
Adenoviridae/genetics , Arenaviridae Infections/genetics , Macrophages/virology , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-12/biosynthesis , Interleukin-12/immunology , Macrophages/immunology , Recombinant Proteins/genetics
20.
Asian Pac J Cancer Prev ; 14(12): 7339-44, 2013.
Article in English | MEDLINE | ID: mdl-24460299

ABSTRACT

To analyze the effects of a new unknown peptide DEF on the growth of tumor cells, a fused polypeptide TAT-DV1-DEF was designed and synthesized. The lung adenocarcinoma cell line GLC-82 treated with TAT- DV1-DEF was analyzed with a cell counting kit 8, and the location of polypeptides in cells was observed under laser confocal microscopy. The efficiency of polypeptide transfection and changes in nuclear morphology were analyzed by flow cytometry and fluorescence microscopy, respectively. Finally, the mechanism of tumor cell growth inhibition was evaluated by Western blotting. We found that TAT-DV1-DEF could significantly inhibit the growth of the lung adenocarcinoma cell line GLC-82, but not the normal human embryonic kidney cell line HEK-293. Polypeptides were found to be mostly localized in the cytoplasm and some mitochondria. The efficiency of polypeptide transfection in the two cell types was approximately 99%. Apoptotic nuclei were observed under fluorescence microscopy upon treatment with polypeptides and DAPI staining. Western blot analyses indicated that the polypeptide inhibition of tumor cell growth was apoptosis dependent. In the present study, we demonstrated that fused polypeptides could induce apoptosis of the lung adenocarcinoma cell line GLC-82, indicating that the new unknown peptide DEF has antitumor effects.


Subject(s)
Adenocarcinoma/pathology , Apoptosis , Lung Neoplasms/pathology , Peptide Fragments/pharmacology , Recombinant Fusion Proteins/pharmacology , Adenocarcinoma/metabolism , Blotting, Western , Cell Nucleus/metabolism , Cell Proliferation , Cells, Cultured , Cytoplasm/metabolism , Flow Cytometry , Gene Products, tat/genetics , HEK293 Cells , Humans , Lung Neoplasms/metabolism , Mitochondria/metabolism
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