Suppression of Heme Oxygenase-1 mRNA Expression by Interferon-γ in Human Glioblastoma Cells

Heme oxygenase is a rate-limiting enzyme in heme catabolism that cleaves heme to form biliverdin, carbon monoxide, and iron. Heme oxygenase-1 is an inducible isozyme and is expressed in many types of cells and tissues. Large amounts of these heme degradation products may be noxious to the host, especially in the brain. We therefore searched for the factors that suppress the expression of heme oxygenase-1. Northern blot analysis showed that treatment with interferon-gamma and with interleukin-1beta for 24 h decreased the expression levels of heme oxygenase-1 mRNA to approximately 20 and approximately 50% of the control levels, respectively, in a human glioblastoma cell line, T98G. Treatment with a combination of these two cytokines additively decreased the expression levels of heme oxygenase-1 mRNA. Western blot analysis showed that the expression level of heme oxygenase-1 protein was also decreased by treatment with interferon-gamma, but not with interleukin-1beta. Moreover, pretreatment with interferon-gamma partially suppressed the induction of heme oxygenase-1 mRNA expression caused by either sodium nitroprusside, cadmium, or hemin. These findings raise the possibility that the expression of heme oxygenase-1 is down-regulated by interferon-gamma in the nervous system.     

Down-Regulation of MicroRNA-223 Promotes Degranulation via the PI3K/Akt Pathway by Targeting IGF-1R in Mast Cells

Background

Mast cells play a central role in allergic and inflammatory disorders by inducing degranulation and inflammatory mediator release. Recent reports have shown that miRNAs play an important role in inflammatory response regulation. Therefore, the role of miR-223 in mast cells was investigated.

Methods

The expression of miR-223 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) in immunoglobulin E (IgE)-mediated mast cells. After successful miR-223 inhibition by transfection, degranulation was detected in IgE-mediated mast cells. The phosphorylation of IκB-α and Akt were examined using western blotting. NF-κB was tested using electrophoretic mobility shift assay. PI3K-inhibitor (LY294002) was used to investigate whether the PI3K/Akt pathway was essential for mast cell activation. The TargetScan database and a luciferase reporter system were used to identify whether insulin-like growth factor 1 receptor (IGF-1R) is a direct target of miR-223.

Results

MiR-223 expression was up-regulated in IgE-mediated mast cells, whereas its down-regulation promoted mast cell degranulation. Levels of IκB-α and Akt phosphorylation as well as NF-κB were increased in miR-223 inhibitor cells. LY294002 could block the PI3K/Akt signaling pathway and rescue the promotion caused by suppressing miR-223 in mast cells. IGF-1R was identified as a direct target of miR-223.

Conclusions

These findings suggest that down-regulation of miR-223 promotes degranulation via the PI3K/Akt pathway by targeting IGF-1R in mast cells.     

尿路上皮癌胚抗原1（UCA1）在人膀胱癌组织及膀胱癌细胞株中特异表达

UCA1（urothelial carcinoma antigen 1）为自主研发的1个尿路上皮癌基因.应用 实时荧光定量PCR检测UCA1 mRNA在2种膀胱癌细胞系、11种非膀胱癌细胞系、18对膀胱 癌组织和配对癌旁正常膀胱组织中的表达,对表达差异进行统计学分析.结果显示, UCA1在2种膀胱癌细胞系中显著高表达,而在其他11种非膀胱癌细胞系中表达水平很低 或者不表达,二者表达差异达14~24 812倍,差异有统计学意义（P<0.001）;在18例膀 胱癌组织中,UCA1的平均表达水平是癌旁正常膀胱组织的12.4倍,表达差异有统计学意 义（P<0.001）. 实时荧光定量PCR使UCA1在膀胱癌细胞系及组织中的特异性高表达得以 量化.实验结果明确了UCA1作为潜在的肿瘤标记物在膀胱癌临床诊断中的意义,为定量 检测尿液UCA1表达并确定诊断膀胱癌的参考值打下基础.     

Arsenic Trioxide (ATO) Influences the Gene Expression of Metallothioneins in Human Glioblastoma Cells

Arsenic trioxide (As₂O₃; ATO, TRISENOX?) is used to treat patients with refractory or relapsed acute promyelocytic leukaemia while its application for treatment of solid cancers like glioblastoma is still under evaluation. In the present study, we investigated the interaction of arsenic trioxide with metallothionein (MT) isoforms as a possible (protective response) resistance of glioblastoma cells to arsenic-induced cytotoxicity. Special attention was focused on MT3, the isoform expressed mainly in the brain. MT3 has low metal inducibility, fast metal binding/releasing properties and outstanding neuronal inhibitory activity. The human astrocytoma (glioblastoma) cell line U87 MG was treated with 0.6, 2 and 6?C7???M arsenic (equivalent to 0.3, 1 and 3?C3.5???M As₂O₃) for 12, 24 or 48?h and gene expression for different MT isoforms, namely MT2A, MT1A, MT1F, MT1X, MT1E and MT3, was measured by real time qPCR using SYBR Green I and Taqman? gene expression assays. TfR, 18S rRNA, GAPDH and AB were tested as reference genes, and the last two evaluated to be appropriate in conditions of low (GAPDH) and high (AB) arsenic exposure. The gene expression of MT3 gene was additionally tested and confirmed by restriction enzyme analysis with PvuII. In the given conditions the mRNAs of six MT isoforms were identified in human glioblastoma cell line U87 MG. Depending on arsenic exposure conditions, an increase or decrease of MT gene expression was observed for each isoform, with the highest increase for isoforms MT1X, MT1F and MT2A mRNA (up to 13-fold) and more persistent decreases for MT1A, MT1E and MT3 mRNA. Despite the common assumption of the noninducibility of MT3, the evident MT3 mRNA increase was observed during high As exposure (up to 4-fold). In conclusion, our results clearly demonstrate the influence of As on MT isoform gene expression. The MT1X, MT1F and MT2A increase could represent brain tumour acquired resistance to As cytotoxicity while the MT3 increase is more enigmatic, with its possible involvement in arsenic-related induction of type II cell death.     

Expression of an Antigen Associated with Basal Bodies of Human Ciliated Epithelial Cells

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000–45,000Mr protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9+0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.     

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background

The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells.

Methodology/Principal Findings

Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity.

Conclusions/Significance

We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.     

逆转录病毒介导的siRNA对宫颈癌细胞CXCR4表达的抑制

目的：观察逆转录病毒介导RNA干扰抑制宫颈癌Caski细胞CXCR4基因表达的效率。方法：人工合成CXCR4特异性小干扰RNA(small interfering RNA,siRNA)片段,装入带有绿色荧光蛋白(GFP)的逆转录病毒载体pSOS,先转染PT67细胞包装成病毒,收获病毒上清,再将其转染Caski细胞,采用实时定量PCR和Western blotting观察CXCR4表达受抑的情况。结果：成功构建pSOS-CXCR4载体,并发现在24、48和72h,CXCR4 mRNA的抑制率分别为29.9%,56.8%和62.8%,CXCR4蛋白的抑制率分别为43.6%,49.6%和62.9%。结论：逆转录病毒介导RNA干扰能有效抑制宫颈癌细胞CXCR4表达。     

IGF-1及IGF-1R在胃癌中的表达及意义

目的探讨胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)及胰岛素样生长因子1受体(insulin-like growth factor-1receptor,IGF-1R)在胃癌及癌旁胃黏膜组织中的表达及意义。方法采用免疫组化MaxVision两步法检测80例胃癌和50例癌旁胃黏膜组织中IGF-1及IGF-1R蛋白的表达,并分析两者与胃癌患者临床病理指标间的关系及其相关性。结果 IGF-1及IGF-1R蛋白在胃癌组织中表达阳性率分别为71.25%和75.00%,在癌旁胃黏膜组织中表达阳性率分别为30.00%和24.00%,差异有统计学意义(Z=-4.942,P0.001;Z=-5.688,P0.001)。IGF-1及IGF-1R蛋白的表达与胃癌的组织分化程度、有无淋巴结转移、浸润深度及临床分期相关(Z=-2.067、-2.837,P0.05;Z=-4.117、-3.579,P0.05;Z=-2.885、-2.836,P0.05;Z=-3.286、-3.313,P0.05)。相关性分析显示两者表达呈正相关。结论 IGF-1和IGF-1R蛋白在胃癌组织中呈高表达,两者可能在胃癌的发生、发展过程中具有协同及相互调节的作用。IGF-1和IGF-1R蛋白可能成为胃癌早期诊断、评估胃癌患者预后的重要指标。     

IGF-1通过SRF结合位点调节SMYD1在C2C12细胞中的表达

SMYD1是组蛋白甲基转移酶,在骨骼肌和心肌中特异表达,是调节心肌和骨骼肌发育的关键因子.虽然SMYD1的生物学功能比较清楚,但细胞外因子调节SMYD1基因表达的机制还没有报导.IGF-1能促进心肌和骨骼肌的发育、加速肌肉的损伤修复过程.通过Western印迹发现,在用IGF-1处理的C2C12细胞中,SMYD1的表达水平随处理时间逐步升高,SRF蛋白和Myogenin的表达也呈现类似的趋势.通过构建不同长度的SMYD1基因启动子荧光素酶报告基因载体,发现SMYD1基因启动子上IGF-1的应答区域位于-620～-110 bp;EMSA实验表明,SRF结合在SMYD1启动子的CArG位点,而IGF-1则能促进SRF与SMYD1启动子的结合;若将启动子上的CArG元件突变,IGF-1对SMYD1启动子的激活效应被削弱.可见IGF-1能够上调SMYD1在C2C12细胞中的表达,并且这种调控作用是部分通过调节SRF与SMYD1启动子上CArG位点的结合而实现的.此外,通过荧光素酶报告基因分析,发现SMYD1能够激活肌肉标志因子肌肉肌酸激酶(MCK)基因活性,而且与MyoD基因存在协同激活效应.因此,SMYD1可能是IGF-1的下游靶基因,SMYD1可能通过与MyoD协同作用,促进肌肉的分化。     

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Background

Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.

Methodology/Principal Findings

We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.

Conclusions/Significance

Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.     

Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression

Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowett (nude) rats has been established as a representative animal model for human GBMs, with a tumor take rate close to 100%. In immunocompetent littermates however, primary human GBM tissue is invariably rejected. Here we show that after repeated passaging cycles in nude rats, human GBM spheroids are enabled to grow in the brain of immunocompetent rats. In case of engraftment, xenografts in immunocompetent rats grow progressively and host leukocytes fail to enter the tumor bed, similar to what is seen in nude animals. In contrast, rejection is associated with massive infiltration of the tumor bed by leukocytes, predominantly ED1+ microglia/macrophages, CD4+ T helper cells and CD8+ effector cells, and correlates with elevated serum levels of pro-inflammatory cytokines IL-1β, IL-18 and TNF-α. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo passaging cycles, characterized by striking attenuation of microglial infiltration. Furthermore, tumor-derived chemokines that promote leukocyte migration and their entry into the CNS such as CXCL-10 and CXCL-12 are down-regulated, and the levels of TGF-β2 increase. We propose that through serial in vivo passaging in nude rats, human GBM cells learn to avoid and or/ suppress host immunity. Such adapted GBM cells are in turn able to engraft in immunocompetent rats without signs of an inflammatory response.     

Knockdown of FRAT1 Expression by RNA Interference Inhibits Human Glioblastoma Cell Growth,Migration and Invasion

Background

FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades.

Methods

In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi.

Results

FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice.

Conclusions

Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.     

miR-195通过下调IGF-1R的表达抑制胶质母细胞瘤的增殖和迁移

目的:探讨miR-195对胶质母细胞瘤(Glioblastoma,GBM)增殖和迁移的影响,并阐明其分子调控机制。方法:采用qRT-PCR检测不同级别胶质瘤中miR-195的表达。将miR-195转染至胶质瘤U251细胞后,应用qRT-PCR验证转染效率,MTT及划痕实验检测U251细胞的增殖及迁移能力的改变,qRT-PCR及Western blot检测胰岛素样生长因子1受体(Insulin-like growth factor 1receptor, IGF-1R)的mRNA和蛋白表达;利用质粒转染过表达miR-195后,同时过表达IGF-1R,再应用MTT及划痕实验检测U251细胞的增殖及迁移能力的变化。结果:随着胶质瘤级别的增加,miR-195的表达逐渐降低,各级别胶质瘤中miR-195的表达差异有统计学意义(P0.05)。体外转染miR-195至U251细胞24、48、72 h后,转染组细胞活力和迁移能力均较对照组显著降低(P0.05),细胞中IGF-1R的mRNA和蛋白的表达也明显减少(P0.05);通过转染IGF-1R过表达质粒可显著逆转miR-195过表达对U251细胞增殖及迁移的抑制作用。结论:miR-195可能通过下调IGF-1R的表达,进而抑制胶质母细胞瘤的增殖和迁移。     

Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

Marginal zone (MZ) B cells, identified as surface (s)IgM^highsIgD^lowCD23^low/−CD21⁺CD38⁻ B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27⁺ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.     

Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNP^IGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNP^TD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNP^IGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNP^IGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.