NGAL基因在永生化食管上皮细胞恶性转化中过表达的研究

为研究NGAL（neutrophil gelatinase-associated lipocalin）基因在永生化食管上皮细胞恶性转化中的表达情况,以永生化食管上皮细胞系SHEE和食管癌细胞系SHEEC互为对照,用cDNA微列阵进行筛选,用RNA印迹和RT-PCR进行鉴定,cDNA克隆测序后与GenBank进行BLAST分析比较.结果表明NGAL基因在SHEEC中出现显著差异过表达,其cDNA序列与小鼠24p3、大鼠NRL（neu-related lipocalin）、人中性粒细胞NGAL和卵巢癌NGAL具有较高的相似性.这提示NGAL基因在永生化食管上皮细胞恶性转化中可能发挥着重要作用,可能是一种新的癌基因或促癌基因.     

SHEEC食管癌细胞中NGAL基因的功能

中性粒细胞明胶酶相关脂质运载蛋白 (neutrophilgelatinase associatedlipocalin ,NGAL)是脂质运载蛋白(lipocalin)家族的一个新成员 ,可能是人类的一种新的癌基因 ,但是在肿瘤中的功能不清楚。以往研究发现NGAL基因在SHEEC食管癌细胞中显著过表达 ,表明该细胞是一种用来揭示NGAL基因在肿瘤中功能的良好模型。采用反义封闭技术 ,同时结合裸鼠成瘤实验等研究了反义封闭NGAL基因转录对SHEEC食管癌细胞的浸润和分裂增殖等行为的影响。结果发现 ,反义封闭NGAL基因转录不但可以有效地降低SHEEC细胞分泌的基质金属蛋白酶 9和基质金属蛋白酶 2的活性 ,而与此同时裸鼠成瘤细胞的浸润行为也相应地受到了明显抑制 ,然而SHEEC细胞端粒的长度、拓扑异构酶II的含量以及细胞增殖指数等未发生明显变化。表明NGAL基因在食管癌细胞SHEEC中的功能可能主要是通过明胶酶在促进肿瘤细胞的浸润中发挥作用 ,而可能与肿瘤细胞分裂增殖的相关性不明显     

NGAL基因不同长度片段的克隆及其顺式作用元件定位分析

NGAL(neutrophilgelatinase-associatedlipocalin)是lipocalin家族的一个新成员,可能是人类的一种新癌基因,但其在肿瘤中的表达调控机制尚不清楚。应用生物信息学手段对NGAL(X99133,包括外显子和内含子)进行分析发现,此区域内含有许多潜在的顺式作用元件,其中主要为增强子元件。对此设计了以下实验:应用PCR技术从食管癌细胞SHEEC基因组DNA中扩增不同长度的NGAL基因片段,将其连接到PGEMT-eacy载体中进行扩增,并测序。NCBI/BLAST分析确认扩增片段为NGAL基因所有,与实验所设计相吻合。在此基础上并应用KEGG/MOTIF检索分析系统对NGAL基因的上述区段进行了顺式作用元件定位分析,结果共鉴定出5个Score值=100分的顺式作用元件位点。这为NGAL基因增强子的鉴定提供了新线索。     

微RNA-101在肿瘤中的作用

微RNA(microRNA)是内源性发挥转录后调控功能的单链小RNA,种类繁多,在细胞周期调控、凋亡调控、肿瘤的进展等方面起重要作用。近期研究发现,微RNA在功能上表现为癌基因与抑癌基因与前列腺癌、膀胱癌、肝癌、乳腺癌等肿瘤的发生、发展关系密切。EZH2基因的生物学行为与癌基因类似,微RNA-101主要通过调控EZH2的表达量和表观遗传学特征调控其功能。微RNA-101的缺失或表达下调促使组织细胞分裂、增殖,最终可能导致肿瘤的发生与浸润。因此,探寻微RNA-101的生物学作用具有重要意义。     

尿中性粒细胞明胶酶相关脂质转运蛋白在狼疮肾炎中的研究进展

中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)已被证明是肾脏疾病一种新的生物标记物。大量证据证明,系统性红斑狼疮肾炎(lupus nephritis,LN)患者尿NGAL(urinary neutrophil gelatinase-associated lipocalin,u NGAL)的特异性和灵敏度均高于抗双链DNA抗体。尽管u NGAL能否作为系统性红斑狼疮肾新生物标记物还存在争议,但临床与基础实验均显示u NGAL能促进LN的发生,且与其严重程度及临床活动性密切相关。本文就u NGAL与LN的关系进行综述。     

自噬与肿瘤

自噬（autophagy）作为细胞一种基本生物学特征,具有独特的形态学改变和特有的调控通路。近年来,自噬在关于对肿瘤的作用的研究已成为热点,在不同的种类肿瘤中,自噬扮演着不同的角色,分为促进和抑制肿瘤两种作用。自噬异常与人类恶性肿瘤的发生、发展联系紧密,其启动及调节与细胞能量代谢、微环境变化、抑癌基因及癌基因家族及复杂的信号调节等有关。清楚了解自噬的特点可为肿瘤治疗提供新的方向。     

MicroRNAs与肿瘤

microRNA(miRNA)是一类对基因具有调控功能的内源性非编码小分子RNA,通过与靶mRNA完全或不完全互补配对,引起mRNA降解或翻译抑制,从而对基因转录后水平进行调控.目前认为miRNA在多种生物学过程中起着至关重要的作用,包括细胞增殖、分化、凋亡等.研究显示miRNA的表达异常能导致疾病甚至肿瘤的发生,有类似于抑癌基因或癌基因的功能.本文就miRNA在肿瘤发生和诊断方面的研究进展作一综述.     

MicroRNA与肿瘤相关的信号转导通路

信号转导通路在细胞代谢、生长、增殖、应激、发育和凋亡等生命活动中具有极为重要的作用。干扰这些通路将可能影响细胞的正常发育, 甚至导致肿瘤。MicroRNA(miRNA)是近年来在真核生物中发现的、在转录后水平负调节基因表达的一类长度约22个核苷酸的非编码小RNA, 其靶基因数目众多, 生物学功能广泛。在多种肿瘤中发现了miRNA的异常表达, 提示后者与肿瘤发生有关, 可能机制为调控癌基因或肿瘤抑制基因的表达。此外亦发现miRNA的靶基因有许多作用于肿瘤相关的信号转导通路。miRNA在肿瘤发生过程中的重要调控功能预示其将成为人类癌症诊断和治疗方面的新星。     

陷窝蛋白-1在肿瘤发生发展中的双重性

陷窝蛋白-1(caveolin-1)是陷窝(caveolae)的主要结构成分,在细胞内吞、胆固醇运输、信号传导、肿瘤发生中发挥重要作用。陷窝蛋白-1在肿瘤中发挥抑癌作用还是促癌作用一直存在争论:在乳腺癌、肺癌、卵巢癌中发挥抑癌基因样作用,而在前列腺癌中则发挥癌基因样作用。这一现象提示,陷窝蛋白-1在不同肿瘤中发挥作用可能不同,其生物学作用具有双重性。本文将对陷窝蛋白-1的结构、分布、表达及与肿瘤的关系作一综述。     

LZTS2 抑癌基因在肿瘤中的作用机制

亮氨酸拉链肿瘤抑制因子2(leucine zipper tumor suppressor 2,Lzts2)是一种新发现的抑癌基因,位于人类染色体10q24.3,在多种肿瘤中异常表达,且对肿瘤的发生、增殖和迁移中均发挥重要作用。LZTS2拥有亮氨酸拉链结构域(LZ),可以通过该结构域结合DNA调节基因转录。LZTS2还可以参与Wnt/β-catenin信号通路调控β-catenin的表达及细胞内分布,与NF-κB亦拥有广泛的相互作用,从而调控细胞增殖和凋亡。在微管系统中,LZTS2也有重要的调节作用,可抑制细胞的有丝分裂和迁移,增加肿瘤细胞的药物敏感性。LZTS2作为新的抑癌基因,与肿瘤的大小和淋巴结转移等生物学特性密切相关,可能成为新的诊断标志物,判断肿瘤的预后,亦可能为肿瘤的基因治疗潜在方向。然而关于LZTS2及其作用机制的研究尚为不足,本文将对LZTS2在肿瘤中的作用机制作简要综述。     

Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.     

Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin

Neutrophil gelatinase associated lipocalin (NGAL), a constituent of neutrophil granules, is a member of the lipocalin family of binding proteins. NGAL can also be highly induced in epithelial cells in both inflammatory and neoplastic colorectal disease. NGAL is proposed to mediate inflammatory responses by sequestering neutrophil chemoattractants, particularly N-formylated tripeptides and possibly leukotriene B(4) and platelet activating factor. The crystal structures of NGAL display a typical lipocalin fold, albeit with an unusually large and atypically polar binding site, or calyx. The fold of NGAL is most similar to the epididymal retinoic acid-binding protein, another lipocalin, though the overall architecture of the calyces are very different. The crystal structures also reveal either sulfate ions or an adventitiously copurified fatty acid bound in the binding site. Neither ligand is displaced by added N-formylated tripeptides. The size, shape, and character of the NGAL calyx, as well as the low relative affinity for N-formylated tripeptides, suggest that neither the copurified fatty acid nor any of the proposed ligands are likely to be the preferred ligand of this protein. Comparisons between the crystal structures and the recently reported solution structure of NGAL reveal significant differences, in terms of both the details of the structure and the overall flexibility of the fold.     

The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer

Neutrophil gelatinase associated lipocalin (NGAL), also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2, is a 24kDa secreted glycoprotein originally purified from a culture of mouse kidney cells infected with simian virus 40 (SV-40). Subsequent investigations have revealed that it is a member of the lipocalin family of proteins that transport small, hydrophobic ligands. Since then, NGAL expression has been reported in several normal tissues where it serves to provide protection against bacterial infection and modulate oxidative stress. Its expression is also dysregulated in several benign and malignant diseases. Its small size, secreted nature and relative stability have led to it being investigated as a diagnostic and prognostic biomarker in numerous diseases including inflammation and cancer. Functional studies, conducted primarily on lipocalin 2 (Lcn2), the mouse homologue of human NGAL have revealed that Lcn2 has a strong affinity for iron complexed to both bacterial siderophores (iron-binding proteins) and certain human proteins like norepinephrine. By sequestering iron-laden siderophores, Lcn2 deprives bacteria of a vital nutrient and thus inhibits their growth (bacteriostatic effect). In malignant cells, its proposed functions range from inhibiting apoptosis (in thyroid cancer cells), invasion and angiogenesis (in pancreatic cancer) to increasing proliferation and metastasis (in breast and colon cancer). Ectopic expression of Lcn2 also promotes BCR-ABL induced chronic myelogenous leukemia in murine models. By transporting iron into and out of the cell, NGAL also regulates iron responsive genes. Further, it stabilizes the proteolytic enzyme matrix metalloprotease-9 (MMP-9) by forming a complex with it, and thereby prevents its autodegradation. The factors regulating NGAL expression are numerous and range from pro-inflammatory cytokines like interleukins, tumor necrosis factor-α and interferons to vitamins like retinoic acid. The purpose of this review article is to examine the expression, structure, regulation and biological role of NGAL and critically assess its potential as a novel diagnostic and prognostic marker in both benign and malignant human diseases.     

Neutrophil Gelatinase-associated Lipocalin in Normal and Neoplastic Human Tissues. Cell Type-specific Pattern of Expression

Neutrophil gelatinase-associated lipocalin (NGAL) has recently been identified in myeloperoxidase-negative neutrophil granules. Members of the lipocalin family are thought to bind and transport small lipophilic molecules such as retinoids and roles in cell regulation have been proposed. Recently, NGAL has also been demonstrated in the colonic mucosa in certain pathologic conditions.The aim of this study was to examine the distribution of NGAL in normal and neoplastic tissues by immunohistochemistry. Interestingly, NGAL was found in a variety of normal and pathological human tissues. A cell type-specific pattern of expression was seen in bronchus, stomach, small intestine, pancreas, kidney, prostate gland, and thymus. The comparative analysis of the putative rat homologue neu-related lipocalin showed a very similar pattern of expression with the exception of pancreas and kidney. Neoplastic human tissues showed a very heterogeneous expression of NGAL protein. High NGAL levels were found in adenocarcinomas of lung, colon and pancreas. In contrast, renal cell carcinomas of various subtypes and prostate cancers contained low NGAL levels. Lymphomas and thymic tumours were negative for NGAL immuno-labeling. Knowledge about the location of NGAL in normal cells and in disease states provides the first clues towards understanding its biological function.     

The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition

First identified as a neutrophil granule component, neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, or neu-related lipocalin) is a member of the lipocalin family of binding proteins. Putative NGAL ligands, including neutrophil chemotactic agents such as N-formylated tripeptides, have all been refuted by recent biochemical and structural results. NGAL has subsequently been implicated in diverse cellular processes, but without a characterized ligand, the molecular basis of these functions remained mysterious. Here we report that NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions. We therefore propose that NGAL participates in the antibacterial iron depletion strategy of the innate immune system.     

A novel alternative spliced variant of neutrophil gelatinase-associated lipocalin receptor in oesophageal carcinoma cells

Recent studies suggest that NGAL (neutrophil gelatinase-associated lipocalin) is a novel iron transporter with functions distinct from that of transferrin and mediates a new iron-delivery pathway. To get a better understanding of NGAL function in oesophageal carcinoma, we analysed the expression of NGAL receptors in oesophageal carcinoma cells and identified a novel spliced variant designated NgalR-3. When expressed in a heterologous system, the protein produced from this novel spliced variant exhibits the biochemical characteristics of interaction and co-localization with NGAL protein in vivo. This new finding suggests that NgalR-3 may act as a potential NGAL receptor and play a role in NGAL-mediated iron transport in oesophageal carcinoma.     

Neutrophil Gelatinase-associated Lipocalin, a Siderophore-binding Eukaryotic Protein

NGAL (neutrophil gelatinase-associated lipocalin) also known as lcn2 or siderochalin is constitutively expressed in myelocytes and stored in specific granules of neutrophils. It is highly induced in a variety of epithelial cells during inflammation. Analysis of the crystal structure of NGAL expressed in E.coli showed that NGAL has the ability to bind catecholate type siderophores and in this way prevent bacteria from acquisition of siderophore-bound iron. NGAL (or 24p3 as the highly homologous murine orthologue is named) knock out mice have a profound defect in defense against E.coli after intraperitoneal injection. This defect can be mimicked in wild-type mice by providing siderophore iron, which cannot be sequestered by NGAL, testifying to the specific role of NGAL as a siderophore binding protein in innate immunity. Megalin, a scavenger receptor functions as a receptor for NGAL and mediates uptake into endosomes, but other NGAL receptors are likely to exist.     

Structural characterization of glycoprotein NGAL, an early predictive biomarker for acute kidney injury

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising new renal biomarker that can reduce the time to diagnose acute kidney injury (AKI). There is little information available about complex glycans on NGAL. Detailed structural characterization of NGAL is necessary to understand the structural variability of NGAL used as a standard in the NGAL immunoassay. This study demonstrated that 7-9% of mutant (C87S) recombinant NGAL was N-glycosylated and no O-glycosylation was detected. The NGAL sequence was confirmed by nanoLC/MS/MS following in gel and in solution trypsin digestion, and the N-glycosylation site was localized by MS/MS. Six different mutant recombinant NGAL samples (samples A-F) were analyzed in this study; however, these samples demonstrated two different glycan patterns. Forty-one N-glycans were detected in sample A and the more abundant N-glycans were unsialylated. Forty-three N-glycans were detected in sample F and the more abundant N-glycans were sialylated. Each of the other four samples (B-E) had a similar N-glycan pattern as sample F.     

Neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinases as novel stress markers in children and young adults on chronic dialysis

Phenomena related to chronic kidney disease, such as atherosclerosis, aggravate with the introduction of dialysis. Matrix metalloproteinases (MMP) and factors modifying their activity, such as their tissue inhibitors (TIMP) or neutrophil gelatinase-associated lipocalin (NGAL), take part in the matrix turnover and the endothelial damage characteristic for atherogenesis. However, there are no data on the associations between these parameters and other known pro-atherogenic factors, or on the impact of various dialysis modalities on them. The aim of our study was to assess the serum concentrations of NGAL, MMP-7, MMP-9, and TIMP-1, as well as their correlations with human heat shock proteins (Hsp90α, anti-Hsp60), endothelial dysfunction (sE-selectin), and inflammation (hsCRP) in pediatric patients chronically dialyzed. Twenty-two children on automated peritoneal dialysis (APD), 17 patients on hemodialysis (HD) and 24 controls were examined. The serum concentrations of NGAL, MMP-7, MMP-9, TIMP-1, Hsp90α, anti-Hsp60, and sE-selectin were assessed by enzyme-linked immunosorbent assay (ELISA). The median values of NGAL, MMP-7, MMP-9, TIMP-1, and MMP-9/NGAL ratio were significantly elevated in all dialyzed children vs. controls and were higher in HD than in APD. The values of MMP-9/TIMP-1 and MMP-7/TIMP-1 ratios in the HD subjects were lower than those in the APD children. Hsp90α and anti-Hsp60 predicted the values of NGAL, MMPs, and TIMP-1. Additionally, sE-selectin was a predictor of NGAL levels, whereas NGAL predicted the MMP and TIMP-1 concentrations. The increased concentrations of examined parameters indicate the dysfunction of MMP/TIMP/NGAL system in the dialyzed children, more pronounced on hemodialysis. The discrepancies between dialysis modalities and correlations with heat shock proteins (HSPs) suggest that NGAL may be considered a novel stress protein, whereas MMP-7, MMP-9, and TIMP-1 may be regarded as indicators of stress response in the pediatric population on chronic dialysis.