GP73与消化系统疾病的研究进展

高尔基体是一个非常重要的细胞器,最近研究表明,它除了蛋白加工外,还能参与细胞分化、细胞间信号传导和细胞凋亡,其功能障碍也许和疾病的发生、发展有着某种联系。随着蛋白组学技术的发展,使得筛选新的肿瘤标志物成为可能,其中高尔基体蛋白73（GP73）被认为是最值得期待的血清标志物之一,尤其是早期肝癌的血清标志物。本文对近年来有关GP73结构、表达分布以与及消化系统疾病的研究进展进行综述。     

高尔基体蛋白73与肝癌诊断

肝癌是我国最常见的恶性肿瘤之一,血清甲胎蛋白(α-fetoprotein,AFP)检查是目前肝癌诊断最常采用的手段,但血清AFP对肝癌诊断的灵敏度和特异性均不理想,因此,临床上迫切需要更理想的肝癌标志物。高尔基体蛋白73(Golgi protein 73,GP73)是一种II型高尔基体跨膜糖蛋白,与肝炎、肝硬化和肝癌等肝病的发生、发展密切相关,GP73对肝癌诊断的灵敏度和特异性为65%和90%,而核心岩藻糖化GP73对肝癌诊断的灵敏度和特异性则高达90%和100%。GP73有可能成为一种更理想的肝癌临床诊断标志物,而核心岩藻糖化的GP73在肝癌的早期诊断中可能具有更重要的意义。     

GP73及其相关miRNA在肝癌中的研究进展

高尔基体蛋白73 (Golgi protein 73, GP73)是位于顺式高尔基体膜上的糖基化跨膜蛋白,其在肿瘤的发展进程中具有重要作用,是肿瘤治疗的潜在靶标。目前研究表明, GP73可作为辅助诊断肝细胞癌(hepatocellular carcinoma, HCC)的血清学标志物。随着GP73的深入研究,与GP73有关的微RNA (microRNA, mi RNA)也逐渐被挖掘出来。GP73相关mi RNA与多种肿瘤的发生、发展密切相关,其中mi R-212、mi R-27a等mi RNA能抑制HCC的侵袭及转移, GP73与mi R-27b、mi R-493-5p等能作为HCC患者预后的生物标志物。因此, GP73相关mi RNA用于肝癌的诊治是有前景的。本文总结了GP73及其相关mi RNA在肝癌发展中的作用以及机制,希望为肝癌的机制研究和诊疗提供思路。     

原发性肝癌患者血清高尔基蛋白73检测临床价值

目的探讨血清高尔基蛋白73(GP73)检测对原发性肝癌的临床意义。方法采用酶联免疫法检测60例健康者为正常对照组,60例肝良性肿瘤组,132例原发性肝癌治疗前组,82例原发性肝癌有效治疗后组的血清GP73水平,分析比较各组GP73水平,以及将原发性肝癌治疗前组的不同临床参数的GP73水平进行比较分析。结果正常对照组、良性肿瘤组、原发性肝癌治疗前组、原发性肝癌有效治疗后组的血清中GP73水平比较,差异有显著统计学意义(P0.01)。原发性肝癌治疗前组的GP73水平明显高于正常对照组及肝良性肿瘤组,差异有统计学意义(P0.01);原发性肝癌有效治疗后组的GP73水平明显低于原发性肝癌治疗前组(P0.01)。肝癌患者血清GP73水平与合并肝硬化及临床分期有关(P0.01)。结论 GP73作为一种新的血清肿瘤标志物,对原发性肝癌的早期诊断、疗效监测、临床分期均具有重要的临床意义。     

高尔基体磷酸化蛋白GOLPH2的研究及其潜在的应用价值

高尔基体磷酸化蛋白（Golgi phosphoprotein2,GOLPH2又称GP73）是一种11型高尔基体跨膜糖蛋白,与肝炎、肝硬化和肝癌等的发生发展密切相关。GP73是一个可靠的诊断HCC及监测复发的血清标记物,其敏感性和特异性均高于传统的AFP。GP73在目前的肝癌早期诊断和治疗中的显示出重要的作用,有可能成为临床上新的肝癌血清标记物。而且部分研究发现GP73对肝癌的转移潜能和预后有重要的预测价值,抑制其表达可显著降低肝癌细胞的恶性表型,这似乎提示GP73可以成为肝癌治疗的新靶点。     

GP73参与肝癌索拉菲尼获得性耐药的初步研究

目的:探讨高尔基蛋白73(GP73)是否参与调控肝癌索拉菲尼获得性耐药。方法:构建索拉菲尼获得性耐药细胞株;采用蛋白免疫印迹技术检测野生型HepG2细胞及索拉菲尼获得性耐药细胞株中GP73的表达水平;在野生型HepG2细胞中转染GP73,或索拉菲尼耐药细胞株中敲低GP73,24 h后加入不同浓度的索拉菲尼处理细胞,MTT法检测细胞存活。结果:GP73在肝癌索拉菲尼耐药细胞株中高表达;HepG2细胞中过表达GP73能够降低细胞对索拉菲尼的敏感性;耐药细胞株中敲低GP73之后能够升高细胞对索拉菲尼的敏感性。结论:GP73的高表达能够降低HepG2细胞对拉菲尼的敏感性。     

小鼠GP73蛋白真核表达质粒的构建及其对mTOR转录水平的影响

目的:构建小鼠高尔基蛋白73(m GP73)的真核表达质粒,转染HEK293T细胞株验证重组质粒活性,并检测m GP73过表达情况下对小鼠肝癌细胞中mTOR m RNA水平的影响。方法:以实验室保存的H22小鼠肝癌细胞c DNA文库为模板,采用PCR技术扩增获得m GP73序列,将其插入pc DNA3.1-Flag-vector载体构建成重组质粒,转染HEK293T细胞后用蛋白免疫印迹检测融合蛋白的表达,并用q PCR检测在GP73过表达情况下H22细胞中mTOR转录水平的变化。结果:菌液PCR、重组质粒的双酶切结果及测序均表明重组质粒构建成功,蛋白免疫印迹表明m GP73蛋白在HEK293T细胞中获得表达,q PCR结果表明在m GP73过表达时mTOR的转录水平随之上升。结论:构建了pc DNA3.1-Flag-m GP73真核表达质粒,m GP73 m RNA过表达时mTOR的m RNA水平也上升,这为进一步研究m GP73在小鼠肝脏肿瘤转移过程中的作用奠定了基础。     

转化生长因子β上调肝癌中GP73表达的分子机制

目的:研究转化生长因子β(TGF-β)在翻译水平、转录水平对肝癌细胞表达高尔基蛋白73(GP73)的影响。方法:用不同时间(0、1、2、4、8 h),不同浓度(0、1、2 ng/mL)的TGF-β1刺激转染了pcDNA3-Flag-GP73、pcDNA3-Flag-V质粒的HepG2细胞,分别用免疫印迹检测细胞内GP73蛋白水平变化,荧光定量PCR检测细胞内GP73 mRNA表达水平变化;双萤光素酶报告基因检测TGF-β1能否激活GP73基因启动子。结果:在TGF-β1的刺激下,细胞GP73蛋白及mRNA水平均明显上调;TGF-β能激活GP73启动子且使其活性增强6倍左右,但对GP73启动子突变体无作用。结论:TGF-β能在蛋白水平、mRNA水平影响GP73的表达,GP73是TGF-β作用的靶分子。     

GP73及其糖基化修饰对肝癌细胞HepG2炎症相关分子信号通路的影响

目的:通过构建高尔基体膜蛋白73(GP73)氨基酸序列109、144位糖基化位点双突变真核表达质粒,研究GP73及其糖基化修饰对肝癌细胞炎症相关分子信号通路的影响。方法:根据GP73的DNA序列,设计合成2对针对GP73氨基酸序列109、144位糖基化位点突变的PCR引物,以本实验室构建的野生型质粒pc DNA3-Flag-GP73为模板,构建GP73的109、144位糖基化位点双突变质粒pc DNA3-Flag-GP73(DM);用脂质体将此双突变质粒转染293T细胞,用糖蛋白染色和免疫印迹检测该质粒在细胞中的表达情况,用双萤光素酶报告基因实验检测GP73及其糖基化修饰对Hep G2细胞中NF-κB转录激活的影响。结果:糖蛋白染色和免疫印迹结果证实构建的双突变质粒pc DNA3-Flag-GP73(DM)能够表达GP73双糖基化位点突变的蛋白,且糖基化位点的突变使GP73的糖基化修饰完全缺失;双萤光素酶报告基因实验结果表明,野生型GP73能够激活Hep G2细胞中NF-κB的转录活性,而双糖基化位点突变会使GP73失去此激活作用。结论:构建了GP73双糖基化位点突变的真核表达质粒pc DNA3-Flag-GP73(DM)。GP73参与激活肝癌细胞炎症信号通路,糖基化修饰对于GP73发挥此作用是必不可少的。     

小鼠GP73敲低质粒的构建

目的:用p SUPER-NEO空载体构建可以敲低小鼠高尔基蛋白73(m GP73)表达的重组质粒p SUPER-m GP73-si RNA,并鉴定其功能。方法:合成靶向m GP73基因区的3条小干扰RNA(si RNA)m GP73-si RNA1、m GP73-si RNA2和m GP73-si RNA3,通过细胞实验挑选干扰效率大于60%的si RNA,用于合成能够敲低m GP73的重组质粒;重组载体经双酶切和测序鉴定正确后转染4T1细胞,检测转染质粒后细胞内m GP73的m RNA水平;将重组质粒p SUPER-m GP73-si RNA经尾部静脉注射小鼠体内,测定小鼠胃组织中m GP73的m RNA水平。结果:构建了p SUPER-m GP73-si RNA重组质粒;在转染该载体后,小鼠乳腺癌4T1细胞m GP73的m RNA表达下调43%;小鼠体内注射该载体后,胃组织中m GP73的m RNA表达受抑制率为33%。结论:构建了针对m GP73敲低的p SUPER-m GP73-si RNA质粒,可以较明显地下调鼠细胞系和小鼠胃组织m GP73的表达。     

Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption

The cis-Golgi protein GPP130 reversibly redistributes to endosomes upon pH disruption, but the identity of the endosomes and the involved cycling route are unknown. It is also unknown whether any other early Golgi proteins participate in this pathway. Here, we analyze GPP130 and the structurally related Golgi protein GP73. Unlike the TGN marker TGN38/46, GPP130 and GP73 colocalized in the early Golgi and redistributed to the ER after brefeldin A treatment. Nevertheless, after pH disruption by monensin, GPP130 and GP73 redistributed to endosomes containing redistributed TGN38/46, but not other endosomal markers. In common with TGN38/46, the redistribution involved transient appearance on the plasma membrane, and upon monensin washout, the proteins moved back to the Golgi along a microtubule- and PI3 kinase-independent route. Although GP73 did not associate with GPP130, its steady-state Golgi targeting was also mediated by a lumenal predicted coiled-coil stem domain. These findings indicate that at least two early Golgi proteins, each containing stem domain Golgi targeting determinants, cycle to the cell surface and back along the late endosome independent TGN38/46 pathway.     

Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GP73 produces marker for hepatocellular carcinoma

Serum GP73 levels are significantly increased in patients with hepatocellular carcinoma (HCC), potentially providing a marker for early detection. However, GP73 is an integral membrane protein localized to the cis Golgi and is not known to be secreted. Based on its presence in sera, we sought to determine whether GP73 might normally be released from cells and to elucidate the mechanism of this release. Indeed, a soluble form of GP73 was released from cultured cells and compared with the Golgi-localized full-length protein, the molecular weight was slightly reduced, suggesting that cleavage releases the GP73 ectodomain. Sequence analysis revealed a proprotein convertase (PC) consensus site, and, indeed, the ubiquitous PC furin was capable of cleaving purified GP73. Further, alanine substitutions in the PC site blocked both the in vitro and the in vivo cleavage of GP73. Using a cleavage-specific antibody, cleaved GP73 was found in the trans Golgi network and endosomes, suggesting that GP73 cleavage occurs as GP73 cycles distal to the early Golgi. We conclude that the endosomal trafficking of GP73 allows for PC-mediated cleavage, resulting in GP73 secretion, and provides a molecular mechanism for its presence as a serum biomarker for HCC.     

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans‐cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis‐Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.     

Golgi protein 73 and its diagnostic value in liver diseases

Golgi protein 73 (GP73, also referred to as Golph 2) with 400 amino acids is a 73 kDa transmembrane glycoprotein typically found in the cis‐Golg complex. It is primarily expressed in epithelial cells, which has been found upregulated in hepatocytes in patients suffering from both viral and non‐viral liver diseases. GP73 has drawn increasing attention for its potential application in the diagnosis of liver diseases such as hepatitis, liver cirrhosis and liver cancer. Herein, we reviewed the discovery history of GP73 and summarized studies by many groups around the world, aiming at understanding its structure, expression, function, detection methods and the relationship between GP73 and liver diseases in various settings.     

Diagnostic Value of Immunohistochemical Staining of GP73, GPC3, DCP,CD34, CD31, and Reticulin Staining in Hepatocellular Carcinoma

It has been reported that Golgi protein-73 (GP73), glypican-3 (GPC3), and des-γ-carboxy prothrombin (DCP) could serve as serum markers for the early detection of hepatocellular carcinoma (HCC). This study aimed to evaluate a panel of immunostaining markers (including GP73, GPC3, DCP, CD34, and CD31) as well as reticulin staining to distinguish HCC from the mimickers. Our results revealed that CD34 immunostaining and reticulin staining were highly sensitive for the diagnosis of HCC. A special immunoreaction pattern of GP73—a diffuse coarse-block pattern in a perinuclear region or a concentrated cluster-like or cord-like pattern in a certain part of the cytoplasm—was observed in HCC cells, in contrast to the cytoplasmic fine-granular pattern in surrounding non-tumor cells and non-malignant nodules. This coarse-block pattern correlated significantly with less differentiated HCC. In comparison, GPC3 displayed a good advantage in diagnosing well-differentiated HCC. In our study, DCP and CD31 showed little diagnostic value for HCC as an immunostaining marker. When GP73, GPC3, and CD34 were combined, the specificity improved to 96.6%. Our findings demonstrate for the first time that the immunohistochemical panel of GP73, GPC3, and CD34 as well as reticulin staining is highly specific for the pathological diagnosis of HCC.     

Diagnostic Value of the Combination of Golgi Protein 73 and Alpha-Fetoprotein in Hepatocellular Carcinoma: A Meta-Analysis

Conflicting results have been widely reported on the use of Golgi protein 73 (GP73) as a serum biomarker for diagnosing hepatocellular carcinoma (HCC). This study evaluated the accuracy of GP73, alpha-fetoprotein (AFP), and GP73 + AFP for diagnosing HCC. The meta-analysis was performed on 11 studies that were selected by means of a comprehensive systematic literature review. Summary diagnostic accuracy, meta-regression analysis for heterogeneity and publication bias, and other statistical analyses were performed using Meta-Disc (version 1.4) and Stata (version 12.0). Pooled sensitivity, specificity, and diagnostic odds ratio were 0.77 (95% CI: 0.75–0.79), 0.91 (95% CI: 0.90–0.92), and 12.49 (95% CI: 4.91–31.79) for GP73; 0.62 (95% CI: 0.60–0.64), 0.84 (95% CI: 0.83–0.85), and 11.61 (95% CI: 8.02–16.81) for AFP; and 0.87 (95% CI: 0.85–0.89), 0.85 (95% CI: 0.84–0.86), and 30.63 (95% CI: 18.10–51.84) for GP73 + AFP. The area under the curve values were 0.86, 0.84, and 0.91 for GP73, AFP, and GP73 + AFP, respectively. These results indicate that for HCC diagnosis, the accuracy of GP73 was higher than that of AFP, and that GP73 + AFP exhibited significantly higher diagnostic accuracy than did GP73 or AFP alone.     

Golgi phosphoprotein 2 (GOLPH2/GP73/GOLM1) interacts with secretory clusterin

Golgi phosphoprotein 2 (GOLPH2/GP73/GOLM1), a type-II Golgi transmembrane protein of unknown function, is up-regulated in many cancers. Its Golgi luminal domain is potentially the major functional domain. The goal of this study is to identify the proteins interacting with GOLPH2. Using secretory GOLPH2 (sGOLPH2, amino acid residues 55–401) as bait, secretory clusterin (sCLU) was identified as one interacting candidate by yeast two-hybrid screening, and the coiled-coil domain of GOLPH2 was found to be sufficient for interaction with sCLU. The interaction between GOLPH2 and sCLU was confirmed intracellularly and extracellularly. The intracellular co-localization of GOLPH2 and sCLU in Golgi was also shown. These results can help in understanding the biological and pathological significance of GOLPH2.