Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues

Purpose: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). Methods: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. Results: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. Conclusion: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.     

Profiling the potential tumor markers of pancreatic ductal adenocarcinoma using 2D-DIGE and MALDI-TOF-MS: Up-regulation of Complement C3 and alpha-2-HS-glycoprotein

Background/aimsPancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with an increasing incidence worldwide. Due to lack of early diagnosis and poor prognosis, it is rather critical to improve the early diagnosis of PDAC. A comparative proteomic method was used to analyze serum proteins to find a new potential specific marker.MethodsComparative analysis of the pancreatic peripheral blood protein profiling from 40 pancreatic cancer patients, 10 pancreatic benign tumor patients, 10 chronic pancreatitis patients and 40 cancer-free controls. The samples were carried out by 2D-differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Two up-regulated proteins were further validation by real time RT-PCR, Western blot analysis and Immunohistochemistry (IHC).ResultsWe identified fourteen differently expressed proteins in PDAC group compared with cancer-free control group, including 9 up-regulation and 5 down-regulation proteins. Increased Complement C3 and alpha-2-HS-glycoprotein (AHSG) were further confirmed by real time RT-PCR, Western blot analysis and IHC. The expressions of Complement C3 and AHSG were higher in PDAC than that in other groups.ConclusionsThese results suggest that Complement C3 and AHSG might be the potential tumor markers in PDAC screening and diagnosis. The finding of inflammation mediated factor Complement C3 revealed that inflammation might be closely related with the occurrence and development process of PDAC.     

杜氏利什曼原虫无鞭毛体表达抗原的Western blot和MALDI-TOF/TOF分析

目的鉴定杜氏利什曼原虫无鞭毛体特异表达抗原。方法培养杜氏利什曼原虫前鞭毛体并体外转化无鞭毛体,其总蛋白经2-DE电泳后以小鼠抗杜氏利什曼原虫无鞭毛体血清进行Western blot,对前鞭毛体与无鞭毛体特异表达抗原蛋白进行MALDI-TOF/TOF串联质谱鉴定。重组表达无鞭毛体特异表达抗原编码基因,以Western blot法对重组蛋白进行鉴定。结果等量的杜氏利什曼原虫前鞭毛体与无鞭毛体蛋白经2-DE电泳均可呈现680~742个蛋白点,Western blot及MALDI-TOF/TOF-MS分析甘油醛3-磷酸脱氢酶与延伸因子2为杜氏利什曼原虫前鞭毛体特异表达抗原,核苷二磷酸激酶为无鞭毛体特异表达抗原。重组核苷二磷酸激酶编码基因表达产物经Western blot证实为杜氏利什曼原虫无鞭毛体特异表达强抗原。结论杜氏利什曼原虫前鞭毛体与无鞭毛体抗原表达存在差异,核苷二磷酸激酶为杜氏利什曼原虫无鞭毛体特异表达强抗原。     

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

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

Identification and verification of transthyretin as a potential biomarker for pancreatic ductal adenocarcinoma

Purpose

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide and is difficult to detect at its early stages when treatment is most effective. Therefore, we performed a comparative proteomic study to identify new biomarkers for the detection of PDAC.

Methods

Serum samples from patients with PDAC, chronic pancreatitis and normal controls were compared using two-dimensional difference gel electrophoresis (2D-DIGE). Differentially expressed separated proteins were subsequently identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF–MS). Then, transthyretin (TTR), one of the differentially expressed proteins, was validated through real-time PCR, western blot and immunohistochemistry. Finally, enzyme-linked immunosorbent assays (ELISA) were employed to confirm the levels of transthyretin in the sera.

Results

A total of 21 protein spots showed greater than 1.5-fold changes in expression level in the sera from PDAC patients compared with the normal controls. Among the identified proteins, validation experiments verified the differential expression of transthyretin in PDAC tissue, confirming the proteomic data showing that transthyretin was significantly elevated in patients with PDAC. The ELISA results revealed that the sensitivity and specificity for TTR and CA19-9 in distinguishing PDAC patients from normal individuals were 90.5, 47.6, 66.7 and 85.7 %, respectively, and 81.0 and 85.7 % for their combination.

Conclusions

These results suggest that the level of transthyretin is elevated in patients with PDAC. In combination with CA19-9, transthyretin may provide additional information for the detection of PDAC and should be further investigated.     

大鼠酒精性肝纤维化肝非实质细胞蛋白质组学研究

目的本文研究酒精对肝非实质细胞蛋白质表达的影响,以探讨酒精性肝纤维化的发病机制。方法大鼠酒精灌胃导致其发生肝纤维化。采用James染色法检测大鼠肝脏的病理学变化,通过Percoll密度梯度离心富集肝非实质细胞,再通过二维凝胶电泳(2DE)分离非实质细胞的蛋白质,经考马斯亮蓝(G250)染色,采用液相色谱串联质谱鉴定差异表达的蛋白质,并对部分差异蛋白质采用实时定量RT-PCR和免疫印迹的方法进行验证。对于2DE胶上的蛋白质点,采用两样本t检验的方法,对于RT-PCR分析,采用Mann-Whitney U检验进行统计分析。结果建立了酒精性肝纤维化大鼠模型,通过Percoll密度梯度离心纯化的非实质细胞中淋巴细胞、Kupffer细胞和内皮细胞分别富集了1.5、3.2和3.7倍。采用二维凝胶电泳法检测到了800多个蛋白质点,检测到具有2倍以上的差异蛋白质有26个,采用LC-MS法鉴定了21个非冗余蛋白质,对其中7个蛋白质的RT-PCR分析发现:ANXA3、CES3、ATPA和NDUFV2的mRNA水平和蛋白质组研究结果一致。结论本研究鉴定了一批与酒精性肝纤维化相关的蛋白质,可能为了解酒精性肝纤维化发病机制提供一些新线索。     

胰腺癌伴糖尿病的蛋白质组学分析

目的 应用蛋白质组学方法 分析胰腺癌伴或不伴糖尿病的差异蛋白质,试图阐明糖尿病在胰腺癌发生、发展过程中的分子机制.方法 研究标本分为四组:胰腺癌组及其癌旁组织组,伴有糖尿病的胰腺癌组及其癌旁组织组,每组5份.利用二维电泳和基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS/MS)技术研究四组间的蛋白表达差异.Western印迹法对巨噬细胞加帽蛋白(CapG)在癌组织中的表达进行验证.结果 在胰腺癌伴糖尿病组,有7个蛋白表达明显上调(P＜0.05),而在其他三组间的表达则差异无统计学意义(P＞0.05),这些蛋白参与了细胞运动、能量代谢、氧化应激等,其中CapG蛋白可能参与了这类患者的癌细胞浸润及转移过程.结论 该研究初步支持糖尿病参与胰腺癌进展的假说.     

An insight into the Phlebotomus perniciosus saliva by a proteomic approach

Sand fly saliva is known to play an important role in the establishment of Leishmania spp. infection. As a consequence, identifying antigenic salivary proteins of different leishmaniasis vectors has currently become a major task in the field of anti-Leishmania vaccine development. The purpose of this work was to improve the knowledge of Phlebotomus perniciosus salivary proteins by combining two-dimensional gel electrophoresis (2DE) methodology, mass spectrometry and Western blotting (WB). Salivary protein profiles of three P. perniciosus colonies from different geographic origins in Spain were compared through SDS-PAGE, leading to a similar pattern with no qualitatively noticeable differences. A gradual increase of the protein content was significantly detected with the age of sand flies, reaching the complete salivary protein profiles at day four. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). More spots rather than protein bands (19 versus 11) were visualized by 2DE and 1DE, respectively, suggesting the presence of either protein isoforms or posttranslational modifications. Sera of mice and hamsters immunized through exposure to sand fly bites following different immunization schedules showed elevated anti-saliva IgG levels. These sera allowed the detection of 5 bands and 16 immunogenic spots in 1DE and 2DE, respectively, followed by WB. These antigens were identified by MALDITOF/TOF as SP03, SP03B, SP08, SP01, SP01B, SP04, SP04B, SP02, Phlebotomus ariasi SP16, and Phlebotomus argentipes SP13. This work is assumed to be the first attempt to establish 2DE proteomic maps of P. perniciosus saliva. All spots were identified as salivary proteins, confirming this technology as an interesting tool to improve sand fly salivary knowledge.     

特发性间质性肺炎蛋白质组学研究及Annexin Ⅱ表达验证

目的 应用蛋白质组学方法筛选出特发性间质性肺炎(idiopathic interstitial pneumonia,IIP)相关蛋白质,为阐明IIP的发病机制和预后提供理论依据.方法 收集8例通过局部麻醉小切口开胸肺活检获取的新鲜肺组织及5例远离肺部良性病变的肺组织,采用双向凝胶电泳(2-DE)分离组织总蛋白,运用Imagemaster 2D 5.0图像分析软件识别差异表达的蛋白质点,应用基质辅助电离解析飞行时间质谱获取肽质量指纹图谱,检索数据库鉴定差异表达的蛋白质点,明确其生物学功能.利用Western blot和RT-PCR技术检测差异蛋白在IIP患者肺组织和正常肺组织的表达.结果 建立双向电泳图谱,鉴定出7个表达量有明显差异的蛋白质点,包括热休克蛋白70、AnnexinⅡ和Haptoglobin等.Western blot和RT-PCR技术证实IIP组AnnexinⅡ表达下调,与2-DE结果一致.结论 建立重复性较好的IIP患者肺组织与正常肺组织双向电泳图谱,并鉴定出一些与IIP发病机制相关的蛋白质.本实验所证实的AnnexinⅡ差异表达蛋白质可能通过引起纤溶系统的失衡来参与IIP的发生和发展,从而为进一步阐明IIP的发病机制提供重要方法和新线索.     

Serological Proteomics of Gastritis: Degradation of Apolipoprotein A-I and Alpha1-Antitrypsin Is a Common Response to Inflammation Irrespective of Helicobacter pylori Infection

Proteomic technology was employed to analyze serum samples from healthy subjects (10 cases) and gastritis patients with negative and positive Helicobacter pylori (Hp) infection (15 cases each). The serum proteins were separated by two-dimensional (2-D) gel electrophoresis and analyzed by a computer-aided program. The altered proteins in expression were then identified by mass spectrometry and validated by Western blotting. Compared to those in normal control, proteins in at least six areas of 2-D gels were found to significantly increase their expression levels in both Hp-negative and Hp-positive serum samples. These proteins were identified by mass peptide fingerprinting and confirmed by Western blotting to be the truncated or cleaved protein fragments of apolipoprotein A-I and alpha-1 antitrypsin, two well-known acute-phase proteins. We conclude that the degradation or metabolization of acute-phase proteins, apolipoprotein A-I, and alpha1-antitrypsin, is a common response to gastric inflammation irrespective of Hp infection.     

Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance

AIM: To examine fibroblast activation protein (FAP) expression in pancreatic ductal adenocarcinoma (PDAC) and to analyze its relationship with the clinicopathology of PDAC.METHODS: FAP expression was examined in 134 PDAC specimens by immunohistochemistry, and in four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) by Western blotting assay. We also analyzed the association between FAP expression in PDAC cells and the clinicopathology of PDAC patients.RESULTS: The results showed that the FAP was ex-pressed in both stromal fibroblast cells (98/134, 73.1%) and carcinoma cells (102/134, 76.1%). All 4 pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 88-kDa seprase subunit were identified. Higher FAP expression in carcinoma cells was associated with tumor size (P < 0.001), fibrotic focus (P = 0.003), perineural invasion (P = 0.009) and worse clinical outcome (P = 0.0085).CONCLUSION: FAP is highly expressed in carcinoma cells and fibroblasts in PDAC tissues, and its expression is associated with desmoplasia and worse prognosis.     

Proteomic identification of tumor biomarkers associated with primary gallbladder cancer

AIM:To identify potential biomarkers of primary gallbladder cancer(PGC).METHODS:Fresh PGC,cholecystitis and normal gallbladder tissue specimens collected from 10 patients,respectively,were subjected to comparative proteomic analysis.The proteomic patterns of PGC were compared with those of cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis(2-DE).The differentially expressed proteins were then identified using a MALDI-TOF mass spectrometer(MS)and database searches.To further validate these proteins,20 samples of PGC tissues and normal tumoradjacent tissues were collected for Western blot,quantitative real-time PCR,and immunohistochemical staining assay.RESULTS:Seven differentially expressed protein spots were detected by 2-ED analysis by comparing the average maps of PGC,cholecystitis and normal gallbladder tissues.Six of the seven differentially expressed proteins were identified using MALDI-TOF MS,with three overexpressed and three underexpressed in PGC tissue.Protein levels of annexin A4(ANXA4)were significantly elevated,and heat shock protein90-beta(Hsp90β)and dynein cytoplasmic 1 heavy chain 1(Dync1h1)were decreased in PGC tissues relative to the normal tumor-adjacent tissues as shown by Western blot analysis.However,levels of actin,aortic smooth muscle and gamma-actin were unchanged.In addition,the mRNA levels of all 5 proteins showed similar changes to those of the protein levels(P<0.01).Further validation by immunohistochemical analysis showed the upregulated expression of ANXA4 and decreased expression of Hsp90βand Dync1h1 in the cytoplasm of PGC tissues relative to the normal tumoradjacent tissues.CONCLUSION:Three proteins are identified as potential biomarkers of PGC using proteomic analysis.The functions of these proteins in the carcinogenesis of PGC remain to be studied.     

晚发型阿尔茨海默病蛋白质组学中触珠蛋白的表达

目的建立晚发型阿尔茨海默病(LOAD)蛋白质组学技术体系,寻找并鉴定与LOAD发生有关的生物标记物。方法选择8例经病理证实为LOAD患者的颞叶脑皮质为LOAD组,另选择5例尸体解剖为正常老年人颞叶脑皮质为对照组。分别采用固相pH梯度双向凝胶电泳分离总蛋白质,凝胶经银染显色后,用图像分析软件进行比较分析、确定差异蛋白质点。将选取的差异蛋白质点进行胶内酶切,采用基质辅助激光解析-电离-飞行时间质谱及电喷雾电离串联质谱法进行质谱分析;搜索数据库鉴定蛋白质。结果分别获得两组脑组织双向电泳蛋白质组表达图谱,筛选并鉴定出表达有明显差异的蛋白质点11个,其中蛋白质点2触珠蛋白在LOAD组有明显上调。结论筛选并鉴定表达有明显差异的蛋白质点11个,可能成为具有临床诊断意义的LOAD潜在标记物,从而为阿尔茨海默病的早期诊断和治疗提供理论和实验依据。     

人肝细胞性肝癌组织转移相关分子的比较蛋白质组学研究

目的 应用比较蛋白质组学方法研究肝细胞性肝癌(HCC)转移相关的关键蛋白分子。方法 用双向凝胶电泳(2DE)对有转移的HCC组织和未发生转移的HCC组织总蛋白进行分离，两组间差异蛋白点用质谱和数据库搜索鉴定，并在蛋白质和mRNA水平上进一步检测验证。结果 鉴定出l6个差异表达蛋白，包括S100钙结合蛋白(S100)、热休克蛋白27(HSP27)、细胞角蛋白18(CK18)等。对在转移性HCC组织中表达显著增高的HSP27，应用western blot分析在蛋白水平上验证了2DE结果，RT-PCR检测出两组问mRNA水平也存在差异，但不显著，免疫组织化学检测显示HSP27主要定位于肝细胞胞浆内。结论 HCC转移与多种蛋白表达相关，其中HSP27高表达可能在转移中发挥作用，可能为潜在的判断HCC转移的分子标记或控制转移的治疗靶点。     

Proteomic Analysis of Pancreatic Ductal Adenocarcinoma Compared with Normal Adjacent Pancreatic Tissue and Pancreatic Benign Cystadenoma

Background: Dual expression of potential biomarkers in both benign and malignant pancreatic tumors was a major obstacle in the development of diagnostic biomarkers of early pancreatic cancer. Methods: To better understand the limitations of potential protein biomarkers in pancreatic cancer, we employed two-dimensional difference gel electrophoresis technology and tandem mass spectrometry to study protein expression profiles in pancreatic cancer tissues, benign pancreatic adenoma and normal adjacent pancreas. Seven differently expressed proteins were selected for validation by Western blot and/or immunohistochemistry. Results: 21 spots were overexpressed and 24 spots were downexpressed in pancreatic cancer compared with benign and normal adjacent tissues. Our study demonstrated that three candidate pancreatic ductal adenocarcinoma biomarkers identified in previous studies, fructose-bisphosphate aldolase A, α-smooth muscle actin and vimentin, were also overexpressed in pancreatic cystadenoma, which might lower their further utility as biomarkers for pancreatic cancer. Aflatoxin B₁ aldehyde reductase (AKR7A2) was confirmed to be only highly expressed in pancreatic cancer, not in normal adjacent pancreas and benign tumors. Conclusions: The protein profile pattern of pancreatic cystadenoma was more similar to normal adjacent pancreas than pancreatic cancer. We identified panels of the upregulated proteins in pancreatic cancer, which have not been reported in prior proteomic studies. AKR7A2 may be a novel potential biomarker for pancreatic cancer.     

Immunohistochemical profiling of liver metastases and matched-pair analysis in patients with metastatic pancreatic ductal adenocarcinoma

BackgroundThe purpose of the current study was to investigate the immunohistochemical (IHC) profile of liver metastases (LM) in patients with pancreatic ductal adenocarcinoma (PDAC).MethodsExpression of 15 IHC markers in liver biopsies from 77 patients with PDAC, who were diagnosed between 2010 and 2014, were evaluated. In a separate subgroup analysis (n = 12), paired samples (LM and primary tumor) from the same patient were investigated for IHC profile differences.ResultsLM samples were classified as pancreatobiliary-type (PB-type) in 72 patients (93.5%), intestinal-type (INT-type) in four patients (5.2%), and squamous in one patient (1.3%). There was no significant difference in overall survival (OS) between LM of the PB-type or INT-type (p = 0.097). In a multivariate analysis, age <70 years (p = 0.047), absence of SMAD4 mutation (p = 0.026), absence of CDX2 expression (p = 0.003), and well to moderate differentiation were significant prognostic factors for better OS in patients with LM (p = 0.031). Analysis of paired tissue samples from LM and the primary tumor revealed a difference in CDX2 (50% increase, p = 0.125) and SMAD4 (33% loss of SMAD4, p = 0.375).ConclusionsCDX2 expression and SMAD4 mutation indicate a poor outcome in patients with LM of PDAC. Matched-pair analysis revealed differences in distinct IHC marker expression.     

应用免疫蛋白质组学方法鉴定日本血吸虫虫卵诊断抗原

目的虫卵可溶性抗原（soluble egg antigen,SEA）是目前日本血吸虫病免疫诊断中最常用的抗原。本研究联合应用双向凝胶电泳（2-DE）、Western blot和MALDI-TOF质谱等技术,鉴定SEA中诊断抗原蛋白质。方法 SEA经2-DE分离蛋白质后进行银染,或应用日本血吸虫感染兔血清Western blot筛选抗原蛋白点,用MALDI-TOF/TOF串联质谱鉴定每一抗原蛋白质分子。结果虫卵可溶性蛋白分子经2-DE分离后转印PVDF膜上,与感染兔血清孵育出现29个特异性阳性反应点,与健康兔血清出现3个非特异的阳性反应点;29个特异性抗阳性反应点中,从银染2-DE胶图上找到21个匹配的抗原蛋白质点;MALDI-TOF/TOF质谱鉴定和NCBI数据库检索,13个点（61.9%）获得匹配蛋白质,4个点（19.0%）获得匹配EST,4个点（19.0%）未能从数据库中找到相匹配的信息。重组表达筛选出的SjCHGC06040和SjP40两个抗原蛋白质,Western blot结果显示reSjCHGC06040、reSjP40均能与感染兔血清抗体发生特异性反应,具有潜在的应用价值。结论 2-DE、MALDI-TOF质谱联合Western blot的免疫蛋白质组学研究方法,用于筛选、鉴定SEA中诊断抗原蛋白质,是切实可行的;但该方法存在一定的局限性,有待进一步改进。     

Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using two-dimensional differential gel electrophoresis

Distant metastasis represents the major lethal cause of breast cancer. To understand the molecular mechanisms of breast cancer metastasis and identify markers with metastatic potential, we established a highly metastatic variant of parental MDA-MB-231 cells (MDA-MB-231HM). Using two-dimensional electrophoresis (2-DE), we performed a proteomic comparison of the two kinds of cells. As much as 51 protein spots were differentially expressed between the selected variant and its parental counterpart in at least 3 experiments. Ten unique proteins were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and database searching software. Among them, nine proteins were up-regulated in MDA-MB-231HM cells, including Macrophage-capping protein (CapG), Galectin-1, Chloride intracellular channel protein 1, Endoplasmic reticulum protein ERp29 precursor, Stathmin-1 (STMN1), Isoform 1 of uridine–cytidine kinase 2(UCK2), Rho GDP-dissociation inhibitor 2 (ARHGDIB), isocitrate dehydrogenase [NADP] cytoplasmic (IDH1), and N-myc downstream regulated gene 1 (NDRG1) protein. Only transgelin-2 was down-regulated. Differential expression was confirmed for three proteins including CapG, STMN1, and transgelin-2 by Western blotting analysis. Transgelin-2 was chosen for further verification by immunohistochemistry. The results suggested that 2-DE would be an efficient way to screen the proteins responsible for specific biological function. Furthermore, the findings imply that different proteins may be involved in the metastatic process in breast carcinomas.     

Identification of Annexin A1 protein expression in human gastric adenocarcinoma using proteomics and tissue microarray

AIM:To study the differential expression of Annexin A1(ANXA1)protein in human gastric adenocarcinoma.This study was also designed to analyze the relationship between ANXA1 expression and the clinicopathological parameters of gastric carcinoma.METHODS:Purified gastric adenocarcinoma cells(GAC)and normal gastric epithelial cells(NGEC)were obtained from 15 patients with gastric cancer by laser capture microdissection.All of the peptide specimens were labeled as18O/16O after trypsin digestion.Differential protein expressions were quantitatively identified between GAC and NGEC by nanoliter-reverse-phase liquid chromatography-mass/mass spectrometry(nanoRPLC-MS/MS).The expressions of ANXA1 in GAC and NGEC were verified by western blot analysis.The tissue microarray containing the expressed ANXA1 in 75 pairs of gastric carcinoma and paracarcinoma specimens was detected by immunohistochemistry(IHC).The relationship between ANXA1 expression and clinicopathological parametes of gastric carcinoma was analyzed.RESULTS:A total of 78 differential proteins were identified.Western blotting revealed that ANXA1 expression was significantly upregulated in GAC(2.17/1,P<0.01).IHC results showed the correlations between ANXA1protein expression and the clinicopathological parameters,including invasive depth(T stage),lymph node metastasis(N stage),distant metastasis(M stage)and tumour-lymph node metastasis stage(P<0.01).However,the correlations between ANXA1 protein expression and the remaining clinicopathological parameters,including sex,age,histological differentiation and the size of tumour were not found(P>0.05).CONCLUSION:The upregulated ANXA1 expression may be associated with carcinogenesis,progression,invasion and metastasis of GAC.This protein could be considered as a biomarker of clinical prognostic prediction and targeted therapy of GAC.