RESUMO
Human epididymis protein 4 (HE4) is a recognized biomarker in ovarian and endometrial cancer and over-expressed in pancreatic adenocarcinoma. The diagnostic value of HE4 in pancreatic adenocarcinoma remains unknown. Here we elucidate mRNA, protein and serum level of HE4 in pancreatic adenocarcinoma. HE4 mRNA level in tumor adjacent tissues and pancreatic adenocarcinoma tissues were tested by real time-PCR. Tissue microarray containing normal, adenocarcinoma, and adjacent pancreatic tissue was tested by immunohistochemistry (IHC). Serum level of HE4, carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 15-3 (CA15-3) and carbohydrate antigen 125 (CA125) were detected by ELISA assay in control and tumor patients. Further we compared the sensitivity and specificity of determining HE4, CA19-9, CA15-3, and CA125 for diagnosis of pancreatic adenocarcinoma and assessed the complementary diagnostic value of HE4, CA19-9, CA15-3 and CA125. Real time PCR showed significantly increased HE4 mRNA level in pancreatic adenocarcinoma compared with control. Result of IHC showed that HE4 significantly higher expressed in the human pancreatic carcinoma tissues than in both normal and adjacent non-tumorous pancreatic tissues, and the staining intensity is inversely correlated with the clinical stage. HE4 was highly expressed in early stage of pancreatic adenocarcinoma. Serum HE4 level is higher in cases with pancreatic adenocarcinoma than in the controls. Serum HE4 levels could research to a sensitivity of 45.83% and specificity of 93.75% when the Cutoff was set at 4.59 ng/mL. The Combined HE4 and CA19-9 increased the sensitivity to 83.33%; and interestingly, the combination of HE4 with CA15-3 led to the most powerful sensitivity of 87.5%. Combined with CA19-9 and CA15-3, HE4 could be a potential biomarker to improve the diagnostic power for pancreatic adenocarcinoma.
Assuntos
Adenocarcinoma/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Proteínas/análise , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antígenos Glicosídicos Associados a Tumores/sangue , Área Sob a Curva , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Antígeno Ca-125/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and ß-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.
Assuntos
Eletroforese/métodos , Eritrócitos , Hemoglobina A/metabolismo , Sequência de Aminoácidos , Extratos Celulares/química , Membrana Eritrocítica/metabolismo , Eritrócitos/química , Eritrócitos/metabolismo , Hemoglobina A/química , Hemoglobina A2/química , Hemoglobina A2/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Espectrometria de Massas em TandemRESUMO
In this paper, we aimed to introduce a newly established red blood cells (RBCs) electrophoresis method hemoglobin release test (HRT) and tried to determine its significance. Human blood samples from beta-thalassemia patients and healthy controls were analyzed with HRT, which was carried out on starch-agarose mixed gel. First, the whole blood samples were electrophoresed for 2 h, then paused for 15 min and ran for 15 min by turns. This "pause-run-pause" experiment was performed for several turns and the total electrophoresis time lasted for about 6 h. The results showed that some other hemoglobin (Hb) components were released from the origin of each sample during the HRT, and the samples from beta-thalassemia patients released more Hb than the healthy controls. This finding demonstrates that Hb may exist differently associated in RBCs, and it may have an important theoretical and clinical significance in Hb and RBC research.