| Background and AimBiliary tract cancers(BTC)are a heterogeneous group of carcinomas with features of cholangiocyte differentiation originating from the epithelium of the bile ducts and gallbladder,which are composed of cholangiocarcinoma(CCA),gallbladder carcinoma(GBC)and ampullary cancer.BTC remains as one of the most lethal cancers.Curative resection(R0 tumor resection)is the only approach leading to optimistic outcomes.Whereas,diagnosis of BTC at an early stage is challenge,and the chance of surgical resection is low.CA19-9 is one of the most commonly utilized biomarkers in diagnosis of BTC,and so far it is still regarded as the most promising diagnostic and prognostic marker in BTC.However,increased levels of CA19-9 also can be observed in patients with benign diseases,such as cholestasis or cholangitis.It may be a disturbance to specific malignant diagnosis.Thus,it urged us to explore new diagnostic biomarkers of BTC at early stage.Although blood appears to be the most common test object,10 most abundant proteins in blood account for >90% of the total protein contained.These most abundant proteins contain little information regarding the status of an organ and greatly inhibit the accurate detection of less abundant proteins that potentially contain more information.Moreover,proteins may be released and /or shed directly into the bile by tumor cells.Therefore,higher levels of secreted or shed markers may be contained in the bile than serum,and bile is expected to be rich in glycoproteins.In this study,bile is regarded as the major test object.This study aims to investigate cancer-related biomarkers,which could be helpful for early diagnosis of BTC and further provide a potential targets for adjuvant therapy,comparing differential expressing proteins in the bile between malignant and benign biliary disease.MethodProteomics that is a qualitative and quantitative analysis of proteins and peptides of samples,has become a new subject for investigating protein expression and activities.It can observe the change of protein expression in the process of tumor development and metastasis comprehensively,dynamically,and quantitatively.Isobaric tags for relative and absolute quantitation(iTRAQ)in combination with two-dimensional liquid chromatography tandem MS(2D-LC-MS/MS)analysis is emerging as one of the more powerful policy for proteomics methodologies in the search for tumor biomarkers.ITRAQ is a quantitative proteomic technique,which can analysis up to eight samples in one experiment.Due to the high precision,it has been widely used in the field of quantitative proteomics.KEGG is the main public database of pathway,which can be used to determine the most important biochemical pathways and signal transduction pathway.GO is an internationally standardized gene function classification system that provides a set of dynamically updated standard vocabularies to fully characterize the properties of genes and gene products in organisms.GO has three ontologies that describe molecular function(MF),cellular component(CC)and Biological Process(BP),respectively.Bile and serums were obtained from patients with histopathologically proven malignant or benign biliary diseases.Aliquoted bile and serum samples were frozen at-80℃ after centrifugated until use,and the clinical data of patients were recorded.Eight bile samples were selected randomly.Total proteins were extracted,and stable isotope labeled peptides using reagent after enzymatic digestion.Combined labeled digests were separated by off-line 2D-LC,and then detected by Triplet TOF 5600.Next,these were qualitative and quantitative analyzed by Mascot and scaffold.Finally,bioinformatic analysis was carried out,such as KEGG pathway analysis,GO analysis and PPI protein interaction network.Meanwhile,western blot(WB)and enzyme-linked immunosorbent assay(ELISA)was used to validate the results in the bile and serums of 100 patients with malignant or benign biliary disease.The sensitivity and specificity of the diagnosis of high expression protein were determined by ROC curve to confirm the diagnosis value.Kaplan-Meier survival and Cox proportional hazards analysis was used to assess the prognostic value of potential biomarkers in malignant and benign biliary disease.ResultTen differential protein profiles were detected using 2D LC-MS/MS in four protein groups(FDR < 1%).Six proteins were down regulated,while four proteins were up regulated.Among them,Haptoglobin(Hp)and Complement Factor H(CFH)was found to be evaluated in the bile of patients with malignancy.Western blot analysis demonstrated that Hp was highly expressed in the bile and serums of the malignancy group.ELISA indicated that the level of Hp and CFH in the bile was higher than that of serum(P = 0.004).In the bile,the expression of Hp and CFH in malignant group was increased(P = 0.006 and 0.014,respectively).However,different types of malignant biliary diseases have no significant difference.In serum samples,the concentrations of Hp and CFH in patients with CCA or GBC were significantly increased,while those of duodenal papilla carcinoma were obviously low(P = 0.033 and 0.004,respectively).When the concentration of Hp was 57.237 ng/ml,the sensitivity was 0.909 and specificity was 0.667.When the concentration of CFH was 148.1807 ng/ml,the sensitivity was 0.636 and specificity was 0.8.According to the cut-off value of ROC curve,Hp and CFH in the serums of patients with malignant biliary tract cancer was divided into two categories: positive and negative.Kaplan-Meier curve showed that the survival of patients was closely related to the negative expression of Hp and CFH(P= 0.001 and 0.002,respectively).ConclusionThe expression of Hp and CFH in patients with malignant biliary tract cancer was evaluated,and closely related to the prognosis of the diseases.Moreover,the diagnosis sensitivity of Hp was higher than that of CA19-9 and CEA,these may be a potential biomarker and therapeutic targets of BTC.It is needed further studies to confirm whether evaluated Hp is glycosylated,or site and linkage on haptoglobin in patients with various types of cancer is similar. |
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