Proteomic Analysis Of A Candidate Tumor Suppressor Protein 14-3-3σ Associated Protein In Esophageal Squamous Cell Carcinoma

Background:Among the sixth most common cancer worldwide, esophageal carcinoma (EC) is characterized by the remarked regional distribution evidenced by a large body of epidemiological studies. The incidence rate between high and low incidence area for EC can be as high as 500-fold difference. Long time survival after confirmed diagnosis of EC is very poor and the five-year survival is approximately 10%. In northern China, Linzhou has long been known for its highest incidence and mortality in world. In the past decades, scientists put forward that EC is a multi-stage process involved a variety of abnormalities after comprehensive and multi-displinary investigations. The incidence and mortality rate of EC in this high area, however, haven't ameliorated yet and EC still persists one of the main causes of cancer-related deaths. The main cause consists of the following. First, lack of the exact pathogenic mechanisms of EC constrains the development of effectively preventive strategies and measures. Second, molecular mechanisms of EC remain elusive and biomarkers for early diagnosis, high-risk population surveillance are in dire need. Five-year survival rate for patients with carcinoma in situ and intra-mucosal carcinoma localized in esophagus can reach 100% and 95%, respectively. Therefore, improvement of efficacy of EC treatment relies on early diagnosis and treatment.14-3-3σ, a new candidate tumor suppressor gene, has the ability to modulate cell proliferation, differentiation, and apoptosis through binding to protein targets. It has been reported that down-regulation of 14-3-3σprotein correlated with many forms of tumor formation and development, e.g. breast, bladder, prostate gland, liver, lung, etc. However, studies concerning 14-3-3σand esophageal cancer is not clear. Previous studies in our research group have found that 14-3-3σwas down-related in cancer cell lines and tumor tissue of esophagus and also correlated with squamous cell differentiation. The precise molecular mechanisms of 14-3-3σin esophageal carcinogenesis remain elusive yet.In present study, the whole length of 14-3-3σcoding sequence was cloned from esophageal immortal cell lines NE6 followed by sequencing confirmation of its authority. The open reading frame of 14-3-3σwas inserted into eukaryotic expression vector pcDNA3.2 and selected by culture medium. Esophageal cancer cell line 9706 was transfected with pcDNA3.2-14-3-3σ-TAP(C) and two daughter cell lines, pcDNA3.2- BioEase- TEV-His - V5-14-3-3σ(NT) and pcDNA3.2-14-3-3σ(CT)-V5-His-TEV-BioEase, express 14-3-3σprotein, in which the N-terminus and carboxyl-terminus of 14-3-3σwas integrated with pcDNATM3.2/capTEVTM /V5-DEST. Tandem affinity purification (TAP) and High Definition Mass Spectrometry (HDMS) were used to purify, identified the protein components associated with 14-3-3σprotein. The present results provide more insights related with the molecular mechanisms of tumor inhibitive abilities of 14-3-3σin the multi-stage process esophageal neoplastic pathogenesis. Furthermore, the identified proteins targeted by 14-3-3σare useful for biomarker discovery of screening and early diagnosis of high-risk subjects for esophageal cancer.Aims:To illuminate the suppressive molecular mechanisms of 14-3-3σin the multi-step process of esophageal carcinogenesis through identification of protein components targeted by 14-3-3σprotein and provides theoretic basis for biomarker discovery of high-risk subjects screening and early diagnosis of esophageal cancer.Methods:Reverse transcriptase PCR (RT-PCR) was used to clone the coding sequence (747bp) using cDNA as template derived from immortalized esophageal cell line NE6. After comparison with Medline cDNA database, Stratagene's QuikChange site directed mutagenesis kits were employed to correct the two mutation sites, 119 and 686 bp, respectively. Orientated insertion was performed to construct the eukaryotic expression vector pcDNA3.2 followed by sequencing confirmed authenticity. The constructed pcDNA3.2- BioEase- TEV-His - V5-14-3-3σ(NT) and pcDNA3.2-14-3-3σ(CT)-V5-His-TEV-BioEase were transfected into esophageal cancer cell line 9706 mediated by lipid. NP-40 mediated multi-freeze-thaw cell lysis was carried out to extract total protein from daughter cell line expressing infused 14-3-3σ-TAP (C) protein and control cell line null for 14-3-3σfollowed by TAP isolation and purification of 14-3-3σprotein complexes. The extracted 14-3-3σcomplex protein was fractionated by SDS-PAGE electrophoresis, revealed by silver staining. The differential protein bands showing on SDS-PAGE gel was cut out subjected to in-gel trypsin digestion, HDMS analysis, and database mining to identify the protein components associated with 14-3-3σcomplex.Results:1. The coding sequence of 14-3-3σwas cloned from immortalized esophageal cell line NE6 by RT-PCR followed by authenticity confirmation using 14-3-3σsequencing;2. Lipid-mediated transfection of esophageal cancer cell line 9706 was performed to establish two daughter cell lines expressing 14-3-3σinfused-NT/-CT protein, i.e. pcDNA3.2- BioEase- TEV-His - V5-14-3-3σ(NT), pcDNA3.2-14-3-3σ(CT)-V5-His-TEV-BioEase and vector control cell line pcDNA3.2- BioEase- TEV-His - V5;3. 14-3-3σprotein complex was isolated and purified by TAP technique, separated by SDS-PAGE electrophoresis;4. Subtractive method was used to identify the 57 specific protein components associated with 14-3-3σfollowing differential protein bands excised from SDS-PAGE gel.Conclusions:The present study using proteomic techniques identified 57 proteins associated with 14-3-3σ, which are useful for illumination of molecular mechanisms of 14-3-3σimplicated in the multi-stage process of esophageal carcinogenesis and provide more candidate biomarker for high-risk population screening and early diagnosis of EC patients.