An Experimental Study On Early Diagnostic Serum Biomarkers Of Non-small Cell Lung Cancer By Proteomics Technique

Background and objective:About75%-85%of lung cancer are non-small cell lung cancer (NSCLC). The existing tumor markers cannot be applied to early diagnosis of NSCLC yet. Therefore, looking for lung cancer related serum marker is currently one of research hot spots. Proteomics technique based on two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has the characteristics of high throughput, sensitiveness and automation. So it has been one of the important technical method to current research on tumor markers. This topic is plan to screening and identification of differentially expressed serum proteins of early stage NSCLC by Proteomics technique. Meanwhile, combined the traditional experiment methods, including Western blot, fluorescent quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay, we aimed at finding serum biomarkers for early detection of NSCLC.Methods:1.Sera from three groups, including lung cancer group (n=11), benign control group (n=12), and normal control group (n=30), were collected, balanced pooled intra-group and high abundance proteins were removed from the pooled samples.2DE and MALDI-MS were employed to separate, screen and identify the differentially expressed proteins related to stage I NSCLC.2. Differentially expressed proteins closely related to lung or other cancer were selected as candidates. Western blot were conducted in the pooled samples of the three groups to confonn the accuracy of MS results and screen the significant differentially expressed proteins.3.The screened proteins were tested via fluorescent quantitative PCR at the genetic level, and immunohistochemistry and enzyme-linked immunosorbent assay at the protein level, to determine the biomarkers for early diagnosis of NSCLC.Results:1. Clear2DE protein expression profiling were obtained from the three groups. PDquest8.0image analysis software was used to scan and analyze images. Total38protein spots were considered as differential spots. 2.In contrast to the two control groups,12differentially expressed proteins were identified via PMF using MALDI-MS. Six proteins (hemoglobin beta, cold agglutinin chain A, carbonic anhydrase I, carboxypeptidase N catalytic chain precursor, complement C3precursor, and complement C4A) were up-regulated in the lung cancer group. The other six proteins (apolipoprotein J, haptoglobin, transthyretin precursor, complement C8beta propetide, complement C1s subcomponent precursor, and hemopexin precursor) were down-regulated in the lung cancer group.3. Differentially expressed proteins closely related to lung or other cancer, including hemoglobin beta, carbonic anhydrase I, complement C3precursor, apolipoprotein J, transthyretin precursor and hemopexin precursor, were selected to be tested via Western blot. As a result, the6kinds of proteins appeared clear bands of protein expression, which confirmed the accuracy of the finding of MS. In contrast to the two control groups, the expression of carbonic anhydrase Ⅰ in the lung cancer group was significantly up-regulated, whereas, that of transthyretin precursor was down-regulated.4. Carbonic anhydrase I and transthyretin precursor were effective biomarker for early detection of NSCLC via validation of fluorescent quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay.Conclusion:1.As an accessible experimental materials, serum specimen is effective and feasible research object. The results of study can be directly applied to clinical practice, which is of great clinical significance and practical value.2. Application of proteomics technique, combined with traditional experiment method, can serve as a kind of effective strategies for developing new serum biomarkers of lung cancer.3. Carbonic anhydrase I and transthyretin precursor are serum markers in early diagnosis of NSCLC.