Quantitative Proteomic Analyses Of The Secretome Of NSCLC

Non-small cell lung cancer (NSCLC) is one of the highest morbidity and mortality of malignant tumors in the world, non-small cell lung cancer (NSCLC) accounts for about 80-85% of the total number of lung cancer, which seriously influence the quality of people’s lives. At present, the pathogenesis of this kind of deadly disease is still not very clear, the molecular mechanism of its occurrence and development needs to be elucidated. Proteomic technology has been developing rapidly in recent years, providing powerful tools for cancer research. In This experiment, we used isobaric tags in a relative and absolute quantitation (iTRAQ) proteomic approach and SWATHTM quantification approach to analyze the secretome of an isogenic pair of highly metastatic and low metastatic NSCLC cell lines. We identified the differently expressed secretory proteins for screening of NSCLC metastasis related molecules.Part Ⅰ:Serum prtoteome analysis method was preliminary established. We established the method for removing high-abundance proteins in serum proteomics research and evaluated the removal efficiency of the high-abundance protein. Human 14 Multiple Affinity Removal System of Agilent Technologies was applied to eliminate the high-abundance proteins, and showed good efficiency for removing serum high-abundance proteins which provides basis for further strategies in proteomics research.Part Ⅱ:Quantitative analysis of secretory proteins which associated with NSCLC metastasis. We used iTRAQ-based proteomic approach and SWATHTM quantification approach to analyze the secretome of an isogenic pair of highly and low metastatic NSCLC cell lines. Results showed that 326 proteins were repeatedly quantified by the two methods, and 110 proteins were differently expressed. Moreover,96% of these proteins were predicted that could secrete into conditioned media (CM). We applied qRT-PCR and western blot method to detect the level of several differently expressed proteins, and the results showed that the differences between the results were consistent with the quantitative trend of mass spectrometry.In addition, we analyzed the early-stage lung cancer patients group (phase Ⅰ and Ⅱ) and late-stage lung cancer patients group (phase Ⅲ and Ⅳ) for screening the differently expressed serum proteins with same method which was used in Part Ⅰ. As a result, we identified 71 serum proteins as differently expressed proteins. ELISA assay was used to detect Complement C3 in serum of the serum samples, and the result showed that Complement C3 was up-regulated in the serum of late-stage NSCLC patients. Combined with the screening results of serum and CM, we found that CD 109 was highly expressed in advanced lung cancer patients and also in high metastasis potential lung cancer cell line. This indicted that this protein may play an important role in lung cancer development.Part Ⅲ:Detection of CD109 expression level in cancer and preliminary study of its biological function. qRT-PCR were applied to test CD 109 in tissues of 72 patients with lung cancer and their adjacent carcinoma tissue, and resulted in significantly high in cancerous tissues, and high in poorly differentiated lung cancer tissues. ELISA assay was used to detect CD 109 in serum of 19 cases of normal people and 30 cases of lung cancer patients. Results showed that CD 109 was highly expressed in serum in lung cancer patients, and increased significantly in the patients of late-stage. To explore CD 109 molecular biology function, we used siRNA knockdown CD 109 expression in lung cancer cell lines, and detected cell proliferation, invasion, migration ability and the change of cell cycle. The results showed knockdown of CD 109 had a significant effect on the cell cycle, inhibition of cell proliferation, but no significant effect on cell invasion and migration ability. These results indicated that CD 109 may be associated with NSCLC development, and can be used as potential therapeutic target.