| BAKEGROUND & OBJECTIVE: Nowadays, lung cancer has become one of the malignant tumors that have the highest incidence and mortality. As a grand disease which is threatening the whole human society, its mortality has reached as high as 13%, and is still increasing gradually. According to the WHO's estimation, China will become one of the countries that have the relatively high incidence of lung cancer in the 21century. Lung cancer comprises a broad spectrum of tumors that include squamous carcinoma, adenocarcinoma, large-cell carcinoma, and small-cell carcinoma according to histological diagnosis. Squamous carcinoma is the most common type, accounting for 30-50 % in all of lung cancer patients. The clinical statistic study showed that in Chinese urban, the number of squamous carcinoma patients was rapidly increasing, which occupied 50-70% in all of lung cancer patients. Carcinogenesis of lung squamous carcinoma is a complex process involving multiple events and steps. Though some molecular pathogenesis studies on human lung cancer have been undertaken successfully on the gene (DNA) and transcription (mRNA) levels, the carcinogenic mechanism is still unclear. At present, there is also no the special lung cancer molecular marker for an "early-stage" diagnosis and prognosis evaluation. Furthermore, functional complexity of normal and neoplastic tissues is not dictated solely by the encoding genomic information, but also determined by execution molecules, proteins. The proteomic study of lung cancer may directly and comprehensively explore the carcinogenic mechanism of lung cancer. Thus, the proteomic approach is essential to understand pathogenesis and development of lung cancer,it is expected to obtain some clues to further study of the carcinogenic mechanisms, diagnosis, treatment and new drug exploitation of lung squamous carcinoma. On the one hand, this study was designed to optimize the research methods for proteome, to establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression proteins. On the other hand, the western-blot imaging films reacted with the patient autologous serum and with control serum were compared and tumor-associated antigens were identified by using Serologic Proteome Analysis (SERPA), an approach which combines conventional proteome analysis with serological screening. METHODS: In the first place, comparative proteome analysis with twenty human lung squamous carcinoma tissues and the paired normal bronchial epithelial tissues adjacent to tumors were carried through. Namely, the total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were seperated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE) and silver staining. The differential expression proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: (1) The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained. For tumor tissue, average spots of 3 gels were 1567 ±46, 1436 ± 54 spots were matched with an average matching rate of 91.6 %. For control, average spots of 3 gels were 1349 ± 58, and 1228 ± 35 spots were matched with an average matching rate of 91.03 %. The average positiondeviation of matched spots was 0.924±0.128 mm in IEF direction, and 1.022±0.205 mm in SDS-PAGE direction. (2) A total of 1178 ± 56 spots were matched between the electrophoretic maps of 20 human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue. Seventy-six differentially expressed proteins were screened. (3) Sixty-eight differential proteins were identified by PMF, some proteins were the products of oncogenes, and others involve in the regulation of cell cycle and signal transduction. (4) In order to validate the reliability of the identified results, the expressions of three proteins mdm2,c-jun and EGFR , which correlate with lung squamous carcinoma, were detected by immunohistochemical staining and Western blot analysis. The results displayed that mdm2,c-jun and EGFR were up-regulated in lung squamous carcinomas, whereas they are down-regulated in adjacent normal bronchial epithelial tissues, normal lung tissues and inflammatorypseudotumor, which is consistent with our proteome analysis results. These data suggest that these proteins may play roles in the carcinogenesis of lung squamous carcinoma. In this study, Serologic Proteome Analysis (SERPA) of ten human lung squamous carcinoma tissues were also performed. Methods: Three gels were run simultaneously with equal amounts of proteins from the same lung squamous carcinoma by IPG -2D PAGE. After separation, the proteins of one gel (replica gel) was visualized by silver-based staining technique. The other two gels were used for western blot analysis. Proteins are transferred to NC membranes, and then reacted with autologous patient serum and with control normal serum, respectively. The western blot imaging films were obtained and the differential reacting protein spots were recognized, then the differential reactingprotein spots in the replica gel by matching analysis. RESULTS: (1) Well-resolved, reproducible 2-DE western blot imaging films of human lung squamous carcinoma reacted with autologous patient sera and with control sera were obtained. (2) 36 ±8 differentially expressed proteins which only were reactive with lung squamous carcinoma patient sera were found. (3) Eleven proteins, which are lung squamous carcinoma–associated antigens, were identified by PMF. Six of them such as Alpha enolase, Pre-B cell enhancing factor precursor, Triosephosphate isomerase, Phosphoglycerate mutase 1, Fructose-bisphosphate aldolase A, Guanine nucleotide-binding protein beta subunit-like protein are as same as proteins that had been identified in the above comparative proteomic study, which further supports that these lung squamous carcinoma–associated antigens may be the molecular candidate biomarkers for the diagnosis and therapy of lung squamous carcinoma. CONCLUSION: In this study, the well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were established and sixty-eight differential proteins were characterized by applying comparative proteome analysis successfully. And fourteen tumor-associated antigens were characterized by using Serologic Proteome Analysis (SERPA). These results will provide scenticfic foundation for screening the molecular biomarker used to diagnose and treat lung squamous carcinoma, as well as to elevate the patient's prognosis and provide new clue for the research of lung squamous carcinogenic mechanism.
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