Identification And Analysis Of Differentially Expressed Proteins Of Gastric Adenocarcinomas Tissue

It has been well established that cancer development possesses complicated molecular mechanism, which usually involves multiple genetic changes and effects from the environment. This complicated interplay of factors leading to neoplastic progression is ultimately reflected in the protein expression profiles of cells. In this study, we tried to identify the change of protein expression profiles of gastric adenocarcinomas (GA) with the purpose to find differentially expressed proteins between cancer and normal mucosa tissue using a proteomics approach, thereby to identify a cancer associated protein which was few investigated previously for further study. The further study included to validate it as a potential candidate of GA biomarker using both western blot and immunohistochemistry assays, and to preliminarily investigate its role in the carcinogenesis of GA using a gene cloning techniques by constructing its mammalian expression vector, and then using a gene transfection technique to introduce it into cells.In the first part of this study, ten protein expression profiles of GA and paired non-neoplastic mucosa tissues were analyzed by 2-dimensional gel electrophoresis (2-D PAGE) . Forty-two protein spots that were differentially expressed by 2-fold or greater between cancer and normal mucosa tissue were excised and identified by MALDI-TOF mass spectrometry(MS). This generated 42 distinct proteins that were differentially expressed at least two-fold between the tissues. 29 of these proteins displayed decreased expression in cancer tissue, such as PACAP protein; while 13 were over expressed in the cancer tissue, such as vimentin.In the second part of this study, human proteasome activator PA28 suunit beta (PA28β) which was decreased expression in GA tissue observed by 2-D PAGE and identified by MS was chosen for further verification using both western blot and immunohistochemistry assays. 40 cases GA and paired non-neoplastic tissues were examined by both the assays with an anti PA28βantibody. PA28βwas found down-expressed in 28 samples of cancer tissue (28/40, 70.0%) when detected by western blot. The results of immunohistochemistry confirmed that PA28βwas expressed in 38 cases of paired non-neoplastic tissues with positive cytoplasmic staining in gastric gland cells of lamina propria of gastric mucosa of these samples, and was indeed down-expressed or absent in 29 cases of GA (29/40, 72.5%). The preliminary results indicate PA28βmay be used as a novel biomarker for GA.In the third part of this study, the role of PA28βin the carcinogenesis of GA was investigated. The full length cDNA sequences of PA28βwere obtained by RT-PCR. pcDNA3.1-PA28βmammalian expression vector was constructed, and was transfected into MKN-45 gastric adenocarcinomas cells. The activity of cell proliferation before and after transfected with PA28βgene was detected by MTT assay, Brdu labeling assay and colony formation assay. The tumorigenicity of the MKN-45 cells before and after transfected with PA28βgene was tested by the colony formation in soft agar assay. The results showed that both the proliferation activity and the tumorigenicity of MKN-45 cells were decreased after the cells had been transfected with PA28βgene. The analystical results of statistics indicated the discrepancy was very significant between MKN-45 cells transfected with pcDNA3.1-PA28βvector and pcDNA3.1/hygro(+) empty vector (P<0.01), and also between MKN-45 cells transfected with pcDNA3.1- PA28βvector and untransfected MKN-45 cells(P<0.01). The preliminary results indicate PA28βmight cause the changes of cell proliferation activity and tumorigenicity, and it participates the carcinogenesis of GA with an unclear molecular mechanism which needs to be further investigated.