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Studies on the Effects and Mechanism of TSPYL5 Anti-oncogene on Development and Progress in Gastric Carcinoma

Author: DiFu
Tutor: XiaJianGuo
School: Nanjing Medical University
Course: Department of General Surgery
Keywords: TSPYL5 β-catenin Wnt signaling pathway Tumor suppressor gene Methylation
CLC: R735.2
Type: Master's thesis
Year: 2010
Downloads: 30
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Abstract


Objective: To investigate the testis-specific Y-like protein-5 (testis-specific Y-like protein 5, TSPYL5) the expression on the biological behavior of gastric cancer. The study TSPYL5 in the development of gastric cancer possible mechanism of action of Wnt / β-catenin signaling pathway. Methods: RT-PCR, quantitative PCR, immunofluorescence technique in gastric carcinoma and adjacent normal tissue TSPYL5 expression. 2. MTT, colony formation of experimental detection TSPYL5 role in gastric cancer cell lines AGS. 3. Luciferase reporter gene, western blot, immunohistochemistry, immunofluorescence the technology research TSPYL5 whether a key molecule β-catenin by Wnt / β-catenin signaling pathway play a role in the results: 1. Selected 46 cases of gastric carcinoma and phase corresponding adjacent normal tissue RT-PCT, immunofluorescence showed that, compared with the corresponding cancer adjacent normal tissue, the presence of gastric tissue the TSPYL5mRNA expression down or missing. Also show that quantitative PCR analysis of 23 cases of cancer and adjacent normal tissue, 25 cases of gastric carcinoma, TSPYL5 expression in gastric cancer tissues are significantly lowered, and a statistically significant (P lt; 0.05). These results prompted after a statistical analysis TSPYL5 mRNA expression and tumor size, histological grade, depth of invasion, and lymph node involvement were not statistically significant between TNM stage Ⅱ, Ⅲ of as well as non-celiac and far The distant metastases TSPYL5mRNA significantly lower (P lt; 0.05). MTT proliferation assay results showed that the pc-DNA-TSPYL5 transfected group from the first day and compared to the control group at all time points cell proliferation was significantly inhibited absorbance values ??to the fifth day pc-DNA-TSPYL5 transfected group 0.48, pc-DNA idling transfected group absorbance value of 0.75, the empty control group absorbance value of 1.20, the difference was statistically significant (P lt; 0.05). Colony formation assay. Greater than 10 cells microscope (low magnification) count the number of clones, calculate the rate of colony forming P group was 100%, AGS cells transfected TSPYL5 strains of colony formation rate of about was 18.6%, the results showed that transfection TSPYL5 AGS cell line growth was inhibited. 3 for 46 pairs of gastric cancer and adjacent normal tissue immunohistochemical analysis of β-catenin, the result shows that about 47.8% (22/46) of gastric cancer tissue samples show abnormal positive β-catenin expression. At the same time, we will collect 46 cases of gastric cancer and adjacent normal tissue samples for western blotting analysis TSPYL5 and β-catenin expression was found in relatively early gastric cancer and adjacent normal tissue (I, II period) TSPYL5 down significantly, at the same time β-catenin upregulation. AGS cell lines transfected with empty plasmid and TSPYL5 plasmid, cultured 48 h, cells were collected, immunofluorescence results showed that transfection of the total protein in the TSPYL5 the experimental group than in the empty plasmid control group β-catenin downregulation . Function tests determined by luciferase reporter analysis system detects pcDNA-TSPYL5 transfected group than pc-DNA empty vector transfected group luciferase activity decreased, the difference was statistically significant (P lt; 0.05) shows that transfection of eukaryotic expression vector the pcDNA-TSPYL5 can inhibit the Wnt signaling pathway. We also was detected by Western blotting TSPYL5 before and after transfection AGS cells, total protein, cytoplasmic protein and nucleoprotein β-catenin expression results showed that the AGS cell nuclear β-catenin expression significantly decreased TSPYL5 after transfection, the results further prompted TSPYL5 Wnt pathway inhibition of the targets may be reduced in the β-catenin conclusions: 1. TSPYL5 expression related to the development of gastric carcinogenesis. 2. TSPYL5 possibly through inhibition of the key molecule β-catenin in the Wnt / β-catenin signaling pathway to inhibit the activation of the Wnt / β-catenin signaling pathway to play its inhibitory effect.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Gastric neoplasms
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