Studies On The Effects And Mechanism Of Tspyl5 Anti-oncogene On Development And Progress In Gastric Carcinoma

Objective:To investigate the effect of TSPYL5 (Testis-Specific Protein, Y-encoded Like protein 5) on the biobehavion of gastric cancer and study its potential molecular mechanism of Wnt/β-catenin signaling passway in gastric cancer.Methods:1. Detection the expression of TSPYL5 in gastric carcinoma and adjacent normal tissues by RT-PCR, Q-PCR, immunofluorescence technique.2. Checking out the effection of TSPYL5 in AGS cells by MTT, clone formation.3. To study whether the TSPYL5 suppressed Wnt/β-catenin signaling transcriptional activity through inhibition ofβ-catenin protein expression by Luciferase Reporter Assay, western blot, immunohistochemisty, immunofluorescence technique. Result:1. RT-PCR, immunofluorescence technique was used to detect the 46 gastric cancer and adjacent normal tissues, we found that the expression of TSPYL5 mRNA is frequently down-regulated in human gastric cancers. Furthermore ,the result of Q-PCR show the positive loss expression of TSPYL5mRNA in gastric carcinoma (P<0.05).In one word, the expression of TSPYL5 mRNA has significantly decreased in TNM and none metastasis but negative situation in tumor size, Tumor-node-metastasis stage, depth of invasion and lymph node metastases.2. The result of MTT shows that the pc-DNA-TSPYL5 inhibited the proliferation in AGS cells from first day to fifth day. The absorbance ratios were 0.48, 0.75, 1.20 in pc-DNA-TSPYL5, pc-DNA, mock, respectively on fifth day (P<0.05). In the clone formation assays, the cloning efficiency were 100% in control group and 18.6% in TSPYL5-AGS group, which indicated that the TSPYL5 inhibited the proliferation in AGS cells.3. According to the immunohistochemisty, the significantly positive expression ofβ-catenin protein was about 47.8%(22/46) in gastric carcinoma. In the same time, we also used western blotting to detect the expression TSPYL5 andβ-catenin protein, which indicated that TSPYL5 was down-regulated, especially in gastric cancers showing concomitant accumulation ofβ-catenin. In AGS cells ,the tested group proved theβ-catenin protein down-regulated compared with control group by immunofluorescence technique. In the functional experiment, we used Luciferase Reporter Assay and confirmed that the luciferase activity in the pcDNA-TSPYL5 group significantly decreased than the pc-DNA group and the difference has significance in statistics (P<0.05). It shows that the Wnt signaling passway can be inhibited by transecting eukaryotic expression vector pcDNA-TSPYL5.We also tested the expression ofβ-catenin in AGS total protein, plasmosin and nucleoprotein before and after the transfection of TSPYL5 by Western blotting. The result showed that the expression ofβ-catenin in nucleus of AGS strain cells decreases. The result suggests that TSPYL5 has an effect on Wnt signaling passway and the target may beβ-catenin protein.Conclusion:1. The down-regulation of TSPYL5 may be had a significance effect on development and progress in gastric cancer.2. TSPYL5 may inhibit tumor growth by inhibitting the key moleculeβ-catenin in Wnt/β-catenin signaling passway to inhibit the activation of Wnt/β-catenin signaling passway.