The Study Of Par-4 Effect On Tlomerase Activity

Objective: To investigate the protein expression of par-4 in tumor tissue ,and its correlation with hTERT and the activity of Telomerase. To study the affection and mechanism of the sense and antisense par-4 gene on biological characteristic,expression of par-4 and hTERT and telomerase activity in vitro culture tumor cells. Materials and Methods: 1. The expression of par-4 in 46 cases of tumor tissue and 46 cases of normal tissue were determined by immunohistochemistry, western blot, and RT-PCR. The expression of hTERT in 46 cases of tumor tissue and 46 cases of normal tissue were determined by the methods mentioned above, and the telomerase activitiy in the 46 cases of tumor tissue were determind by TRAP PCR-ELISA。Then the correlation among the expression par-4, hTERT and the telomerase activitiy was analysed. 2. Molecular cloning technique was used to construct the sense and antisense par-4 eukaryotic expressiion vector pClneo/par-4 and pcDNA3.1(-)/par-4. And then stably transfecting the sense and antisense par-4 eukaryotic expression vector and contral vector pClneo and pcDNA3.1(-) into vitro culture tumor cells such as SGC-7901, A549, HCT-116 and HepG2 through G418 screening. 3. The cell biological characteristic and cell growth curve after transfected with pClneo/par-4 and pcDNA3.1(-)/par-4 were observed. Meanwhile the changes of the expression level of par-4 ,hTERT and telomerase were detected by immunohistochemistry, western blot, RT-PCR and TRAP PCR-ELISA. The correlation among the expression of par-4 ,hTERT and telomerase activity were analysed.Results: 1. Par-4 was widely expressed in tumor tissue and normal tissue of stomach, liver, colon and lung. And it's expression level decreased excessively in tumor tissue vs normal tissue.2. par-4 was chiefly in gland cell, vascular endotheliocyte, fibroblast of normal tissue. Par-4 was expressed in plasma of the positive cells. Par-4 was expressed mainly in plasma, but also in cellular nucleus.3. The expression of hTERT was accordant with the telomerase activity of the tumor tissue and vitro cultured tumor cells.4. The sense and antisense par-4 eukaryotic exprssion vector pClneo/par-4 and pcDNA3.1(-)/par-4 was constructed.5. The various eukaryotic exprssion vector such as pClneo/par-4, pcDNA3.1(-)/par-4 was transfected into 4 kinds of vitro tumor culture cells, such as SGC-7901, A549, HCT-116 and HepG2. The foreign gene was expression. The expression of par-4 mRNA and par-4 protein increased 1-2 times in the cells transfected with pClneo/par-4; and that decreased to 20%-30%in the cells transfected with pcDNA3.1(-)/par-4.6. Of the cells stably transfected with various vector after G418 screening, the cells transfected with pClneo/par-4 has nodifference in morphology and growth, but growth of the cell transfected with pcDNA3.1(-)/par-4, such as A549, HCT-116, HepG2 slowered.7. The expression of par-4 has no obvious correlation with the expression of hTERT and the activity of telomerase.Conclusion:1. par-4 expression was widely expressed in normal tissue, and the expression excessively decressed in tumor tissue. The decreased expression of par-4 is correlated with tumor tissue.2. The sense and antisense par-4 eukaryotic expression vector pClneo/par-4 and pcDNA3.1(-)/par-4 was constructed, and those vector were transefected into various vitro culture tumor cells. The foreign gene was expressed availably in the cells transfected with sense and antisense gene.3. The expression of par-4 has no obvious correlation with the expression of hTERT and the activity of telomerase.