The Study Of T-STAR Effect On Telomerase Activity

Objective: To investigate the protein expression of T-STAR in tumor tissue, and its correlation with hTERT and the activity of Telomerase. To study the affection and mechanism of the sense and antisense T-STAR gene on biological characteristic, expression of T-STAR and hTERT and telomerase activity in vitro culture tumor cells.Materials and methods: 1. The expression of T-STAR 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 T-STAR, hTERT and the telomerase activitiy was analysed. 2. Molecular cloning technique was used to construct the antisense T-STAR eukaryotic expressiion vector pneo-STAR. And then stably transfecting the sense and antisense T-STAR eukaryotic expression vector and control vector pCl-neo and pEGFP-cl 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 pEGFP-cl/T-STAR and pneo-STAR were observed. Meanwhile the changes of the expression level of T-STAR, hTERT and telomerase activity 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. T-STAR was widely expressed in tumor tissue and normal tissue of lung, liver, stomach, colon and recta, and the expression in tumor tissue was much higher than in normal tissue.2. Of the positive expression cell, T-STAR was chiefly expressed in gland cells. T-STAR was expressed mainly in cytoplasma, but also in nucleus.3.The antisense T-STAR eukauyotic expression vector pneo-STAR was constructed successfully.4.The various eukaryotic expression vectors such as pEGFP-cl/T-STAR, pneo-STAR,pEGFP-cl and pcl-neo were transfected into 4 kinds of vitro cultured tumor cells, such as SGC-7901, A549, HepG2 and HCT-116. Those vector was expressed availably in vitro. The expression level of T-STAR protein increased 1-2 times in the cell transfected with pEGFP-c1/T-STAR and decreased 75-85% in the cells transefected with pneo-STAR.5. Of the positive colon stably transfected with various vectors after G418 screeing, the colon transfected with pEGFP-c1/T-STAR transfomated phaenotype decreased , such as growth slowered, the colons reduced, karyoplasmic ratio increased , the cells transfected with pneo-STAR changed reversely.6. The expression of hTERT was accordant with the telomerase activity through the study of tumor tissue and vitro cultured tumor cells.7. The expression of T-STAR was positively correlated with the expression of hTERT and telomerase activity. That means, the upregulation of T-STAR expression can leads to the increasing activity of the telomerase.Conclusion:1. T-STAR was widely expressed in tumor tissue and normal tissue of lung, liver, stomach, colon and recta, and the expression in tumor tissue was much higher than in normal tissue.2.The antisense T-STAR eukaryotic expression vector pneo-STAR was constructed successly. The sense and antisense T-STAR gene eukaryotic expression vector (pEGFP-cl/T-STAR, pneo-STAR) and the control vector (pEGFP-cl, pcl-neo) was transfected into 4 kinds of vitro cultured tumor cells such as SGC-7901, A549, HepG2 and HCT-116. Those vector was expressed availably in vitro.3. T-STAR may be involved in the cellular signal transduction pathways; T-STAR can decrease the transformated phaenotypr, and inhibit cell growth and colon formation.4. T-STAR has the positive correlation with the expression of hTERT and telomerase activity. It maybe upregulation telomerase cativity thought phosphorylation the hTERT reversibility or act as a pre-mRNA alternative regulator of hTERT...