Effects Of TIN2 On Telomere And Chromosome Of Human Gastric Epithelial Cell Line GES-1

[Objective]TIN2 is a key member of the telomere protection protein complexs(Shelterin),numerous studies have shown that the changes in the structure of the Shelterin,which can lead to the disorder of telomere function and results in a series of diseases.In this study,we investigated the effect of TIN2 on the expression of telomere and chromosome in human gastric epithelial cell line(GES-1)on the basis of the establishment of stable high TIN2 expression and inhibited TIN2 expression in human gastric epithelial cells.[Methods]To establish a stable high TIN2 expression or inhibited TIN2 expression of human gastric epithelial cell GES-1,Southern blot assay was used to detect the average length of telomere,telomere dysfunction lesions caused damage response(TIFs)detected by immunofluorescence / experimental telomeric peptide nucleic acid fluorescence in situ hybridiz-ation(IF/PNA-FISH).Through the analysis of chromosome,we want to see whether abnormal TIN2 expression can lead to chromosome aberration.And the situation of TRF1,POT1,TRF2 related to DNA were detected by chromatin immunoprecipitation(chip).[Results]1.Successfully constructed a stable TIN2 high expression and inhibited TIN2 expression of human gastric epithelial cells GES-1.2.The results of Southern blot showed that the average telomere length of GES-1 cells was shorter when the TIN2 expression was abnormal;the telomere average length in high TIN2 expression GES-1cells was shorter than that of inhibited TIN2 expression GES-1 cells.3.IF/PNA-FISH method for detection of telomere dysfunctioninduced by TIFs found that GES-1 cells with high expression of TIN2 appeared more than 2 TIFs,GES-1 cell group with inhibited TIN2 expression found TIFs occasionally.Through statistical analysis,it was found that the TIFs expression in GES-1 cells with high expression of TIN2 and low expression of TIN2 increased compared with other control groups.The difference was statistically significant(P<0.05).Compared with low expression of TIN2 in GES-1 cells,the TIFs expression of high expression of TIN2 in GES-1 cells increased significantly,and the difference was statistically significant(P<0.05).4.High expression and low expression of TIN2 can lead to chromosome aberration of GES-1cells.The chromosome aberration in high expression of TIN2 is more than that of inhibited TIN2 expression.The differences were statistically significant(P<0.05).5.The results of chip experiment showed that the binding ability of TRF1,TRF2 and telomere DNA was decreased when the expression of TIN2 was abnormal.However,when TIN2 was highly expressed,the binding ability of TRF2 and telomere DNA was markedly lower than that of inhibited TIN2 expression,and the difference was statistically significant(P<0.05).Compared with other groups,when TIN2 was highly expressed,the binding ability of POT1 to telomere DNA was decreased,and the difference was statistically significant(P<0.05).[Conclusions]In human gastric epithelial GES-1 cells,high expression and low expression of TIN2 resulted in shortening of telomere length and damage of telomere function,and chromosome aberration.High expression of TIN2 leads the above situation more.The high expression of TIN2 may decrease of the binding ability of TRF1,TRF2,POT1 and telomere DNA results in the change and the low expression of TIN2 can only lead to the decrease of the binding ability of TRF1 and TRF2 to telomere DNA results in the change.The results suggest that theabnormal expression of TIN2 may play an important role in the development of gastric cancer.