| Background:Gastric carcinogenesis is a complex,multistep,and multifactorial event in which the accumulation of many genetic changes such as oncogenes,tumor-suppressor genes,DNA mismatching repair genes,cellular adhesion molecular,telomere/telomerase, cell cycle regulators and growth factor/receptor system play important role.The POT1 (protection of telomeres,POT1) is a single-stranded telomeric DNA binding protein, identified first in fission yeast and human by Peter Baumann et al,in 2001,which is proposed to play an important role in regulation of telomere length and stability of chromosome.Down-regulation of POT1 expression levels or disabled POT1 may produce telomere dysfunction and chromosomal aberration,which closely associate with cell aging and carcinogenesis.In order to further investigate expression of the hPOT1(human protection of telomeres,hPOT1) gene in gastric cancer and its relation to gastric carcinogenesis,we detected the expression of hPOT1 in gastric mucosa samples from 57 patients with gastric cancer and 10 normal controls by SP immunohistochemistry.results showed that the expression of hPOT1 was significantly up-regulated in gastric cancer tissue than in normal gastric mucosa,Moreover,we investigated the effect of hPOT1 protein on the proliferation of gastric cancer cell line SGC7901 transfected with the sense and anti-sense eukaryotic expression vectors of hPOT1.The results show that the cells transfected with the antisense gene showed a significant down regulation of the hPOT1 expression and a slowdown of cell growth.Our study provides a new experimental basis for further elucidating hPOT1's biological functions and its relationship with the carcinogenesis and development of gastric cancer.Object:To explore the biological functions of hPOT1 and its relationship with carcinogenesis of gastric cancer by observing the expression of hPOT1 in gastric cancerous tissues and their clinical significance,the effect of hPOT1 protein on the growth and proliferation of gastric cancer cell line SGC7901. Methods:We detected the expression of hPOT1 in gastric mucosa samples from 57 patients with gastric cancer and 10 normal controls by SP immunohistochemistry, constructed the sense and anti-sense eukaryotic expression vectors of hPOT1 by molecular cloning and then identified them by PCR,enzymatic digestion and sequencing.The sense and anti-sense eukaryotic expression vectors of hPOT1 were transfected into gastric cancer cell line SGC7901;Meanwhile,cells transfected with void vectors and cells not transfected were used as controls.The positive clones were screened by the G418 resistance assay and then identified through PCR amplification of the exogenous NeoR gene.hPOT1 protein was detected in transfected SGC7901 cells by Western blotting.Furthermore,we observed cell growth curves by MTT assay,cell cycle and apoptosis by flow cytometry,and cell morphology under transmission electron microscope,respectively.Results:(1)All 57 cases gastric cancer showed positive staining of hPOT1(100%), which was much higher than that in normal gastric mucosa(20%),hPOT1 expression levels were significantly higher in infiltrated plasma membrane tumors than in which didn't infiltrate plasma membrane,in stageâ…¢/â…£tumors than in stageâ… /â…¡tumors,in poorly differentiated tumors than in well-differentiated tumors(P＜0.05).(2)PCR,Restriction enzyme mapping and sequencing assay were performed on DNA from transformed clones, demonstrated the presence of the hPOT1 cDNA insert cloned in the sense and antisense orientation in the pCDNA3.1(-) vector,which were named as pcDNA-s-hPOT1 and pcDNA-as-hPOT1.(3)The sense and antisense eukaryotic expression vectors of hPOT1 were transfected into gastric cancer cell line SGC7901 with LipofectamineTM 2000 and gain clones through G418 selection respectively,which were named as SGC7901s, SGC7901as and SGC7901neo.(4)The transfected cells were identified through PCR amplification of the exogenous NeoR gene.A specific 276bp Neo gene fragment can be found in transfected G418-resistant cells with the sense or anti-sense eukaryotic expression vectors of hPOT1,or void vectors,but negative in the cells without transfection. (5)Compared with SGC7901,SGC7901neo and SGC7901s cells,a significant downregulational expression of the hPOT1 protein(P＜0.05) and an arrest of cell growth were found in SGC7901 as cells which transfected with the antisense eukaryotic expression vectors of hPOT1.(6)Flow cytometric analysis displayed an accumulation of transfected SGC7901as cells in the G2/M phase of cell cycle,the decrease in S phase,increase in apoptosis(15.28±0.8%vs 1.84±1.3%,1.93±1.6%,1.75±0.6%,P＜0.05).No significant difference was found among SGC7901s,SGC7901neo and SGC7901 cells.(7) Observations under the electron microscope revealed apoptotic changes in the cells transfected with the antisense gene,such as increased electron density,cytoplasmic condensation,karyopyknosis,chromatin aggregation,and chromosome margination.Conclusion:Our results showed that hPOT1 is abnormally overexpressed in gastric cancer tissue,and the expression level determined was closely correlated to the differentiation level,infiltration depth,and clinical TNM stage of gastric cancer.Eukaryotic expression vectors of sense and antisense hPOT1 were successfully constructed,and trasfected into gastric cancer cell line SGC7901,respectively.The hPOT1 antisense eukaryotic expression vector pcDNA-as-hPOT1 could block the expression of hPOT1 greatly,inhibit proliferation of Gastric Gancer Cell Line SGC7901,induce their apoptosis.Our results should further provide some clues to hPOT1's biological functions and its relationship with the carcinogenesis and development of gastric cancer.
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