| 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. It has been found that telomere shortening and telomerase activation were involved in the gastric carcinogenesis. In yeast, genes placed near telomeres are silenced, a phenomenon known as telomere position effect. The recent demonstration that human cells exhibit telomere position effect raises the intriguing possibility that genetic instability could be induced by telomere shortening with major implications for cancer. In order to explore the role of telomere length change and telomerase activation in gastric carcinogenesis ,we would seed the exogenous telomere fragment into gastric cancer cells, observe the effect of telomere seeding and telomere position effect on cell biological behaviours, telomere length, telomerase activation, expressions of cell cycle –related genes and the methylation of P16 and hMLH1 gene in gastric cancer cell. Object: To explore the role of telomere seeding and position effect of telomere in gastric carcinogenesis by observing cell biological behaviours, telomere length, telomerase activation, expressions of cell cycle –related genes and the methylation of P16 and hMLH1 gene in gastric cancer cell. Methods: The fluorescence expression plasmid were prepared and transfected to gastric cancer cell SGC7901 by LipofectamineTM 2000,then the plasmid pSX-T2AG3-neo with telomere fragment and the control vector plasmid were transfected to these cells. G418 was used to scan the cell positive clones and PCR was preformed to verify the stable seeding of the exogenous telomere in cell. Telomerase activity was detected by TRAP and telomeric restriction fragment (TRF) was measured by Southern blot. MTT aassay and flow cytometry were used to determine cell growth and cell cycle distribution. Protein expression of p53, p21 and caspase3 was detected by Western blot. Cell morphology by microscope and chromosme change by chromosme karyotype analysis. Expression of endogenous p21 mRNA and caspase3 mRNA were detected with RT-PCR.Methylation status of p16 gene and MLH1 gene in the promoter region were observed with methylation-specific PCR(MSP). At last, the tet-off regulatory system were prepared and then transfected to gastric cancer cell SGC7901 by LipofectamineTM 2000. The fluorescence activity induced by the expression of the target gene (the luciferase gene) which transfected to SGC7901 with tet-off regulatory system were analyzed. Then the luciferase reporter plasmid pBX inserted telomere fragment and its control plasmid pBX-ntf(no teloemere fragment) were co-infected to 7901 cell line with pTet-off regulate plasmid respectively and the luciferase activity was determined with doxycycline's induction. Results:(1) pSX-T2AG3-neo eukaryonic expressing vector interted with telomere TTAGGG fragments and control vector were transfected into gastric cancer cell line SGC7901 with LipofectamineTM 2000 and gain clones through G418 selection respectively, which were named as SGC7901-telo and SGC7901-neo. The cells were examined on DNA level by PCR to determine the correction of transfection and it showed the exogenous telomere could seed into the gastric cancer cell. (2)Relative telomerase activity was significantly lower in 7901-telo than in 7901-neo and 7901(200±2.08 vs 246.33±4.73, 253±2, P<0.05), TRF length in 7901-telo, 7901-neo and 7901 have no significant difference. (3) Compared with 7901 and 7901-neo cells, proliferation in 7901-telo cell was obviously decreased, flow cytometric analysis displayed an accumulation of transfected 7901-telo cell in the G0/G1 phase of cell cycle , the decrease in S phase, the increase in G2M phase and a decreased percentage of proliferation index (PI) ,7901-neo cell had no significant difference compared with 7901 cell. (4) Expressions of p21 in 7901-telo cell was increased ( 0.95±0.026 vs 0.73±0.04,0.68±0.05, P<0.05); p53 decreased (0.59±0.01vs 0.68±0.00,0.69±0.01,P<0.05) and caspase3 no distinct difference(0.60±0.01 vs 0.60±0.03,0.60±0.01, P﹥0.05).(5) After transfection, the number of chromosomes in SGC 7901,7901-neo and 7901-telo cells were all 65-68.(6) Expression of hTERT in 7901-telo cell was decreased ( 0.73±0.00 vs0.95±0.04,0.95±0.02, P<0.05) compared with in 7901-neo and 7901 cells. hTERT mRNA levels decreased significantly in 7901-telo cell compared with gastric carcinoma 7901cell and 7901-neo cell. (0.56±0.01 vs. 0.76±0.02,0.78±0.03, P<0.05). (7) Expression of p21 mRNA in 7901-telo cell was increased (0.99±0.01 vs 0.91±0.01,0.90±0.02,P﹤0.05)compared within 7901-neo and 7901 cells while the expression of caspase-3 had no significant change(0.94±0.01 vs0.94±0.01,0.94±0.01, P﹥0.05). (8) methylation of p16 gene promoter in 7901-telo cell was decreased compared with gastric carcinoma 7901cell and 7901-neo cell.(9) methylation of MLH1 gene promoter had no significant change.(10) with the inducing of Dox, the activity of the fluorescence caused by the expression of the luciferase gene inhabited . With the treatment of deoxycyline to the cell infected pBX and pTet-off or pBX-ntf and pTet-off, the activity of the fluorescence was decreased 4 time compared the front to the later. Conclusion: the pSX-T2AG3-neo eukaryonic expressing vector interted with 1600bp telomere TTAGGG fragments were successfully transfected into gastric cancer cell line SGC7901 with the help of LipofectamineTM 2000. the result showed that telomere seeding decrease the telomerase activity and hTERT expression at mRNA and protein levels, up-regulate the expression of p21 and down-regulate mutant p53 protein, decrease the methylation level of p16 gene promoter and suppress the cell proliferation, but this seeding did not distinctly influence on TRF, hMLH1 promoter methylation and caspase3 mRNA expression, the gastric cancer cell SGC7901 can be transfected by tet-off regulatory system successfully and the expression of luciferase gene could be repressed by Dox. The telomere fragment transfected into gastric cancer cell could decrease the expression of luciferase reporter gene. The upwards research and conclusion further expatiated the effect of chromosome telomere dysfunction in gastric carcinogenesis and to understand the gastric carcinogenesis from a new angle.
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