The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated ...The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.展开更多
AIM： To investigate the effect of telomerase hTERT gene antisense oligonucleotide （hTERT-ASO） on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3. METHODS： MTT assay was used to detect t...AIM： To investigate the effect of telomerase hTERT gene antisense oligonucleotide （hTERT-ASO） on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3. METHODS： MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity, the cells were divided into there groups： Control group （pancreatic cancer cell Bxpc-3）; antisense oligonucleotide （hTERT-ASO） group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay. Cell apoptosis was observed by transmission electron microscope in each group. RESULTS： After treatment with 6 mmollL hTERT- ASO, cell proliferation was inhibited in dose- and time- dependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage. CONCLUSION： Telomerase antisense oligodeoxy- nucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.展开更多
Objective:The aim of the study was to examine the effect of Sp1 on the expression of the human telomerase reverse transcriptase(hTERT) gene in human colorectal carcinoma SW480 cells.Methods:The Sp1 shRNA plasmid was t...Objective:The aim of the study was to examine the effect of Sp1 on the expression of the human telomerase reverse transcriptase(hTERT) gene in human colorectal carcinoma SW480 cells.Methods:The Sp1 shRNA plasmid was transfected into colorectal carcinoma SW480 cells line by liposome mediation for transient expression.After Sp1 shRNA plasmid transfected SW480 cells,the exogenous Sp1 protein expression was determined by the method of Western blot.At same time,hTERT mRNA expression was detected by RT-PCR,telomerase activity was determined by the telomeric repeat amplification protocol(TRAP) assay,and the apoptotic rate of cells was also tested by flow cytometry.Results:The protein expressions of Sp1 gene could be reduce by transfecting of pGenesil-1-Sp1(+) recombinant plasmid into SW480 cells.The apoptotic rate was increased compared with pGenesil-1-Sp1(-)/SW480 and SW480(P < 0.05),which indicated that lowexpression of Sp1 gene could lead to low level of telomerase activity and induce apoptosis.Conclusion:Silencing Sp1 may suppress the activity of telomerase by inhabiting hTERT gene expression.展开更多
To detect the expression of telomerase subunits （human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA） in gastric cancer and to examine the role that different telom...To detect the expression of telomerase subunits （human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA） in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect telomerase suhunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25 %, respectively. The former was significantly higher than the latter （X^2 = 26.4, P〈0.01）. The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 91.7%, respectively and no significant difference was found between them （X^2 =2.1, P〉0.05）. The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100 % and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.展开更多
文摘The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.
文摘AIM： To investigate the effect of telomerase hTERT gene antisense oligonucleotide （hTERT-ASO） on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3. METHODS： MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity, the cells were divided into there groups： Control group （pancreatic cancer cell Bxpc-3）; antisense oligonucleotide （hTERT-ASO） group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay. Cell apoptosis was observed by transmission electron microscope in each group. RESULTS： After treatment with 6 mmollL hTERT- ASO, cell proliferation was inhibited in dose- and time- dependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage. CONCLUSION： Telomerase antisense oligodeoxy- nucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.
基金Supported by grants from the Natural Science Foundation of Shanxi Province, National Natural Science Foundation,and University Technology Development Project of Shanxi Province, China
文摘Objective:The aim of the study was to examine the effect of Sp1 on the expression of the human telomerase reverse transcriptase(hTERT) gene in human colorectal carcinoma SW480 cells.Methods:The Sp1 shRNA plasmid was transfected into colorectal carcinoma SW480 cells line by liposome mediation for transient expression.After Sp1 shRNA plasmid transfected SW480 cells,the exogenous Sp1 protein expression was determined by the method of Western blot.At same time,hTERT mRNA expression was detected by RT-PCR,telomerase activity was determined by the telomeric repeat amplification protocol(TRAP) assay,and the apoptotic rate of cells was also tested by flow cytometry.Results:The protein expressions of Sp1 gene could be reduce by transfecting of pGenesil-1-Sp1(+) recombinant plasmid into SW480 cells.The apoptotic rate was increased compared with pGenesil-1-Sp1(-)/SW480 and SW480(P < 0.05),which indicated that lowexpression of Sp1 gene could lead to low level of telomerase activity and induce apoptosis.Conclusion:Silencing Sp1 may suppress the activity of telomerase by inhabiting hTERT gene expression.
文摘To detect the expression of telomerase subunits （human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA） in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect telomerase suhunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25 %, respectively. The former was significantly higher than the latter （X^2 = 26.4, P〈0.01）. The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 91.7%, respectively and no significant difference was found between them （X^2 =2.1, P〉0.05）. The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100 % and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
