To study the influence of siRNA targeting survivin gene on the biological behavior of hepatocellular carcinoma (HCC), one pair of 21bp reverse repeated motifs of survivin target sequence with 9 spacers were synthesize...To study the influence of siRNA targeting survivin gene on the biological behavior of hepatocellular carcinoma (HCC), one pair of 21bp reverse repeated motifs of survivin target sequence with 9 spacers were synthesized and inserted into plasmid psilencer2.1 to generate siRNA eukaryotic expression vector. After stable transfection into HepG2 cells, the biological behaviors of the survivin siRNA transfected HCC cells were observed. After the recombinant plasmid Psilence(+)-survivin was successfully constructed, survivin mRNA and protein expression inhibition ratio reached 73 % and 75 % respectively compared to control groups. Transfected cells with survivin siRNA demonstrated significantly inhibited cell growth and increased apoptosis. Subsequent study in nude mouse model demonstrated lower succeeding rate in cells transfected with survivin siRNA and slow growth rate. The results elucidated the siRNA targeting survivin gene could specially suppress its expression in HepG2 cells and inhibit tumor cells growth both in vivo and in vitro. This provides a theoretical basis to turn the drug resistance in tumor cells.展开更多
Survivin, a newly identified member of lAP family, is a powerful apoptosis-inhibiting factor. It is expressed in embryonic tissues as well as in the majority of human cancers, but not in most normal adult tissues. The...Survivin, a newly identified member of lAP family, is a powerful apoptosis-inhibiting factor. It is expressed in embryonic tissues as well as in the majority of human cancers, but not in most normal adult tissues. The cancer-specific expression of survivin makes it a potential target for cancer treatment. A survivin-specific small inhibitory RNA （siRNA） was introduced into hepatocellular carcinoma cells to investigate its effect on cancer cell apoptosis, growth and sensitivity to chemotherapeutic drugs. It was found that expressions of survivin protein and proliferation index （PI） in siRNA groups were significantly decreased, the apoptosis index （AI） of siRNA groups was significantly higher than those of others groups, and the growth inhibition rate （GIR） of chemotherapeutic drugs in siRNA groups were significantly higher than those of other groups. Our study suggests that the expression of survivin may be significantly decreased in hepG2 cell after siRNA transfection. siRNA targeting survivin could induce cell apoptosis, inhibit cell proliferation and sensitize hepatocarcinoma cells to chemotherapy. Our findings provide preliminary evidence for the therapeutic use of survivin-targeted RNA interference for human tumors that express high levels of this molecule.展开更多
Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA re...Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis. Results Restriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treatin~ alone （P〈0.05）. Conclusion The siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5- fluorouracil.展开更多
Objective： Survivin is one of the apoptosis inhibitor genes and is rarely expressed in adult tissues. However, survivin expression has been detected in various human cancers and correlations have been recognized betw...Objective： Survivin is one of the apoptosis inhibitor genes and is rarely expressed in adult tissues. However, survivin expression has been detected in various human cancers and correlations have been recognized between the level of expression of this gene in tumors and prognosis. In this study, we investigated the effect of Survivin-siRNA on the drug sensitivity of osteosarcoma cell line MG-63. Methods： Two siRNAs （Survivin-siRNA1, Survivin-siRNA2） specifically targeting Survivin gene were chemically synthesized and transfected into MG-63 ceils. The Survivin mRNA level was detected by reverse transcription-polymerase chain reaction （RT-PCR）. The survivin protein expression and cell apoptosis rate were analyzed by flow cytometry （FCM）. The 50% inhibition concentration （IC50） of cisplatin （DDP） and adriamycin （ADM） on MG-63 cells was determined by MTT method. Results： Two short siRNA targeting survivin down-regulated the transcription of survivin gene dramatically and elevated apoptosis rate. They increased the drug sensitivity of MG-63 cells to ADM by five-fold and to DDP by nine-fold. Conclusion： Validated Survivin specific siRNA can effectively inhibit Survivin expression in survivin-overexpressing osteosarcoma MG-63 cell line and enhance the drug sensitivity of MG-63 cell line to ADM and DDP. Short survivin-siRNA mediated gene silencing may be a useful therapeutic strategy for osteosarcoma. These results suggest that survivin might be helpful for diagnosis of osteosarcoma and survivin siRNA combined with adriamycin or cisplatin may be a feasible strategy to enhance the effects of chemotherapy in patients with osteosarcoma.展开更多
Objective： To study the inhibition of expression of Survivin gene by synthesized short Survivin-siRNA in osteosarcoma cell line MG-63. Methods： Chemically synthesized short Surviving-siRNA was transfected into osteo...Objective： To study the inhibition of expression of Survivin gene by synthesized short Survivin-siRNA in osteosarcoma cell line MG-63. Methods： Chemically synthesized short Surviving-siRNA was transfected into osteosarcoma cell line MG-63. The Survivin mRNA and protein level were detected by reverse transcription-polymerase chain reaction （RT-PCR） and flow cytometry （FCM）. The biological morphology and growth inhibition of MG-63 were observed with light microscopy and MTT assay, respectively. Results： Short siRNA targeting Survivin down-regulated the transcription and the protein level of Survivin oncogene. The proliferation of osteosarcoma cell line MG-63 was inhibited after transfection. Conclusion： Chemically synthesized short Survivin-siRNA can effectively inhibit Survivin expression and cell proliferation inosteosarcoma cell line MG-63 Survivin-siRNA mediated Survivin gene silencing may be a useful therapeutic strategy for osteosarcoma.展开更多
Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA o...Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA oligonucleotides were designed and synthesized artificially. This siRNA展开更多
Background:Cancer is the leading cause of death in Canada.One of the major challenges in the cancer therapy is chemoresistance,which plays a critical role in cancer dissemination,relapse and patient clinical outcome.I...Background:Cancer is the leading cause of death in Canada.One of the major challenges in the cancer therapy is chemoresistance,which plays a critical role in cancer dissemination,relapse and patient clinical outcome.In retinoblastoma,the most common cancer affecting children eyes,refractory cases are treated by enucleation in order to avoid brain metastasis.Overexpression of the oncogene survivin is recurrent in many cancer types,including in retinoblastoma.Survivin upregulation results in apoptosis inhibition,cell mitosis stimulation and DNA repair.Therefore,survivin silencing through small-interfering RNA(siRNA)technology has emerged as a powerful approach in cancer therapy to overcome chemoresistance.However,the efficacy of siRNA mainly relies on its successful delivery,limited by its highly hydrophilic nature and rapid nucleases degradation.We have recently developed lipid nanoparticle able to protect siRNA in the systemic circulation and release it within the cell in a pH-sensitive fashion.Here we propose to downregulate the survivin oncogene,in vitro,through a liposome-mediated siRNA delivery to improve drug’s effectiveness in a retinoblastoma cell line model(Y79).Methods:Firstly,we formulated a survivin-targeted nanocomplex(STN)composed of pH-sensitive liposomes and survivin-targeted siRNA.STN was characterized regarding size,polydispersity index and zeta potential.The survivin silencing efficiency in retinoblastoma cells(Y79)was assessed through RT-qPCR and Western blotting assays.Then,we evaluate the impact of survivin-silencing on Carboplatin,Topotecan,Teniposide and Melphalan’s effectiveness in Y79 cells through a cell viability assay.Results:A novel STN was formulated within desired nanoscale parameters.Survivin was efficiently depleted at both mRNA(RT-qPCR)and protein levels(western Blot).Furthermore,survivin downregulation synergistically improved chemotherapeutics’effectiveness in vitro in Y79 cells.Interestingly,survivin downregulation did not modify carboplatin’s effect in a non-tumoral human retinal cells(ARPE-19).Conclusions:Those results demonstrate the cancer specific expression of the oncogene survivin and highlight the survivin-silencing as a promising retinoblastoma-specific therapy.This strategy holds a promising clinical translational application,encouraging further experimentation in vivo.展开更多
Objective:The aim of this study was to investigate the molecular mechanism of anti-apoptotic action of survivin to the hepatoma-cellular carcinoma cell line HepG2.Methods:Design and synthesize siRNA gene sequence spec...Objective:The aim of this study was to investigate the molecular mechanism of anti-apoptotic action of survivin to the hepatoma-cellular carcinoma cell line HepG2.Methods:Design and synthesize siRNA gene sequence specifically targeting at HepG2 cell.HepG2 cells cultures were divided into five groups:blank control group,negative control group,low dose group,medium dose group and high dose group.HepG2 cells were treated respectively by pshRNA-survivin-387 of different concentrations.The apoptosis index(AI) was determined by flow cytometry(FCM).Cells were stained with rhodomine-123(Rh123) to detect changes of mitochondrial membrane potentials.The concentration of cytoplasmic cytochrome C(Cyt.C) was continuously determined by ELISA.Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay.Results:Compared with the control group,due to the function of short interference RNAs(SiRNAs) that suppresses the survivin gene expression,the apoptotic index of transfected groups were significantly higher than those of control groups(F = 13568.68,q = 110.47-327.16,P < 0.01),the apoptosis index of high concentration of transfected cells was higher than the low concentration transfected group(q = 39.63-168.22,P < 0.01).The apoptosis index of high concentrations transfected HepG2 cells was 25.54%,higher than that of blank control group,negative control group,low dose group and medium dose group(5.24%,6.61%,12.63% and 15.64%,respectively).HepG2 cells transfected with SiRNA exhibit gradually decreasing mitochondrial membrane potentials,which then lead to the releasing of Cyt C,following it were the activation of caspase-9 and caspase-3.Conclusion:Survivin performs the function of anti-apoptosis to the HepG2 cells via modulating the apoptosis of mitochondrial.HepG2 cells transfected with SiRNA survivin can significantly induce apoptosis.展开更多
基金This project was supported by a grant from National Na ture Sciences Foundation of China (No. 30271242)
文摘To study the influence of siRNA targeting survivin gene on the biological behavior of hepatocellular carcinoma (HCC), one pair of 21bp reverse repeated motifs of survivin target sequence with 9 spacers were synthesized and inserted into plasmid psilencer2.1 to generate siRNA eukaryotic expression vector. After stable transfection into HepG2 cells, the biological behaviors of the survivin siRNA transfected HCC cells were observed. After the recombinant plasmid Psilence(+)-survivin was successfully constructed, survivin mRNA and protein expression inhibition ratio reached 73 % and 75 % respectively compared to control groups. Transfected cells with survivin siRNA demonstrated significantly inhibited cell growth and increased apoptosis. Subsequent study in nude mouse model demonstrated lower succeeding rate in cells transfected with survivin siRNA and slow growth rate. The results elucidated the siRNA targeting survivin gene could specially suppress its expression in HepG2 cells and inhibit tumor cells growth both in vivo and in vitro. This provides a theoretical basis to turn the drug resistance in tumor cells.
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No 30471533)
文摘Survivin, a newly identified member of lAP family, is a powerful apoptosis-inhibiting factor. It is expressed in embryonic tissues as well as in the majority of human cancers, but not in most normal adult tissues. The cancer-specific expression of survivin makes it a potential target for cancer treatment. A survivin-specific small inhibitory RNA （siRNA） was introduced into hepatocellular carcinoma cells to investigate its effect on cancer cell apoptosis, growth and sensitivity to chemotherapeutic drugs. It was found that expressions of survivin protein and proliferation index （PI） in siRNA groups were significantly decreased, the apoptosis index （AI） of siRNA groups was significantly higher than those of others groups, and the growth inhibition rate （GIR） of chemotherapeutic drugs in siRNA groups were significantly higher than those of other groups. Our study suggests that the expression of survivin may be significantly decreased in hepG2 cell after siRNA transfection. siRNA targeting survivin could induce cell apoptosis, inhibit cell proliferation and sensitize hepatocarcinoma cells to chemotherapy. Our findings provide preliminary evidence for the therapeutic use of survivin-targeted RNA interference for human tumors that express high levels of this molecule.
基金Supported by National High Technology Research and Development Program of China (2001AA218051)Educational Commission of Hubei Province of China (2005A304B09)
文摘Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis. Results Restriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treatin~ alone （P〈0.05）. Conclusion The siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5- fluorouracil.
文摘Objective： Survivin is one of the apoptosis inhibitor genes and is rarely expressed in adult tissues. However, survivin expression has been detected in various human cancers and correlations have been recognized between the level of expression of this gene in tumors and prognosis. In this study, we investigated the effect of Survivin-siRNA on the drug sensitivity of osteosarcoma cell line MG-63. Methods： Two siRNAs （Survivin-siRNA1, Survivin-siRNA2） specifically targeting Survivin gene were chemically synthesized and transfected into MG-63 ceils. The Survivin mRNA level was detected by reverse transcription-polymerase chain reaction （RT-PCR）. The survivin protein expression and cell apoptosis rate were analyzed by flow cytometry （FCM）. The 50% inhibition concentration （IC50） of cisplatin （DDP） and adriamycin （ADM） on MG-63 cells was determined by MTT method. Results： Two short siRNA targeting survivin down-regulated the transcription of survivin gene dramatically and elevated apoptosis rate. They increased the drug sensitivity of MG-63 cells to ADM by five-fold and to DDP by nine-fold. Conclusion： Validated Survivin specific siRNA can effectively inhibit Survivin expression in survivin-overexpressing osteosarcoma MG-63 cell line and enhance the drug sensitivity of MG-63 cell line to ADM and DDP. Short survivin-siRNA mediated gene silencing may be a useful therapeutic strategy for osteosarcoma. These results suggest that survivin might be helpful for diagnosis of osteosarcoma and survivin siRNA combined with adriamycin or cisplatin may be a feasible strategy to enhance the effects of chemotherapy in patients with osteosarcoma.
文摘Objective： To study the inhibition of expression of Survivin gene by synthesized short Survivin-siRNA in osteosarcoma cell line MG-63. Methods： Chemically synthesized short Surviving-siRNA was transfected into osteosarcoma cell line MG-63. The Survivin mRNA and protein level were detected by reverse transcription-polymerase chain reaction （RT-PCR） and flow cytometry （FCM）. The biological morphology and growth inhibition of MG-63 were observed with light microscopy and MTT assay, respectively. Results： Short siRNA targeting Survivin down-regulated the transcription and the protein level of Survivin oncogene. The proliferation of osteosarcoma cell line MG-63 was inhibited after transfection. Conclusion： Chemically synthesized short Survivin-siRNA can effectively inhibit Survivin expression and cell proliferation inosteosarcoma cell line MG-63 Survivin-siRNA mediated Survivin gene silencing may be a useful therapeutic strategy for osteosarcoma.
文摘Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA oligonucleotides were designed and synthesized artificially. This siRNA
文摘Background:Cancer is the leading cause of death in Canada.One of the major challenges in the cancer therapy is chemoresistance,which plays a critical role in cancer dissemination,relapse and patient clinical outcome.In retinoblastoma,the most common cancer affecting children eyes,refractory cases are treated by enucleation in order to avoid brain metastasis.Overexpression of the oncogene survivin is recurrent in many cancer types,including in retinoblastoma.Survivin upregulation results in apoptosis inhibition,cell mitosis stimulation and DNA repair.Therefore,survivin silencing through small-interfering RNA(siRNA)technology has emerged as a powerful approach in cancer therapy to overcome chemoresistance.However,the efficacy of siRNA mainly relies on its successful delivery,limited by its highly hydrophilic nature and rapid nucleases degradation.We have recently developed lipid nanoparticle able to protect siRNA in the systemic circulation and release it within the cell in a pH-sensitive fashion.Here we propose to downregulate the survivin oncogene,in vitro,through a liposome-mediated siRNA delivery to improve drug’s effectiveness in a retinoblastoma cell line model(Y79).Methods:Firstly,we formulated a survivin-targeted nanocomplex(STN)composed of pH-sensitive liposomes and survivin-targeted siRNA.STN was characterized regarding size,polydispersity index and zeta potential.The survivin silencing efficiency in retinoblastoma cells(Y79)was assessed through RT-qPCR and Western blotting assays.Then,we evaluate the impact of survivin-silencing on Carboplatin,Topotecan,Teniposide and Melphalan’s effectiveness in Y79 cells through a cell viability assay.Results:A novel STN was formulated within desired nanoscale parameters.Survivin was efficiently depleted at both mRNA(RT-qPCR)and protein levels(western Blot).Furthermore,survivin downregulation synergistically improved chemotherapeutics’effectiveness in vitro in Y79 cells.Interestingly,survivin downregulation did not modify carboplatin’s effect in a non-tumoral human retinal cells(ARPE-19).Conclusions:Those results demonstrate the cancer specific expression of the oncogene survivin and highlight the survivin-silencing as a promising retinoblastoma-specific therapy.This strategy holds a promising clinical translational application,encouraging further experimentation in vivo.
文摘Objective:The aim of this study was to investigate the molecular mechanism of anti-apoptotic action of survivin to the hepatoma-cellular carcinoma cell line HepG2.Methods:Design and synthesize siRNA gene sequence specifically targeting at HepG2 cell.HepG2 cells cultures were divided into five groups:blank control group,negative control group,low dose group,medium dose group and high dose group.HepG2 cells were treated respectively by pshRNA-survivin-387 of different concentrations.The apoptosis index(AI) was determined by flow cytometry(FCM).Cells were stained with rhodomine-123(Rh123) to detect changes of mitochondrial membrane potentials.The concentration of cytoplasmic cytochrome C(Cyt.C) was continuously determined by ELISA.Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay.Results:Compared with the control group,due to the function of short interference RNAs(SiRNAs) that suppresses the survivin gene expression,the apoptotic index of transfected groups were significantly higher than those of control groups(F = 13568.68,q = 110.47-327.16,P < 0.01),the apoptosis index of high concentration of transfected cells was higher than the low concentration transfected group(q = 39.63-168.22,P < 0.01).The apoptosis index of high concentrations transfected HepG2 cells was 25.54%,higher than that of blank control group,negative control group,low dose group and medium dose group(5.24%,6.61%,12.63% and 15.64%,respectively).HepG2 cells transfected with SiRNA exhibit gradually decreasing mitochondrial membrane potentials,which then lead to the releasing of Cyt C,following it were the activation of caspase-9 and caspase-3.Conclusion:Survivin performs the function of anti-apoptosis to the HepG2 cells via modulating the apoptosis of mitochondrial.HepG2 cells transfected with SiRNA survivin can significantly induce apoptosis.
