AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell li...AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. METHODS: Three different sites of SECs were constructed by PCR. K1/siRNA,K2/siRNA and K3/siRNA are located at sites 194,491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. RESULTS: Introduction of the Kl/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the Kl/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. CONCLUSION: Kl/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that Kl/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.展开更多
1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Ee...1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.展开更多
Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPa...Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.展开更多
Summary:In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol lo...Summary:In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol loaded by acetyl LDL (ac-LDL), and 9.78 % cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with 100 μg /mL ac-LDL or with 100 μg /mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.展开更多
Objective： MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression ...Objective： MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods： The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUCI 9 to get pU6-MGMTi, co-transfected with pEGFP-CI into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results： hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion： MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.展开更多
文摘AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. METHODS: Three different sites of SECs were constructed by PCR. K1/siRNA,K2/siRNA and K3/siRNA are located at sites 194,491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. RESULTS: Introduction of the Kl/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the Kl/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. CONCLUSION: Kl/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that Kl/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.
基金supported by China Post-Doctorial Foundation(2002031255)Rothamsted International Foundation(2002)of the UKNatural Science Foundation of Zhejiang Province,China(M303081).
文摘1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.
文摘Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.
文摘Summary:In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol loaded by acetyl LDL (ac-LDL), and 9.78 % cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with 100 μg /mL ac-LDL or with 100 μg /mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.
文摘Objective： MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods： The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUCI 9 to get pU6-MGMTi, co-transfected with pEGFP-CI into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results： hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion： MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.
