Calcium phosphate nanoparticles(CaPNPs)have good biocompatibility as gene carriers;however,CaPNPs typically exhibit a low transfection efficiency.Cell penetrate peptide(TAT)can increase the uptake of nanoparticles but...Calcium phosphate nanoparticles(CaPNPs)have good biocompatibility as gene carriers;however,CaPNPs typically exhibit a low transfection efficiency.Cell penetrate peptide(TAT)can increase the uptake of nanoparticles but is limited by its non-specificity.Grafting adhesion peptide adhesion peptide on carriers can enhance their targeting.The Plekho1 gene encodes casein kinase-2 interacting protein-1(CKIP-1),which can negatively regulate osteogenic differentiation.Based on the above,we produced a Mg-CaPNPs-RGD-TAT-CKIP-1 siRNA carrier system via hydrothermal synthesis,silanization and adsorption.The effects of this carrier system on cell endocytosis and biological effects were evaluated by cell culture in vitro.The results demonstrate that CaPNPs with 7%Mg(60 nm particle size,short rod shape and good dispersion)were suitable for use as gene carriers.The carrier system boosted the endocytosis of MG63 cells and was helpful for promoting the differentiation of osteoblasts,and the dual-ligand system possessed a synergistic effect.The findings of this study show the tremendous potential of the Mg-CaPNPs-RGD-TAT-CKIP-1 siRNA carrier system for efficient delivery into cells and osteogenesis inducement.展开更多
AIM: To investigate whether the 7-difluoromethoxyl-5, 4'-di-n-octylgenistein (DFOG), a novel synthetic genistein analogue, affects the growth of gastric cancer cells and its mechanisms. METHODS: A series of genist...AIM: To investigate whether the 7-difluoromethoxyl-5, 4'-di-n-octylgenistein (DFOG), a novel synthetic genistein analogue, affects the growth of gastric cancer cells and its mechanisms. METHODS: A series of genistein analogues were prepared by difluoromethylation and alkylation, and human gastric cancer cell lines AGS and SGC-7901 cultured in vitro were treated with various concentrations of genistein and genistein analogues. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated by DFOG at different concentrations. The growth inhibitory effects were evaluated using MTT and clonogenic assay. The distribution of the phase in cell cycle was analyzed using flow cytometric analysis with propidium iodide staining. The expression of the transcription factor forkhead box M1 (FOXM1) was analyzed by reverse transcription-polymerase chain reaction and Western blotting. The expression levelsof CDK1, Cdc25B, cyclin B and p27KIP1 protein were detected using Western blotting. RESULTS: Nine of the genistein analogues had more effective antitumor activity than genistein. Among the tested analogues, DFOG possessed the strongest activity against AGS and SGC-7901 cells in vitro. DFOG significantly inhibited the cell viability and colony formation of AGS and SGC-7901 cells. Moreover, DFOG efficaciously arrested the cell cycle in G2/M phase. DFOG decreased the expression of FOXM1 and its downstream genes, such as CDK1, Cdc25B, cyclin B, and increased p27KIP1 at protein levels. Knockdown of FOXM1 by small interfering RNA before DFOG treatment resulted in enhanced cell growth inhibition in AGS cells. Up-regulation of FOXM1 by cDNA transfection attenuated DFOG-induced cell growth inhibition in AGS cells. CONCLUSION: DFOG inhibits the growth of human gastric cancer cells by down-regulating the FOXM1 expression.展开更多
Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing sma...Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve, clonogenic assay and xenograft assays. Results: The siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast to the control. The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays, the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2. Conclusion: The si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation of A549 cell in vivo and in vitro.展开更多
Genetic transformation in some plant species, including cotton （Gossypium hirsutum）, is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing （VIGS） by double-stra...Genetic transformation in some plant species, including cotton （Gossypium hirsutum）, is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing （VIGS） by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA （siRNA） in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence： the disruptionenergy of the target （△Gdisruption）, the differential stability of siRNA duplex ends （DSSE）, and the internal stability at positions 9-14 of the siRNA antisense strand （AIS）, which are calculated by Sfold program （http：//sfold.wadsworth. org）. We find that the siRNAs with low mGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold pro- gram can be used to analyze target sequences when car- rying out VIGS design to increase gene-silencing effects in plants.展开更多
基金Project(81571021)supported by the National Natural Science Foundation of ChinaProject(2018zzts944)supported by the Graduate Student Independent Exploration Innovation Fund of the Central South University,China+1 种基金Projects(2015WK3012,2018SK2017)supported by the Hunan Provincial Science and Technology Department,ChinaProject(20160301)supported by New Talent Project of the Third Xiangya Hospital of Central South University,China。
文摘Calcium phosphate nanoparticles(CaPNPs)have good biocompatibility as gene carriers;however,CaPNPs typically exhibit a low transfection efficiency.Cell penetrate peptide(TAT)can increase the uptake of nanoparticles but is limited by its non-specificity.Grafting adhesion peptide adhesion peptide on carriers can enhance their targeting.The Plekho1 gene encodes casein kinase-2 interacting protein-1(CKIP-1),which can negatively regulate osteogenic differentiation.Based on the above,we produced a Mg-CaPNPs-RGD-TAT-CKIP-1 siRNA carrier system via hydrothermal synthesis,silanization and adsorption.The effects of this carrier system on cell endocytosis and biological effects were evaluated by cell culture in vitro.The results demonstrate that CaPNPs with 7%Mg(60 nm particle size,short rod shape and good dispersion)were suitable for use as gene carriers.The carrier system boosted the endocytosis of MG63 cells and was helpful for promoting the differentiation of osteoblasts,and the dual-ligand system possessed a synergistic effect.The findings of this study show the tremendous potential of the Mg-CaPNPs-RGD-TAT-CKIP-1 siRNA carrier system for efficient delivery into cells and osteogenesis inducement.
基金National Natural Science Foundation of China, No. 81172375Hunan Provincial Natural Science Foundation, No. 03JJY5009
文摘AIM: To investigate whether the 7-difluoromethoxyl-5, 4'-di-n-octylgenistein (DFOG), a novel synthetic genistein analogue, affects the growth of gastric cancer cells and its mechanisms. METHODS: A series of genistein analogues were prepared by difluoromethylation and alkylation, and human gastric cancer cell lines AGS and SGC-7901 cultured in vitro were treated with various concentrations of genistein and genistein analogues. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated by DFOG at different concentrations. The growth inhibitory effects were evaluated using MTT and clonogenic assay. The distribution of the phase in cell cycle was analyzed using flow cytometric analysis with propidium iodide staining. The expression of the transcription factor forkhead box M1 (FOXM1) was analyzed by reverse transcription-polymerase chain reaction and Western blotting. The expression levelsof CDK1, Cdc25B, cyclin B and p27KIP1 protein were detected using Western blotting. RESULTS: Nine of the genistein analogues had more effective antitumor activity than genistein. Among the tested analogues, DFOG possessed the strongest activity against AGS and SGC-7901 cells in vitro. DFOG significantly inhibited the cell viability and colony formation of AGS and SGC-7901 cells. Moreover, DFOG efficaciously arrested the cell cycle in G2/M phase. DFOG decreased the expression of FOXM1 and its downstream genes, such as CDK1, Cdc25B, cyclin B, and increased p27KIP1 at protein levels. Knockdown of FOXM1 by small interfering RNA before DFOG treatment resulted in enhanced cell growth inhibition in AGS cells. Up-regulation of FOXM1 by cDNA transfection attenuated DFOG-induced cell growth inhibition in AGS cells. CONCLUSION: DFOG inhibits the growth of human gastric cancer cells by down-regulating the FOXM1 expression.
文摘Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve, clonogenic assay and xenograft assays. Results: The siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast to the control. The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays, the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2. Conclusion: The si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation of A549 cell in vivo and in vitro.
基金supported by Major Program of Joint Funds (Sinkiang) of the National Natural Science Foundation of China (No. U1303282)
文摘Genetic transformation in some plant species, including cotton （Gossypium hirsutum）, is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing （VIGS） by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA （siRNA） in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence： the disruptionenergy of the target （△Gdisruption）, the differential stability of siRNA duplex ends （DSSE）, and the internal stability at positions 9-14 of the siRNA antisense strand （AIS）, which are calculated by Sfold program （http：//sfold.wadsworth. org）. We find that the siRNAs with low mGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold pro- gram can be used to analyze target sequences when car- rying out VIGS design to increase gene-silencing effects in plants.
