目的 :设计小鼠 Retn基因特异性的 si RNAs,并构建一系列能在哺乳动物细胞内稳定表达这些 si RNAs的表达质粒 ,以便为在体外研究 Retn基因的功能打下基础。 方法 :(1 )设计并合成小鼠 Retn基因特异性的一组寡核苷酸片段 ,并克隆到 p Sil...目的 :设计小鼠 Retn基因特异性的 si RNAs,并构建一系列能在哺乳动物细胞内稳定表达这些 si RNAs的表达质粒 ,以便为在体外研究 Retn基因的功能打下基础。 方法 :(1 )设计并合成小鼠 Retn基因特异性的一组寡核苷酸片段 ,并克隆到 p Silencer EM 1 .0 - N eo载体 ;(2 )用电穿孔转染和 G4 1 8筛选等方法建立能稳定表达相应 si RNAs的一组 3T3- L1细胞克隆 ;(3)诱导细胞分化为脂肪细胞 ,并用 RT- PCR分析这些脂肪细胞内 Retn基因的 m RNA水平。 结果 :设计并构建了 4个小鼠Retn基因特异性的 si RNAs表达质粒 ,并证明其中的 2种质粒能在脂肪细胞内稳定表达相应的 si RNAs,显著地抑制了这些脂肪细胞内 Retn基因的 m RNA水平。 结论 :本研究设计并构建出的小鼠 Retn基因特异性的 si RNAs表达载体 ,所表达的 si R-NAs具有较强的 RNA干涉功能 ,为展开更多
Objective To observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in...Objective To observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo. Methods Gene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: nave group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively. Results The siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated. Conclusion Pre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.展开更多
Tomato has emerged as an emblematic model plants for fleshy fruit research and tomato fruit ripening is a complex and highly coordinated developmental process.The many physiology and biochemical processes associated w...Tomato has emerged as an emblematic model plants for fleshy fruit research and tomato fruit ripening is a complex and highly coordinated developmental process.The many physiology and biochemical processes associated with tomato fruit ripening require changes in the expression of hundreds to thousands of genes.Gene expression is regulated by transcriptional and post-transcriptional pathways,one of the recently discovered mechanisms in plants was small RNAs mediated gene silencing at post-transcriptional(PTGS) level.Intriguingly,several mi RNAs and endogenous si RNAs were revealed to be involved in the fruit ripening process which opened a new avenue in the field of fleshy fruit biology.This review compiled the most recent advances made in deciphering the regulation functions of mi RNAs and si RNAs in tomato fruit ripening.It also emphasized the new perspectives now possible in the small RNAs regulation research in tomato fruit ripening and senescence.展开更多
Stability, specificity, and pharmacokinetic properties are some of the challenges facing RNAi therapeutics. In this review, the progresses in chemically modified siRNAs and siRNA conjugates are summarized. The proper ...Stability, specificity, and pharmacokinetic properties are some of the challenges facing RNAi therapeutics. In this review, the progresses in chemically modified siRNAs and siRNA conjugates are summarized. The proper modification of siRNA with nucleoside analogues, construction of siRNA conjugates, and reliable prediction of the property based on those strategies for a given siRNA sequence would certainly be an essential part of the solution to these challenges.展开更多
RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed ...RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.展开更多
Nanog, Oct4 and Sox2 are important transcription factors that are expressed in embryonic stem （ES） cells or embryonic carcinoma （EC） cells, but in most cases they are absent in somatic cells. These factors play a ...Nanog, Oct4 and Sox2 are important transcription factors that are expressed in embryonic stem （ES） cells or embryonic carcinoma （EC） cells, but in most cases they are absent in somatic cells. These factors play a key role to maintain embryonic stem cell self-renew and pluripotency. Down-regulation of Nanog and Sox2 gene expression can change multiple gene expression pattems and signal transduction pathways, and will initiate ES cell differentiation. This study was designed to select the efficient small interfering RNA （siRNA） fragments that inhibit Nanog and Sox2 gene expression in mouse J1 ES cells and P19 EC cells. Among synthesized siRNAs we screened out the siRNA N301 for Nanog and siRNA $720 for Sox2, which not only down- regulated of Nanog and Sox2 gene expression, but also interfered embryoid bodies formation. Our study provided the defined siRNA fragments that could be used to investigate the epigenetic function of Nanog and Sox2 genes.展开更多
RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust an...RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.展开更多
Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microsco...Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy （CLSM） method to evaluate a novel delivery system of 3＇-peptide-siRNA therapeutic, which was named 3＇-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3＇-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3＇-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.展开更多
RNA degradation systems (e.g., RNA decay and RNA interference) and the phytohormone abscisic acid (ABA) are both essential for plant growth, development, and adaptation to stress. Although the interplay between these ...RNA degradation systems (e.g., RNA decay and RNA interference) and the phytohormone abscisic acid (ABA) are both essential for plant growth, development, and adaptation to stress. Although the interplay between these pathways has been recognized, the molecular mechanisms governing their coordination remain poorly understood. In this study, we revealed that mutations in the 5′–3′ RNA-degrading enzyme Ethylene Insensitive 5 (EIN5) result in hypersensitivity to ABA in Arabidopsis, whereas defects in the 3′–5′ RNA turnover machinery (ski mutants) do not. The ABA hypersensitivity of ein5 mutants was mitigated by mutating components of the post-transcriptional gene silencing (PTGS) pathway, including DICER-LIKE 2 (DCL2)/DCL4, RNA-Dependent RNA Polymerase 1 (RDR1)/RDR6, and ARGONAUTE 1 (AGO1). ABA treatment substantially increased the abundance of coding-transcript-derived small interfering RNAs (ct-siRNAs) in ein5, predominantly from two genes, Nitrate Reductase 1 (NIA1) and NIA2. Further analysis suggested that NIA1 and NIA2 negatively regulate both the ABA biosynthesis and signaling pathways. The key transcription factor Abscisic Acid Insensitive 3 (ABI3) represses SKI3 expression by directly binding to its promoter, thereby promoting the production of NIA1/NIA2-derived ct-siRNAs, leading to the ABA hypersensitivity of ein5. Conversely, ABA enhances the accumulation of EIN5 as well as DCL4 and AGO1, pointing to distinct regulation of the mRNA decay and PTGS pathways. Collectively, these findings demonstrate the pivotal roles of NIA1 and NIA2 in plant responses to abiotic stress and provide new insights into the interplay between the ABA response and RNA degradation pathways.展开更多
Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulat...Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulate gene expression is still limited.Based on genome-wide DNA methylation landscapes of Arabidopsis thaliana Ler and C24 ecotypes and their reciprocal F1 hybrids which were obtained in our previous work,we analyzed allele-specific DNA methylation and distinguished cis-and trans-regulated DNA methylation in hybrids.Our study indicated that both cis-and trans-regulated DNA methylation played roles in hybrids,when cis-regulation played a major role in CG methylation and trans-regulation played major roles in CHG and CHH methylation.In addition,we observed correlations between trans-regulated DNA methylation and siRNA densities.Enriched siRNA regions were significantly concurrent with highly trans-regulated DNA methylation regions.Our results illustrated DNA methylation regulation patterns integrated with siRNAs in Arabidopsis hybrids,and shed light on understanding the mechanism of epigenetic reprogramming for hybrid vigor.展开更多
文摘目的 :设计小鼠 Retn基因特异性的 si RNAs,并构建一系列能在哺乳动物细胞内稳定表达这些 si RNAs的表达质粒 ,以便为在体外研究 Retn基因的功能打下基础。 方法 :(1 )设计并合成小鼠 Retn基因特异性的一组寡核苷酸片段 ,并克隆到 p Silencer EM 1 .0 - N eo载体 ;(2 )用电穿孔转染和 G4 1 8筛选等方法建立能稳定表达相应 si RNAs的一组 3T3- L1细胞克隆 ;(3)诱导细胞分化为脂肪细胞 ,并用 RT- PCR分析这些脂肪细胞内 Retn基因的 m RNA水平。 结果 :设计并构建了 4个小鼠Retn基因特异性的 si RNAs表达质粒 ,并证明其中的 2种质粒能在脂肪细胞内稳定表达相应的 si RNAs,显著地抑制了这些脂肪细胞内 Retn基因的 m RNA水平。 结论 :本研究设计并构建出的小鼠 Retn基因特异性的 si RNAs表达载体 ,所表达的 si R-NAs具有较强的 RNA干涉功能 ,为
基金Supported by National Natural Science Foundation of China (30672029, 30872436)Central Committee Foundation of Health Care Program (B2009B076)
文摘Objective To observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo. Methods Gene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: nave group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively. Results The siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated. Conclusion Pre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.
基金Supported by the National Natural Sciences Foundation of China(31401536)the Natural Science Foundation of Beijing(6144020)+1 种基金the Special Fund of China Agriculture Research System(CARS-25)the Special Fund for Agro-scientific Research in the Public Interest(201203095)
文摘Tomato has emerged as an emblematic model plants for fleshy fruit research and tomato fruit ripening is a complex and highly coordinated developmental process.The many physiology and biochemical processes associated with tomato fruit ripening require changes in the expression of hundreds to thousands of genes.Gene expression is regulated by transcriptional and post-transcriptional pathways,one of the recently discovered mechanisms in plants was small RNAs mediated gene silencing at post-transcriptional(PTGS) level.Intriguingly,several mi RNAs and endogenous si RNAs were revealed to be involved in the fruit ripening process which opened a new avenue in the field of fleshy fruit biology.This review compiled the most recent advances made in deciphering the regulation functions of mi RNAs and si RNAs in tomato fruit ripening.It also emphasized the new perspectives now possible in the small RNAs regulation research in tomato fruit ripening and senescence.
基金National NaturalScience Foundation of China (Grant No.2093200 1,20832008)National Basic Research Program of China(Grant No.2009ZX09503)
文摘Stability, specificity, and pharmacokinetic properties are some of the challenges facing RNAi therapeutics. In this review, the progresses in chemically modified siRNAs and siRNA conjugates are summarized. The proper modification of siRNA with nucleoside analogues, construction of siRNA conjugates, and reliable prediction of the property based on those strategies for a given siRNA sequence would certainly be an essential part of the solution to these challenges.
基金The Nation "863" Program of China(2006AA02A226)The Joint Funds of National Science Foundation of China (U0632010)+2 种基金The State KeyLaboratory of Phytochemistry and Plant Resources in West ChinaChinese Academy of Sciences (O807B11211, O807E21211)"211 grant of MOE"
文摘RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.
基金supported by the National Natural Science Foundation of China (30871786)the National Basic Research Program of China (973 Program,2009CB941002)
文摘Nanog, Oct4 and Sox2 are important transcription factors that are expressed in embryonic stem （ES） cells or embryonic carcinoma （EC） cells, but in most cases they are absent in somatic cells. These factors play a key role to maintain embryonic stem cell self-renew and pluripotency. Down-regulation of Nanog and Sox2 gene expression can change multiple gene expression pattems and signal transduction pathways, and will initiate ES cell differentiation. This study was designed to select the efficient small interfering RNA （siRNA） fragments that inhibit Nanog and Sox2 gene expression in mouse J1 ES cells and P19 EC cells. Among synthesized siRNAs we screened out the siRNA N301 for Nanog and siRNA $720 for Sox2, which not only down- regulated of Nanog and Sox2 gene expression, but also interfered embryoid bodies formation. Our study provided the defined siRNA fragments that could be used to investigate the epigenetic function of Nanog and Sox2 genes.
文摘RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.
基金Ministry of Science and Technology of China(Grant No.2012AA022501,2012CB720604)the National Natural Science Foundation of China(Grant No.20932001,81302626)
文摘Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy （CLSM） method to evaluate a novel delivery system of 3＇-peptide-siRNA therapeutic, which was named 3＇-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3＇-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3＇-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.
基金supported by the National Natural Science Foundation of China(grant no.32261160572 to H.G.)the Shenzhen Science and Technology Program(grant nos.KQTD20190929173906742 and ZDSYS20230626091659010 to H.G.and JCYJ20190809163019421to Y.L.)the New Cornerstone Science Foundation(grant no.NCI202235 to H.G.).
文摘RNA degradation systems (e.g., RNA decay and RNA interference) and the phytohormone abscisic acid (ABA) are both essential for plant growth, development, and adaptation to stress. Although the interplay between these pathways has been recognized, the molecular mechanisms governing their coordination remain poorly understood. In this study, we revealed that mutations in the 5′–3′ RNA-degrading enzyme Ethylene Insensitive 5 (EIN5) result in hypersensitivity to ABA in Arabidopsis, whereas defects in the 3′–5′ RNA turnover machinery (ski mutants) do not. The ABA hypersensitivity of ein5 mutants was mitigated by mutating components of the post-transcriptional gene silencing (PTGS) pathway, including DICER-LIKE 2 (DCL2)/DCL4, RNA-Dependent RNA Polymerase 1 (RDR1)/RDR6, and ARGONAUTE 1 (AGO1). ABA treatment substantially increased the abundance of coding-transcript-derived small interfering RNAs (ct-siRNAs) in ein5, predominantly from two genes, Nitrate Reductase 1 (NIA1) and NIA2. Further analysis suggested that NIA1 and NIA2 negatively regulate both the ABA biosynthesis and signaling pathways. The key transcription factor Abscisic Acid Insensitive 3 (ABI3) represses SKI3 expression by directly binding to its promoter, thereby promoting the production of NIA1/NIA2-derived ct-siRNAs, leading to the ABA hypersensitivity of ein5. Conversely, ABA enhances the accumulation of EIN5 as well as DCL4 and AGO1, pointing to distinct regulation of the mRNA decay and PTGS pathways. Collectively, these findings demonstrate the pivotal roles of NIA1 and NIA2 in plant responses to abiotic stress and provide new insights into the interplay between the ABA response and RNA degradation pathways.
基金supported by Ministry of Science and Technology of China(2013CBA01402)Ministry of Science and Technology of China(2012CB910900)+1 种基金Ministry of Agriculture of China(2010ZX08010-003)Peking University 985 Program
文摘Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulate gene expression is still limited.Based on genome-wide DNA methylation landscapes of Arabidopsis thaliana Ler and C24 ecotypes and their reciprocal F1 hybrids which were obtained in our previous work,we analyzed allele-specific DNA methylation and distinguished cis-and trans-regulated DNA methylation in hybrids.Our study indicated that both cis-and trans-regulated DNA methylation played roles in hybrids,when cis-regulation played a major role in CG methylation and trans-regulation played major roles in CHG and CHH methylation.In addition,we observed correlations between trans-regulated DNA methylation and siRNA densities.Enriched siRNA regions were significantly concurrent with highly trans-regulated DNA methylation regions.Our results illustrated DNA methylation regulation patterns integrated with siRNAs in Arabidopsis hybrids,and shed light on understanding the mechanism of epigenetic reprogramming for hybrid vigor.
