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Effects of Transparent Testa8(TT8) gene and Homeobox12(HB12) gene silencing in alfalfa(Medicago sativa L.) on molecular structure spectral profile in relation to energy,degradation,and fermentation characteristics in ruminant systems

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Animal Nutrition 2023年第3期14卷 79-87页

作者： Yaogeng Lei Abdelali Hannoufa Peiqiang Yu Department of Animal and Poultry Science College of Agriculture and BioresourcesUniversity of Saskatchewan51 Campus DriveSaskatoonSK S7N5A8Canada London Research and Development Centre Agriculture and Agri-Food Canada1391 Sandford StreetLondonON N5V 4T3Canada

Alfalfa(Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization.Downregulation of two transcriptional factors, Transparent Testa8(TT8) and Homeobox12(HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8(TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content,bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants(5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control.Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of

关键词： gene silencing Homeobox12(HB12)gene Transparent Testa8(TT8)gene Alfalfa(Medicago sativa L.) Ruminant system Fermentation and degradation

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Elaborately engineering of lipid nanoparticle for targeting delivery of siRNA and suppressing acute liver injury

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Chinese Chemical Letters 2024年第2期35卷 323-328页

作者： Qiu Wang Qikun Jiang Dan Li Zimeng Yang Lin Gao Fan Liu Chang Li Yao Feng Zhonggui He Cong Luo Jin Sun Department of Pharmaceutics Wuya College of InnovationShenyang Pharmaceutical UniversityShenyang 110016China Kangya of Ningxia Pharmaceutical Co.Ltd. Yinchuan 750000China

Small interfering RNA(siRNA)-based gene silencing has been considered as a potential therapy modality against inflammatory diseases.Nevertheless,the effective delivery of siRNA to desired destination still remains challenging due to poor stability,high molecular weight and negative charge.Currently,ionizable lipid nanoparticle(LNP)has been extensively used as vector for effective delivery of siRNA.Herein,we report a mannose-modified LNP(M-MC_(3) LNP@TNFα)loading tumor necrosis factorα(TNFα)siRNA for targeting liver macrophages,achieving effectively inhibit acute liver injury.The M-MC_(3) LNP@TNFαnot only increases the internalization of LNP by macrophages,but also enhances the gene silencing efficiency of TNFαin vitro.Additionally,the M-MC_(3) LNP@TNFαexhibits higher accumulation in liver of healthy mice than that of MC_(3) LNP@TNFα(un-modified LNP)owing to the targeting effect of mannose.As expected,the M-MC_(3) LNP@TNFαsignificantly suppresses the expression of TNFαand ameliorates liver damage in acute liver injury model.Such a LNP targeting siRNA delivery holds great potential for the treatment of diseases associated with liver in the future.

关键词： Lipid nanoparticle Targeting siRNA delivery Acute liver injury gene silencing Tumor necrosis factorα

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Nanomedicine integrating the lipidic derivative of 5-fluorouracil,miriplatin and PD-L1 si RNA for enhancing tumor therapy

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Chinese Chemical Letters 2024年第6期35卷 385-391页

作者： An Lu Yuhao Guo Yi Yan Lin Zhai Xiangyu Wang Weiran Cao Zijie Li Zhixia Zhao Yujie Shi Yuanjun Zhu Xiaoyan Liu Huining He Zhiyu Wang Jian-Cheng Wang Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems State Key Laboratory of Natural and Biomimetic DrugsSchool of Pharmaceutical SciencesPeking UniversityBeijing 100191China Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics School of PharmacyTianjin Medical UniversityTianjin 300070China Department of Immuno-oncology The Fourth Hospital of Hebei Medical UniversityShijiazhuang 050011China Department of Pharmacy Clinical Trial Research CenterChina-Japan Friendship HospitalBeijing 100029China Laboratory of innovative formulations and pharmaceutical excipients Ningbo Institute of Marine MedicinePeking UniversityNingbo 315832China

Immunosuppressive microenvironments present critical problems in clinical chemotherapy.To regulate the tumor immune microenvironment for enhancing antitumor effect,a combination of immune checkpoint inhibitors(ICIs)with chemotherapeutics has been applied clinically.In this study,miriplatin(MiPt),the lipidic derivative of 5-fluorouracil(Fu-OA),as well as the programmed death ligand 1(PD-L1)target si RNA(siPD-L1)were integrated into Lip-Pt/Fu@siPD-L1 nanoparticles(NPs)for chemo-immunotherapy.In vitro results showed that Lip-Pt/Fu@siPD-L1 NPs could exhibit effective siRNA gene silencing and promote the phagocytosis of tumor cells by macrophages.Furthermore,in vivo results revealed that LipPt/Fu@siPD-L1 NPs showed significantly higher anti-tumor efficiency than that of the physical mixing of Mi Pt,5-fluorouracil,and Lip@siPD-L1 NPs(delivery of siPD-L1 by liposomes).The best anti-tumor efficiency of Lip-Pt/Fu@siPD-L1 NPs resulted from the synergistic immunotherapeutic effects of Mi Pt and siPD-L1 based on the inhibition of CD47 expression and the downregulation of PD-L1 in tumor cells,which elicited a robust anti-tumor immune response through the activation of macrophage phagocytosis and immune checkpoint inhibition.The Lip-Pt/Fu@siPD-L1 NPs provide a potential strategy for tumor chemo-immunotherapy.

关键词： Chemo-immunotherapy Immune checkpoint inhibition gene silencing Miriplatin Macrophage phagocytosis Nanotechnology

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gene silencing:Double-stranded RNA mediated mRNA degradation and gene inactivation

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Cell Research 2001年第3期11卷 181-186页

作者： TANG WEI, XIAO YAN LUO, VANESSA SANMUELS North Carolina State University, Forest Biotechnology Groap, Raleigh, NC 27695, USA University of North Carolina, Department of Cell and Developmental Biology, ChaPel Hill, NC 27599, USA North Carolina State University Forest Biotechnology Group Raleigh USA Department of Cell and Developmental Biology University of North Carolina Chapel Hill USA

The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.

关键词： gene silencing double-stranded RNA methylation homologous RNA transgene.

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Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

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Neural Regeneration Research 2014年第8期9卷 806-814页

作者： Zhiyuan Li Zhanxiu Zhang Lili Zhao Hui Li Suxia Wang Yong Shen Department of Joint Orthopedics Hebei Provincial Xingtai People’s Hospital Department of Spinal Orthopedics Third Hospital of Hebei Medical University

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

关键词： nerve regeneration spinal cord injury bone marrow mesenchymal stem cells Nogo-66receptor RNA interference horseradish peroxidase BrdU gene silencing neural regeneration

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Small RNAs,RNAi and the Inheritance of gene silencing in Caenorhabditis elegans

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Journal of genetics and Genomics 2013年第4期40卷 153-160页

作者： Xuezhu Feng Shouhong Guang School of Life Sciences University of Science and Technology of China

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics,e.g.,unpaired DNA or abnormal doublestranded RNA.Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids.Furthermore,they have developed ways to remember this exposure to invaders and transmit the experience to their descendants.The mechanism underlying this inheritance has remained elusive.Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing.Small regulatory RNAs play a variety of crucial roles in organisms,including gene regulation,developmental timing,antiviral defense,and genome integrity,via a process termed as RNA interference（RNAi）.Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing.Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations.Notably,these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations.Thus,RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.

关键词： RNAi Small RNA Inheritance gene silencing nrde

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Molecular engineering of dendrimer nanovectors for siRNA delivery and gene silencing

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Frontiers of Chemical Science and Engineering 2017年第4期11卷 663-675页

作者： Yu Cao Xiaoxuan Liu Ling Peng . Aix-Marseille Université CNRS Centre Interdisciplinaire de Nanoscience de Marseille UMR 7325 Equipe Labellisée Ligue Contre le Caner 13288 Marseille France.. State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases Center of Drug Discovery Center of Advanced Pharmaceuticals and Biomaterials China Pharmaceutical University Nanjing 210009 China

Small interfering RNA （siRNA） therapeutics hold great promise to treat a variety of diseases, as long as they can be delivered safely and effectively into cells. Dendrimers are appealing vectors for siRNA delivery by virtue of their well-defined molecular architecture and multivalent cooperativity. However, the clinical translation of RNA therapeutics mediated by dendrimer delivery is hampered by the lack of dendrimers that are of high quality to meet good manufacturing practice standard. In this context, we have developed small amphiphilic dendrimers that self-assemble into supramolecular structures, which mimic high-generation dendrimers synthesized with cova- lent construction, yet are easy to produce in large amount and superior quality. Indeed, the concept of supramolecular dendrimers has proved to be very promising, and has opened up a new avenue for dendrimer-mediated siRNA delivery. A series of self-assembling supramolecular dendrimers have consequently been established, some of them out-performing the currently available nonviral vectors in delivering siRNA to various cell types in vitro and in vivo, including human primary cells and stem cells. This short review presents a brief introduction to RNAi therapeutics, the obstacles to their delivery and the advantages of dendrimer delivery vectors as well as our bio-inspired struc^rally flexible dendrimers for siRNA delivery. We then highlight our efforts in creating self- assembling amphiphilic dendrimers to construct supramo- lecular dendrimer nanosystems for effective siRNA delivery as well as the related structural alterations to enhance delivery efficiency. The advent of self-assembling supramolecular dendrimer nanovectors holds great pro- mise and heralds a new era of dendrimer-mediated delivery of RNA therapeutics in biomedical applications.

关键词： gene therapy RNAi therapeutics dendrimer,nanovectors gene silencing

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Visualized and cascade-enhanced gene silencing by smart DNAzyme-graphene nanocomplex

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Nano Research 2020年第8期13卷 2165-2174页

作者： Lingjie Ren Xiaoxia Chen Chang Feng Lei Ding Xiaomin Liu Tianshu Chen Fan Zhang Yanli Li Zhongliang Ma Bo Tian Xiaoli Zhu Center for Molecular Recognition and Biosensing School of Life SciencesShanghai UniversityShanghai200444China School of Medical Shanghai UniversityShanghai200444China Lab for Noncoding RNA and Cancer School of Life SciencesShanghai UniversityShanghai200444China State Key Laboratory of Oncogenes and Related genes Shanghai Cancer InstituteRen Ji HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai200032China

BCL-2 gene as well as its products is recognized as a promising target for the molecular targeted therapy of tumors.However,due to certain defense measures of tumor cells,the therapeutic effect based on the gene silencing of BCL-2 is greatly reduced.Here we fabricate a smart response nucleic acid therapeutic that could silence the gene effectively through a dual-targeted and cascade-enhanced strategy.In brief,nano-graphene oxide(GO),working as a nano-carrier,is loaded with a well-designed DNAzyme,which can target and silence the BCL-2 mRNA.Furthermore,upon binding with the BCL-2 mRNA,the enzymatic activity of the DNAzyme can be initiated,cutting a substrate oligonucleotide to produce an anti-nucleolin aptamer AS1411.Nucleolin,a nucleolar phosphoprotein,is known as a stabilizer of BCL-2 mRNA.Via binding and inactivating the nucleolin,AS1411 can destabilize BCL-2 mRNA.By this means of simultaneously targeting mRNA and its stabilizer in an integrated system,effective silencing of the BCL-2 gene of tumor cells is achieved at both the cellular and in vivo levels.After being dosed with this nucleic acid therapeutic and without any chemotherapeutics,apoptosis of tumor cells at the cellular level and apparent shrinkage of tumors in vivo are observed.By labeling a molecular beacon on the substrate of DNAzyme,visualization of the enzymatic activity as well as the tumor in vivo can be also achieved.Our work presents a pure bio-therapeutic strategy that has positive implications for enhancing tumor treatment and avoiding side effects of chemotherapeutics.

关键词： BCL-2 gene gene silencing DNAzyme graphene oxide AS1411

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Post-transcriptional gene silencing Induced by Short Interfering RNAs in Cultured Transgenic Plant Cells

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Genomics, Proteomics & Bioinformatics 2004年第2期2卷 97-108页

作者： WeiTang VanessaSamuels NickiWhitley NicoleBloom TinyaDeLaGarza RonaldJ.Newton DepartmentofBiology HowellScienceComplexEastCarolinaUniversityGreenvilleNC27858-4353USA DepartmentofBiology ShawUniversityRaleighNC27601USA

Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which weredesigned against different specific areas of coding region of the same target green fluorescentprotein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice(Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Eraser fir [Abies fraseri (Pursh) Poir;AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in thebombarded transgenic cells between two siRNAs, and these results were consistent with theinactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis intested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be thesiRNA specific in different plant species. These results indicate that siRNA is a highly specifictool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could bea reliable approach for large-scale screening of gene function and drug target validation.

关键词： gene inactivation gene silencing green fluorescent protein shortinterfering RNAs transgenic plant cells

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Targeted silencing of Heparanase gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2011年第3期31卷 348-352页

作者：范磊吴强邢晓娟刘郁东邵增务 Department of Orthopaedics Union HospitalTongji Medical CollegeHuazhong University of Science and Technology Southeast Forensic Science Service

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pgenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pgenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pgenesil） transfected group and expression vector（pgenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pgenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

关键词： siRNA shRNA heparanase gene silencing osteosarcoma gene expression invasion metastasis

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