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1.
Chinese Journal of Biotechnology ; (12): 915-924, 2022.
Article in Chinese | WPRIM | ID: wpr-927754

ABSTRACT

Group Ⅱ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg2+ concentration. Finally, we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group Ⅱ introns in biotechnology.


Subject(s)
DNA , Eukaryota , Gene Targeting , Introns/genetics , RNA, Catalytic/genetics
2.
Acta Physiologica Sinica ; (6): 482-490, 2021.
Article in Chinese | WPRIM | ID: wpr-887683

ABSTRACT

S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F


Subject(s)
Animals , Bronchoalveolar Lavage Fluid , CRISPR-Cas Systems/genetics , Calgranulin B , Disease Models, Animal , Gene Knockout Techniques , Gene Targeting , Lung , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin , Phenotype
3.
Chinese Journal of Biotechnology ; (12): 784-794, 2019.
Article in Chinese | WPRIM | ID: wpr-771331

ABSTRACT

The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.


Subject(s)
Animals , Disease Models, Animal , Embryonic Stem Cells , Gene Knockout Techniques , Gene Targeting , Homologous Recombination , Humans , Mice , Mice, Knockout
4.
Acta Physiologica Sinica ; (6): 588-596, 2019.
Article in Chinese | WPRIM | ID: wpr-777152

ABSTRACT

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2 mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2 mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.


Subject(s)
Animals , CRISPR-Cas Systems , Female , Gene Knockout Techniques , Gene Targeting , Male , Mice , Mice, Knockout , RNA, Guide , Genetics
5.
Biol. Res ; 51: 56, 2018. graf
Article in English | LILACS | ID: biblio-1011400

ABSTRACT

BACKGROUND: Glioma is the most prevalent malignant tumor in human central nervous systems. Recently, the development of resistance to radiotherapy in glioma patients markedly vitiates the therapy outcome. MiR-153-3p has been reported to be closely correlated with tumor progression, but its effect and molecular mechanism underlying radioresistance remains unclear in glioma. METHODS: The expression of miR-153-3p was determined in radioresistant glioma clinical specimens as well as glioma cell lines exposed to irradiation (IR) using quantitative real-time PCR. Cell viability, proliferation and apoptosis were then evaluated by MTT assay, colony formation assay, Flow cytometry analysis and caspase-3 activity assay in glioma cells (U87 and U251). Tumor forming was evaluated by nude mice model in vivo. TUNEL staining was used to detect cell apoptosis in nude mice model. The target genes of miR-153-3p were predicted and validated using integrated bioinformatics analysis and a luciferase reporter assay. RESULTS: Here, we found that miR-153-3p was down-regulated in radioresistant glioma clinical specimens as well as glioma cell lines (U87 and U251) exposed to IR. Enhanced expression of miR-153-3p promoted the radiosensitivity, promoted apoptosis and elevated caspase-3 activity in glioma cells in vitro, as well as the radiosensitivity in U251 cell mouse xenografs in vivo. Mechanically, B cell lymphoma-2 gene (BCL2) was identified as the direct and functional target of miR-153-3p. Moreover, restoration of BCL2 expression reversed miR-153-3p-induced increase of radiosensitivity, apoptosis and caspase-3 activity in U251 cells in vitro. In addition, clinical data indicated that the expression of miR-153-3p was significantly negatively associated with BCL2 in radioresistance of glioma samples. CONCLUSIONS: Our findings suggest that miR-153-3p is a potential target to enhance the effect of radiosensitivity on glioma cells, thus representing a new potential therapeutic target for glioma.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Radiation Tolerance/genetics , Genes, bcl-2/physiology , MicroRNAs/radiation effects , MicroRNAs/physiology , Glioma/genetics , Time Factors , Down-Regulation , Gene Expression Regulation, Neoplastic , Cell Survival/radiation effects , Blotting, Western , Analysis of Variance , Gene Targeting/methods , Genes, bcl-2/radiation effects , In Situ Nick-End Labeling , MicroRNAs/analysis , Cell Line, Tumor , Cell Proliferation/radiation effects , Caspase 3/analysis , Real-Time Polymerase Chain Reaction , Flow Cytometry , Glioma/radiotherapy
6.
Chinese Journal of Lung Cancer ; (12): 358-364, 2018.
Article in Chinese | WPRIM | ID: wpr-776309

ABSTRACT

BACKGROUND@#It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology.@*METHODS@#In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR.@*RESULTS@#The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue.@*CONCLUSIONS@#The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.


Subject(s)
Animals , CRISPR-Cas Systems , Enhancer of Zeste Homolog 2 Protein , Genetics , Metabolism , Female , Gene Knockout Techniques , Gene Targeting , Humans , Lung Neoplasms , Genetics , Metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Guide
7.
Mem. Inst. Oswaldo Cruz ; 112(1): 1-7, Jan. 2017. tab
Article in English | LILACS | ID: biblio-841758

ABSTRACT

Insects are considered pests globally, implicated in the destruction of agricultural fields and transmission of pathogens that cause deadly human diseases, such as dengue, Zika and malaria. The diversity of the insecticide arsenal has remained stagnant for decades, but the recent rise of insecticide resistance fueled the discovery of novel modes of action, and the power of genomics has reinvigorated this search. This review discusses the importance of comparative and functional insect genomics in the identification of potential gene targets for an insecticidal mode of action with low off-target toxicity. Due to the global participation in the sequencing and annotation of insect genomes, the targeting of specific genes with molecular tools like RNAi and CRISPR/Cas9 for genome engineering and consequent functional identification and validation has become more efficient. While there are multiple avenues to explore for insecticidal candidates, this review identifies G-protein coupled receptors as attractive targets, and hones in on the octopamine and dopamine receptors due to their potential.


Subject(s)
Animals , Gene Targeting/methods , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Insecticide Resistance , Insect Control/methods , RNA Interference , Genome, Insect , Insecticides
8.
Cell Journal [Yakhteh]. 2017; 18 (4): 532-539
in English | IMEMR | ID: emr-185778

ABSTRACT

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid


Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA [sgRNA] specific to the homology region, and either Cas9 or its mutant nickase variant [Cas9n]. Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells [ESCs]


Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus


Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies


Subject(s)
Gene Knock-In Techniques , Embryonic Stem Cells , Gene Targeting , Genetic Engineering/methods , Homologous Recombination/genetics , Mice
9.
Biol. Res ; 49: 1-9, 2016. ilus, graf, tab
Article in English | LILACS | ID: lil-774430

ABSTRACT

BACKGROUND: The aim of this study was to explore epilepsy-related mechanism so as to figure out the possible targets for epilepsy treatment. METHODS: The gene expression profile dataset GES32534 was downloaded from Gene Expression Omnibus database. We identified the differentially expressed genes (DEGs) by Affy package. Then the DEGs were used to perform gene ontology (GO) and pathway enrichment analyses. Furthermore, a protein-protein interaction (PPI) network was constructed with the DEGs followed by co-expression modules construction and analysis. RESULTS: Total 420 DEGs were screened out, including 214 up-regulated and 206 down-regulated genes. Functional enrichment analysis revealed that down-regulated genes were mainly involved in the process of immunity regulation and biological repairing process while up-regulated genes were closely related to transporter activity. PPI network analysis showed the top ten genes with high degrees were all down-regulated, among which FN1 had the highest degree. The up-regulated and down-regulated DEGs in the PPI network generated two obvious sub-co-expression modules, respectively. In up-co-expression module, SCN3B (sodium channel, voltage gated, type III beta subunit) was enriched in GO:0006814 ~ sodium ion transport. In down-co-expression module, C1QB (complement C1s), CIS (complement component 1, S subcomponent) and CFI (complement factor I) were enriched in GO:0006955 ~ immune response. CONCLUSION: The immune response and complement system play a major role in the pathogenesis of epilepsy. Additionally, C1QB, C1S, CFI, SCN3B and FN1 may be potential therapeutic targets for epilepsy.


Subject(s)
Humans , Epilepsy/genetics , Epilepsy/therapy , Gene Expression Profiling/methods , Transcriptome , Databases, Genetic , Down-Regulation , Gene Ontology , Gene Regulatory Networks , Gene Targeting , Protein Interaction Maps , Up-Regulation
10.
Chinese Journal of Virology ; (6): 39-45, 2016.
Article in Chinese | WPRIM | ID: wpr-296219

ABSTRACT

Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.


Subject(s)
Animals , Chick Embryo , Chickens , Down-Regulation , Fibroblasts , Virology , Gene Targeting , Lentivirus , Genetics , Metabolism , Newcastle Disease , Virology , Newcastle disease virus , Genetics , Physiology , Phosphoproteins , Genetics , Metabolism , Poultry Diseases , Virology , RNA Interference , RNA, Small Interfering , Genetics , Metabolism , Viral Proteins , Genetics , Metabolism , Virus Replication
11.
Biol. Res ; 49: 1-9, 2016. ilus, graf, tab
Article in English | LILACS | ID: biblio-950852

ABSTRACT

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS. RESULTS: A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein-protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes. CONCLUSIONS: Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS


Subject(s)
Humans , Respiration Disorders/genetics , Sepsis/complications , Sepsis/genetics , Genetic Association Studies , Transcriptome , Transcription Factors , Down-Regulation , Cell Cycle/genetics , Up-Regulation , Gene Targeting , Gene Expression Profiling , Databases, Genetic , Protein Interaction Maps
12.
Biol. Res ; 49: 1-10, 2016. ilus, graf
Article in English | LILACS | ID: biblio-950836

ABSTRACT

BACKGROUND: Wnt-5a is a member of the WNT family of secreted lipoglycoproteins, whose expression increases during development; moreover, Wnt-5a plays a key role in synaptic structure and function in the adult nervous system. However, the mechanism underlying these effects is still elusive. MicroRNAs (miRNAs) are a family of small non-coding RNAs that control the gene expression of their targets through hybridization with complementary sequences in the 3' UTR, thereby inhibiting the translation of the target proteins. Several evidences indicate that the miRNAs are actively involved in the regulation of neuronal function. RESULTS: In the present study, we examined whether Wnt-5a modulates the levels of miRNAs in hippocampal neurons. Using PCR arrays, we identified a set of miRNAs that respond to Wnt-5a treatment. One of the most affected miRNAs was miR-101b, which targets cyclooxygenase-2 (COX2), an inducible enzyme that converts arachidonic acid to prostanoids, and has been involved in the injury/inflammatory response, and more recently in neuronal plasticity. Consistent with the Wnt-5a regulation of miR-101b, this Wnt ligand regulates COX2 expression in a time-dependent manner in cultured hippocampal neurons. CONCLUSION: The biological processes induced by Wnt-5a in hippocampal neurons, involve the regulation of several miRNAs including miR-101b, which has the capacity to regulate several targets, including COX-2 in the central nervous system


Subject(s)
Animals , Rats , MicroRNAs/physiology , Cyclooxygenase 2/analysis , Wnt Proteins/physiology , Hippocampus/enzymology , Neurons/enzymology , Down-Regulation , Gene Expression , Cells, Cultured , Blotting, Western , Rats, Sprague-Dawley , Gene Targeting , Gene Expression Profiling , Real-Time Polymerase Chain Reaction , Wnt-5a Protein , Hippocampus/chemistry , Neuronal Plasticity , Neurons/chemistry
15.
Salud colect ; 11(1): 9-21, ene.-mar. 2015.
Article in Spanish | LILACS | ID: lil-746681

ABSTRACT

El fortalecimiento mundial de la patente farmacéutica tensiona el acceso a los medicamentos esenciales. De acuerdo a este trabajo ello ha derivado en la colisión del derecho de propiedad intelectual que impulsa el Acuerdo sobre los Aspectos de los Derechos de Propiedad Intelectual relacionados con el Comercio (ADPIC) de la Organización Mundial del Comercio (OMC), con el derecho a la salud previsto en el Pacto Internacional de Derechos Económicos, Sociales y Culturales (PIDESC). Diversas controversias ventiladas en la OMC ilustran el enfrentamiento entre países con una poderosa industria farmacéutica y los intereses de países en desarrollo. Se concluye que las normas ADPIC-plus suscritas en tratados de libre comercio por países en desarrollo, que confieren al titular de la patente farmacéutica más derechos que los previstos en el propio Acuerdo sobre los ADPIC, son incompatibles con las obligaciones que asumen respecto del acceso a medicamentos esenciales en el marco del derecho a la salud del PIDESC.


The strengthening of pharmaceutical patent protection globally puts strains on access to essential medicines. According to the present paper, this process has led to the collision of the intellectual property rights adopted in the World Trade Organization (WTO) Trade-Related Aspects of Intellectual Property Rights (TRIPS) Agreement and the right to health stated in the International Covenant on Economic, Social and Cultural Rights (ICESCR). Several controversies disputed in the WTO illustrate the confrontation between countries with a powerful pharmaceutical industry and the interests of developing countries. It is concluded that the TRIPS-plus rules subscribed to by developing countries in free trade agreements which give the pharmaceutical patent holder more rights than those stipulated in the original TRIPS Agreement are incompatible with the obligations to provide access to essential medicines under the right to health of the ICESCR.


Subject(s)
Animals , Cricetinae , Humans , Mice , Genetic Vectors , Poly(ADP-ribose) Polymerases/genetics , RNA Interference , Cloning, Molecular , DNA , Fibroblasts/enzymology , Gene Targeting , Mice, Knockout , Poly(ADP-ribose) Polymerases/analysis , Skin/cytology
16.
Article in Chinese | WPRIM | ID: wpr-247941

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of RNA interference (RNAi) targeting NRP-1 gene on the growth and proliferation of nasopharyngeal carcinoma (NPC) CNE-2Z cells.</p><p><b>METHODS</b>Short hairpin RNA (shRNA) plasmids targeting NRP-1 were designed and synthesized. These plasmids were respectively transfected into human NPC CNE-2Z cells and xenograft tumors in nude mice. The expressions of Fluorescein-labeled plasmids in NPC CNE-2Z cells and xenograft tumors were observed by fluorescence microscopy. Cell proliferation was detected by MTT assay. The inhibitory effects on target genes were evaluated with RT-PCR and Western blotting, respectively. The inhibitory effect of plasmids with the most effective sequence on xenograft tumors in nude mice was observed.</p><p><b>RESULTS</b>CNE-2Z cell proliferation was significantly inhibited by NRP-1/shRNA silencing. RT-PCR showed NRP-1 mRNA expression was significantly decreased. Western blotting demonstrated the NRP-1/shRNA silencing can effectively inhibit the expression of target proteins in CNE-2Z cells. After six weeks, there were significant differences in the mean tumor volumes in nude mice between plasmid group and negative control group [(0.599±0.002) vs (1.141±0.013) cm(3), P<0.05] or blank control group [(0.599±0.002) vs (1.165±0.308) cm(3), P<0.05], and the inhibitory rate of tumor growth was 48.6%.</p><p><b>CONCLUSION</b>RNA interference targeting NRP-1 can remarkably inhibit the growth of CNE-2Z cells in vitro and in vivo.</p>


Subject(s)
Animals , Apoptosis , Carcinoma , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Gene Targeting , Humans , Mice , Mice, Nude , Nasopharyngeal Neoplasms , Metabolism , Pathology , Neuropilin-1 , Metabolism , Plasmids , RNA Interference , RNA, Small Interfering , Transfection
17.
Acta cir. bras ; 29(11): 696-702, 11/2014. tab, graf
Article in English | LILACS | ID: lil-728643

ABSTRACT

PURPOSE: To explore the mechanism of resistance to IKKβ inhibitor in multiple myeloma (MM) cells and uncover novel therapeutic targets for MM. METHODS: We downloaded the microarray data (GSE8476) from GEO (Gene Expression Omnibus) database. The data were derived from the human MM cells lines (L363 cells) treated with IKKβ inhibitor MLN120b (MLN) for eight, 12 and 24 hours. Furthermore, we applied the Search Tool for the Retrieval of Interacting Genes (STRING) and Expression Analysis Systematic Explorer (EASE) database to construct protein-protein interaction networks and identified over-represented pathway among DEGs (differentially expressed genes). RESULTS: We obtained 108 DGEs in 8h vs. 12h group and 101 ones in 8h vs. 24h group. Most of DGEs were found to be involved in biological regulation. The significant pathways were Ig A pathway and the CAMs pathways. In addition, 24 common DGEs were found in the networks of the two groups such as ICAM 3 and SELL. CONCLUSION: Intercellular adhesion molecule 3 and SELL may be potential targets in multiple myeloma treatment in the future. .


Subject(s)
Humans , Gene Targeting/methods , I-kappa B Kinase/antagonists & inhibitors , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Oligonucleotide Array Sequence Analysis/methods , Cell Adhesion , Cell Line, Tumor , Cluster Analysis , I-kappa B Kinase/metabolism , Multiple Myeloma/metabolism , Reproducibility of Results , Time Factors
18.
Braz. dent. j ; 25(5): 451-456, Sep-Oct/2014. graf
Article in English | LILACS | ID: lil-731051

ABSTRACT

Osteoblastoma is a benign neoplasia and is uncommon in the jaws. In some cases, this lesion presents extremely aggressive local characteristics and is termed aggressive osteoblastoma. Because the clinical, radiographic and histopathologic characteristics are similar to those of a variety of benign and malignant tumors, it poses a diagnostic dilemma. This report presents a case of an aggressive osteoblastoma in the mandible and discusses the differential diagnosis of this lesion. A 13-year-old white male sought the Stomatology Clinic at the State University of Paraíba, Campina Grande, PB, Brazil, complaining of asymptomatic swelling on the left side of his face. Cone-beam computerized tomography showed a multilocular, hypodense bone lesion, located in the body of the left mandible and lower third of the ascending ramus. The initial diagnostic hypothesis was juvenile ossifying fibroma or osteosarcoma. After histopathologic examination, the final diagnosis was aggressive osteoblastoma. Surgical resection with a safety margin was performed. There was no evidence of recurrence after a follow-up period of 4 years.


O osteoblastoma é uma neoplasia benigna e incomum nos maxilares. Em alguns casos esta lesão apresenta características locais extremamente agressivas, sendo denominada osteoblastoma agressivo. Devido às características clínicas, radiográficas e histopatológicas serem similares a uma variedade de tumores benignos e malignos, o seu diagnóstico é um dilema. Este relato apresenta o caso de um osteoblastoma agressivo na mandíbula e discute o diagnóstico diferencial desta lesão. Paciente, branco, 13 anos de idade, foi atendido na Clínica de Estomatologia da Universidade Estadual da Paraíba, Campina Grande, PB, Brasil, queixando-se de aumento de volume assintomático do lado esquerdo de sua face. A tomografia computadorizada de feixe cônico revelou uma lesão óssea hipodensa multilocular, localizada no corpo do lado esquerdo da mandíbula e no terço inferior do ramo ascendente da mandíbula. A hipótese diagnóstica foi de fibroma ossificante juvenil e osteosarcoma. Após exame histopatológico, o diagnóstico final foi osteoblastoma agressivo. Foi realizada ressecção cirúrgica com margem de segurança. Não houve sinais de recorrência após 4 anos de acompanhamento.


Subject(s)
Animals , Humans , Mice , Apoptosis/physiology , Carrier Proteins/metabolism , Mitochondrial Proteins/metabolism , Antibodies/metabolism , Antibodies/pharmacology , /metabolism , B-Lymphocytes/physiology , Caspase 9 , Cells, Cultured , Carrier Proteins/genetics , Caspases/metabolism , Enzyme Activation , Embryo, Mammalian/physiology , Gene Targeting , Intracellular Signaling Peptides and Proteins , Mice, Knockout , Mitochondrial Proteins/genetics , Survival Rate , Stem Cells/cytology , Stem Cells/metabolism , T-Lymphocytes/physiology
19.
Article in Chinese | WPRIM | ID: wpr-312571

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the effect of small interfering RNA (siRNA) targeting mouse epididymis-specific colipase-like (meClps) gene on mouse sperm mobility.</p><p><b>METHODS</b>The eukaryotic expression vector pDsRed2.0-C1-meClps was constructed and transfected into NIH-3T3 cells, and the protein expression was detected with anti-meClps serum. Three interfering sequences targeting meClps (RNAi-251, 224 and 286) were inserted into lentiviral vectors pRNAT-U6.2/lenti, which were co-transfected with pDsRed2.0-C1-meClps into NIH-3T3 cells. The RNA interfering efficiency was confirmed by semi-quantitative PCR and Western blotting. The lentivirus, packed with the lentiviral vector with the highest interfering efficiency, was injected into the caput tissues of mouse epididymis, and its effect on sperm mobility of the cauda epididymis was evaluated.</p><p><b>RESULTS</b>All the 3 lentiviral RNAi vectors targeting meClps could inhibit the mRNA and protein expressions of meClps, among which pRNAT-U6.2/lenti-RNAi-251 had the highest interfering efficiency. The lentivirus packed with pRNAT-U6.2/lenti-RNAi-251 significantly reduced the path velocity of cauda sperm after injection into the caput epididymis of the mice (P<0.05).</p><p><b>CONCLUSION</b>Knock-down meClps expression by lentiviral-mediated RNA interference can lower sperm mobility of mice.</p>


Subject(s)
Animals , Epididymis , Gene Targeting , Genetic Vectors , Lentivirus , Male , Mice , NIH 3T3 Cells , RNA Interference , RNA, Messenger , RNA, Small Interfering , Sperm Motility , Spermatozoa , Transfection
20.
Article in Chinese | WPRIM | ID: wpr-356945

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of silencing histone deacetylases 1 (HDAC1) gene by RNA interference on the proliferation, apoptosis and histone modulation in gastric cancer MGC-803 cell line.</p><p><b>METHODS</b>The optimal segment targeting HDAC1 gene was designed and transfected into MGC-803 cells by Lipofectamine TM2000. HDAC1 mRNA and protein in the transfected cells were detected by RT-PCR and Western blotting, respectively. The growth inhibition of MGC803 cells was evaluated by MTT assay and the cell apoptosis was detected with TUNEL assay. The expression of Bcl-2, procaspase-9, procaspase-3, c-Myc, histone acetylation of H3, H4, and histone methylation of H3K9 was detected by Western blotting.</p><p><b>RESULTS</b>The siRNA targeting HDAC1HDAC1 markedly suppressed mRNA expression, inhibited cell proliferation and induced apoptosis of MGC-803 cells in a concentration manner. Transfection of the cells with HDAC1 siRNA at 0, 30, 60, and 120 nmol/L for 24 h resulted in a cell apoptotic rate of (4.8∓2.7)%, (18.5∓3.5)%, (41.4∓4.3)%, and (59.2∓5.5)%, respectively, and caused down-regulation of the expressions of Bcl-2, proCaspase9, proCaspase3 and c-Myc, upregulation of histone acetylation of H3, H4, and down-regulation of histone methylation of H3K9.</p><p><b>CONCLUSION</b>Silencing HDAC1 gene expression with HDAC1 siRNA can promote histone H3 and H4 acetylation and inhibit histone methylation of H3K9 to suppress the proliferation and induce apoptosis of gastric cancer MGC-803 cells.</p>


Subject(s)
Acetylation , Apoptosis , Cell Line, Tumor , Cell Proliferation , Gene Silencing , Gene Targeting , Histone Deacetylase 1 , Genetics , Histones , Metabolism , Humans , Methylation , RNA Interference , RNA, Small Interfering
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