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Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALS-associated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Estudos de Associação Genética , Predisposição Genética para Doença , Neurônios Motores/metabolismo , Mutação , Fenótipo , Profilinas/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Comportamento Animal , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Dosagem de Genes , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Degeneração Neural/genética , Degeneração Neural/metabolismo , Paralisia/etiologia , Paralisia/metabolismo , Paralisia/patologia , Paralisia/fisiopatologia , Profilinas/metabolismo , Agregação Patológica de ProteínasRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease that causes motor neuron degeneration, progressive motor dysfunction, paralysis, and death. Although multiple causes have been identified for this disease, >95% of ALS cases show aggregation of transactive response DNA binding protein (TDP-43) accompanied by its nuclear depletion. Therefore, the TDP-43 pathology may be a converging point in the pathogenesis that originates from various initial triggers. The aggregation is thought to result from TDP-43 misfolding, which could generate cellular toxicity. However, the aggregation as well as the nuclear depletion could also lead to a partial loss of TDP-43 function or TDP-43 dysfunction. To investigate the impact of TDP-43 dysfunction, we generated a transgenic mouse model for a partial loss of TDP-43 function using transgenic RNAi. These mice show ubiquitous transgene expression and TDP-43 knockdown in both the periphery and the central nervous system (CNS). Strikingly, these mice develop progressive neurodegeneration prominently in cortical layer V and spinal ventral horn, motor dysfunction, paralysis, and death. Furthermore, examination of splicing patterns of TDP-43 target genes in human ALS revealed changes consistent with TDP-43 dysfunction. These results suggest that the CNS, particularly motor neurons, possess a heightened vulnerability to TDP-43 dysfunction. Additionally, because TDP-43 knockdown predominantly occur in astrocytes in the spinal cord of these mice, our results suggest that TDP-43 dysfunction in astrocytes is an important driver for motor neuron degeneration and clinical phenotypes of ALS.
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Esclerose Lateral Amiotrófica/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Modelos Animais de Doenças , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Sistema Nervoso Central/fisiopatologia , Proteínas de Ligação a DNA/genética , Camundongos , FenótipoRESUMO
Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration and paralysis. No treatment can significantly slow or arrest the disease progression. Mutations in the SOD1 gene cause a subset of familial ALS by a gain of toxicity. In principle, these cases could be treated with RNAi that destroys the mutant mRNA, thereby abolishing the toxic protein. However, no system is available to efficiently deliver the RNAi therapy. Recombinant adenoassociated virus (rAAV) is a promising vehicle due to its long-lasting gene expression and low toxicity. However, ALS afflicts broad areas of the central nervous system (CNS). A lack of practical means to spread rAAV broadly has hindered its application in treatment of ALS. To overcome this barrier, we injected several rAAV serotypes into the cerebrospinal fluid. We found that some rAAV serotypes such as rAAVrh10 and rAAV9 transduced cells throughout the length of the spinal cord following a single intrathecal injection and in the broad forebrain following a single injection into the third ventricle. Furthermore, a single intrathecal injection of rAAVrh10 robustly transduced motor neurons throughout the spinal cord in a non-human primate. These results suggested a therapeutic potential of this vector for ALS. To test this, we injected a rAAVrh10 vector that expressed an artificial miRNA targeting SOD1 into the SOD1G93A mice. This treatment knocked down the mutant SOD1 expression and slowed the disease progression. Our results demonstrate the potential of rAAVs for delivering gene therapy to treat ALS and other diseases that afflict broad areas of the CNS.
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Esclerose Lateral Amiotrófica/terapia , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Medula Espinal , Transdução Genética/métodos , Esclerose Lateral Amiotrófica/genética , Animais , Callithrix , Sistema Nervoso Central/fisiologia , Líquido Cefalorraquidiano/fisiologia , Feminino , Terapia Genética/métodos , Vetores Genéticos/farmacocinética , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Injeções Espinhais , Camundongos , Camundongos Transgênicos , Interferência de RNA , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , TransgenesRESUMO
All pseudouridines identified in RNA are considered constitutive modifications. Here, we demonstrate that pseudouridylation of Saccharomyces cerevisiae U2 snRNA can be conditionally induced. While only Ψ35, Ψ42 and Ψ44 are detected in U2 under normal conditions, nutrient deprivation leads to additional pseudouridylation at positions 56 and 93. Pseudouridylation at position 56 can also be induced by heat shock. Detailed analyses have shown that Pus7p, a single polypeptide pseudouridylase known to modify U2 at position 35 and tRNA at position 13, catalyses Ψ56 formation, and that snR81 RNP, a box H/ACA RNP known to modify U2 snRNA at position 42 and 25S rRNA at position 1051, catalyses Ψ93 formation. Using mutagenesis, we have demonstrated that the inducibility can be attributed to the imperfect substrate sequences. By introducing Ψ93 into log-phase cells, we further show that Ψ93 has a role in pre-mRNA splicing. Our results thus demonstrate for the first time that pseudouridylation of RNA can be induced at sites of imperfect sequences, and that Pus7p and snR81 RNP can catalyse both constitutive and inducible pseudouridylation.
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Pseudouridina/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Temperatura Alta , Dados de Sequência Molecular , Precursores de RNA/metabolismo , Splicing de RNARESUMO
Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ~30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.
Assuntos
Esclerose Lateral AmiotróficaRESUMO
A large number of box H/ACA RNAs have been identified in human cells, and have been predicted to account for nearly all pseudouridylation sites in human rRNAs. However, the function of these mammalian H/ACA RNAs in directing pseudouridylation has been verified experimentally in only two cases. In this study, we used three in vitro reconstitution systems, including yeast and mammalian systems, to test the function of seven H/ACA RNAs guiding16 pseudouridylation sites. Our results verified 12 of these sites; four predictions were incorrect. Further analyses indicated that three components, including the stability of the hairpin structure harboring the pseudouridylation pocket, the stability of guide sequence-target RNA base-pairing interaction, and the distance between the target uridine and the box H or ACA, were critical for the guide function, as changes in these components were sufficient to alter the functionality and specificity of the pseudouridylation pocket. The dynamic functional changes in response to changes in these three important components were further tested in vivo, and the results were completely consistent with the in vitro results. Finally, we compared our results with predictions made by two computer programs, as well as predictions made by human experts using visual inspection. We found that the predictions of one program (snoGPS) agreed with our experimental results with 100% sensitivity (12/12) and 75% specificity (3/4).
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Pareamento de Bases , Sequência de Bases , Genoma Humano , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Pseudouridina/química , Pseudouridina/metabolismo , Precursores de RNA/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Especificidade por Substrato , Pequeno RNA não TraduzidoRESUMO
Researchers hold the view that PLAGL2 is overexpressed in many malignancies and that it can promote tumor proliferation, migration, invasion and selfrenewal; however, there is no evidence revealing a relationship between PLAGL2 and colorectal cancer (CRC). In the present study, genes that are overexpressed in CRC were screened using the COSMIC database and GEPIA database and the expression of PLAGL2 in carcinoma tissues and pericarcinomatous tissues was detected by RTqPCR and western blot assays. A Cell Counting Kit8 assay, a cell cycle analysis experiment and a xenograft model were used to explore the influence of PLAGL2 on CRC after knocking down PLAGL2 expression in HCT116 and SW480 cells. Using ChIP assays and DualLuciferase Reporter assays, the promoter regions to which PLAGL2 binds were discovered. It was observed that PLAGL2 was overexpressed in colorectal cancer and that it influenced the colorectal cancer cell cycle and promoted colorectal cancer proliferation in vivo and in vitro. The expression of some genes in the Wnt/ßcatenin pathway, were downregulated after knocking down the expression of PLAGL2; Wnt6 was altered the most. PLAGL2 could bind to the promoter region of Wnt6 and promote its expression. These results indicated that PLAGL2 was overexpressed in CRC as a protooncogene and that it could active the Wnt/ßcatenin pathway as a transcription factor by binding with the promoter region of Wnt6. PALGL2 was revealed to play an important role in colorectal cancer and may be a new therapeutic target for targeted medicine.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Adulto , Idoso , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1) is a core factor in RNA methylation modification. Recent studies have shown that m6A is closely related to multiple tumors, thus YTHDF1 may also play a role in tumorigenesis. This study, aimed to explore the role of YTHDF1 in the colorectal cancer (CRC). In this study, we identified YTHDF1 as being highly expressed at the mRNA and protein levels in TCGA, GEO CRC and primary CRC. Furthermore, the YTHDF1 gene copy number was positively correlated with YTHDF1 mRNA expression in CRC. Knocking down the expression of YTHDF1 significantly inhibited the CRC cell's tumorigenicity in vitro and murine xenograft tumor growth in vivo. Furthermore, silencing of YTHDF1 inhibited the colonosphere formation ability in vitro. Mechanistically, we found that silencing YTHDF1 significantly inhibited Wnt/ß-catenin pathway activity in CRC cells. Together, YTHDF1 is overexpressed in CRC and plays a vital oncogenic role in CRC, and this novel finding may provide a potential therapeutic target for CRC.
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BACKGROUND: Cancer stem cells (CSCs) are responsible for all important characteristics of tumors. DEAD-box helicase 27 (DDX27) is a member of the DEAD-box RNA helicase family, and there have been only a few studies on DDX27 function in cancer cells. This study is aimed at exploring whether DDX27 has any relation to tumorigenesis of colorectal cancer (CRC) and elucidating the potential mechanism. METHODS: Data from Catalog Of Somatic Mutations In Cancer, Gene Expression Omnibus, and The Cancer Genome Atlas databases reveal that DDX27 is overexpressed in CRC tissues. qRT-PCR and Western blots were used to evaluate the expression level of DDX27 in 40 paired clinical CRC samples. DDX27 was knockdown in HT29 and HCT116 cell line with shRNA. Then CCK-8, colony formation assay and flow cytometry assay were performed to examine proliferative ability, cell cycle and sensitivity to 5-fluorouracil. Sphere-formation assay and in vivo subcutaneous tumor-formation assay were used to assess self-renewal in vitro and vivo as well as the tumor-initiating potential. RESULTS: DDX27 is upregulated in CRC tissues and downregulation of DDX27 inhibits proliferation of colorectal cancer cell and promotes sensitivity to 5-fluorouracil. Downregulation of DDX27 can downregulate the gene expression of known CSC markers in CRC cells, inhibit sphere-formation ability, and promote colonosphere differentiation. Downregulation of DDX27 in CSCs can decrease the tumor-initiating ability of CRC cells in vivo. CONCLUSION: DDX27 may play a tumorpromoter role of CRC by regulating the stem cell-like activity of CRC cells.
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Research on DNA methylation offers great potential for the identification of biomarkers that can be applied for accurately assessing an individual's risk for cancer. In this article, we try to find the ideal epigenetic genes involved in colorectal cancer (CRC) based on a CRC database and our CRC cohort. The top 20 genes with an extremely high frequency of hypermethylation in CRC were identified in the latest database. Remarkably, 3 HOXA genes were included in this list and ranked at the top. The percentage of methylation in the HOXA5, HOXA2, and HOXA6 genes in CRC were up to 67.62, 58.36, and 31.32%, respectively, and ranked first in CRC among all human tumor tissues. Paired colorectal tumor samples and adjacent non-tumor colorectal tissue samples and four CRC cell lines were selected for MethylTarget™ assays. The results demonstrated that CRC tissues and cells had a stronger methylation status around the 3 HOXA gene promoter regions compared with adjacent non-tumor colonic tissue samples. The Receiver operator characteristic curve (ROC) curves for HOXA genes show excellent diagnostic ability in distinguishing tissue from healthy individuals and CRC patients, especially for Stage I patients (AUC = 0.9979 in HOXA2, 0.9309 in HOXA5, and 0.8025 in HOXA6). An association analysis between the methylation pattern of HOXA genes and clinical indicators was performed and found that HOXA2 methylation was significantly associated with age, N, stage, M, lymphovascular invasion, perineural invasion, lymph node number. HOXA5 methylation was associated with age, T, M, stage, and tumor status, and HOXA6 methylation was associated with age and KRAS mutation. Notably, we found that the highest methylation of HOXA5 and HOXA2 occurs in the early stages of colorectal cancer tissues such as stage I, N0, MO, and non-invasive tissues. The methylation levels declined as tumors progressed. However, methylation level at any stage of the tumor was still significantly higher than in normal tissues (p < 0.0001). The mRNA of the 3 HOXA genes was downregulated in early tumor stages due to hypermethylation of CpG islands adjacent to the promoters of the genes. In addition, hypermethylation of HOXA5 and HOXA6 mainly occurred in patients < 60 years old and with MSI-L, MSS, CIMP.L and non-CIMP tumors. Together, this suggests that epigenetic silencing of 3 adjacent HOXA genes may be an important event in the progression of colorectal cancer.
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BACKGROUND: Our previous study revealed that PLAGL2 or POFUT1 can promote tumorigenesis and maintain significant positive correlations in colorectal cancer (CRC). However, the mechanism leading to the co-expression and the underlying functional and biological implications remain unclear. METHODS: Clinical tumor tissues and TCGA dataset were utilized to analyze the co-expression of PLAGL2 and POFUT1. Luciferase reporter assays, specially made bidirectional promoter vectors and ectopic expression of 3'UTR were employed to study the mechanisms of co-expression. In vitro and in vivo assays were performed to further confirm the oncogenic function of both. The sphere formation assay, immunofluorescence, Western blot and qRT-PCR were performed to investigate the effect of both genes in colorectal cancer stem cells (CSCs). FINDINGS: PLAGL2 and POFUT1 maintained co-expression in CRC (râ¯=â¯0.91, pâ¯<â¯.0001). An evolutionarily conserved bidirectional promoter, rather than post-transcriptional regulation by competing endogenous RNAs, caused the co-expression of PLAGL2 and POFUT1 in CRC. The bidirectional gene pair PLAGL2/POFUT1 was subverted in CRC and acted synergistically to promote colorectal tumorigenesis by maintaining stemness of colorectal cancer stem cells through the Wnt and Notch pathways. Finally, PLAGL2 and POFUT1 share transcription factor binding sites, and introducing mutations into promoter regions with shared transcription regulatory elements led to a decrease in the PLAGL2/POFUT1 promoter activity in both directions. INTERPRETATION: Our team identified for the first time a bidirectional promoter pair oncogene, PLAGL2-POFUT1, in CRC. The two genes synergistically promote the progression of CRC and affect the characteristics of CSCs, which can offer promising intervention targets for clinicians and researchers. FUND: National Nature Science Foundation of China, the Hunan province projects of Postgraduate Independent Exploration and Innovation of Central South University.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Fucosiltransferases/genética , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Negative elongation factor complex member C/D (NELFCD), mapped to chromosome 20q13.32, has been found to be significantly overexpressed in colorectal cancer (CRC) by our previous research. However, whether its overexpression contributes to CRC development is unknown. We aimed to explore the biological and clinical roles of NELFCD in CRC. MATERIALS AND METHODS: The expression of NELFCD was detected by qRT-PCR and Western blot. The biological function of NELFCD on CRC cell proliferation, migration, invasion, and apoptosis was detected by cell counting kit-8, plate colony formation assay, transwell migration and invasion assays, and flow cytometry in vitro and by murine xenograft tumor growth in vivo. Moreover, we evaluated the correction between its expression level and clinicopathologic parameters. RESULTS: We found NELFCD was overexpressed in 50 pairs of CRC tissues in comparison to the adjacent nontumor tissues (P<0.05). Knockdown of NELFCD significantly impaired cell proliferation, migration and invasion abilities, facilitated cell apoptosis in vitro, and inhibited tumorigenesis of CRC cells in vivo. NELFCD levels were remarkably connected with tumor location in CRC patients. CONCLUSION: NELFCD is overexpressed and plays an oncogenic role in CRC. Targeting NELFCD may provide a potential therapeutic option for NELFCD-amplified tumors.
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Pleomorphic adenoma gene like-2 (PLAGL2) is a zinc finger protein transcription factor, which is upregulated and serves an oncogenic function in multiple human malignancies, including colorectal cancer (CRC). First, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of PLAGL2 in CRC tissues and normal tissues. Then, bioinformatics analysis, RT-qPCR, western blotting, luciferase reporter assays and RNA-binding protein immunoprecipitation assays were performed to explore whether the underlying mechanisms, including copy number variation (CNV), microRNAs (miRNAs/miRs) and RNA-binding proteins (RBPs) led to the abnormal expression of PLAGL2. Finally, cell counting kit-8 assays, Transwell assays and xenograft models were used to detect carcinogenesis-associated characteristics based on the 3'-untranslated region (3'-UTR) of PLAGL2. In the present study, PLAGL2 was revealed to be upregulated in CRC tissues compared with normal CRC tissues. CNV was one of the causes leading to the upregulation of PLAGL2. miRNA, including downregulated miR-486-5p, and RBPs, including upregulated human antigen R (HuR), were other key underlying causes. In addition, PLAGL2 3'-UTR was revealed to promote the progression of CRC in vitro and in vivo, and to regulate the expression of C-MYC and CD44. To conclude, these results suggested that high expression of PLAGL2 in CRC was associated with CNV, miR-486-5p and HuR expression, whose 3'-UTR may promote colon carcinogenesis and serve as a novel potential biomarker for CRC therapies.
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Copy number variations (CNVs) are key drivers of colorectal cancer (CRC). Our previous studies revealed that protein O-fucosyltransferase 1 (POFUT1) overexpression is driven by CNVs during CRC development. The potential role and underlying mechanisms of POFUT1 in CRC were not investigated. In this study, we analyzed the expression of POFUT1 in CRC from cosmic and TCGA databases and confirmed that POFUT1 is highly expressed in CRC. We used well characterized CRC cell lines, including SW620 and HCT116 to establish a model POFUT1 knockdown cell line. Using these cells, we investigated the role of POFUT1 in CRC. Our data revealed that silencing POFUT1 in CRC cells inhibits cell proliferation, decreases cell invasion and migration, arrests cell cycle progression, and stimulates CRC cell apoptosis in vitro. We further demonstrate that POFUT1 silencing dramatically suppresses CRC tumor growth and transplantation in vivo. We additionally reveal new mechanistic insights into the role of POFUT1 during CRC, through demonstrating that POFUT1 silencing inhibits Notch1 signaling. Taken together, our findings demonstrate that POFUT1 is a tumor activating gene during CRC development, which positively regulates CRC tumor progression through activating Notch1.
Assuntos
Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Fucosiltransferases/metabolismo , Receptor Notch1/metabolismo , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Bases de Dados Genéticas , Fucosiltransferases/genética , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Modelos Animais , Invasividade Neoplásica , Transfecção , Carga TumoralRESUMO
The Long arm of chromosome 20 (20q) is closely related to the development of colorectal cancer, so identifying the expression profile of genes on 20q through a comprehensive overview is indispensable. In this article, preliminar experimental data, several available databases and bioinformatics tools such as the Cancer Genome Atlas, the Encyclopedia of DNA Elements, the JASPAR database and starBase were combined to analyze the correlation between genes and chromosomal aberrations, microRNA and transcription factors, as well as to explore the expression feature and potential regulative mechanism. The results showed that the most frequently unregulated genes in colorectal cancer arelocated on chromosome 20q, present a significant CNA-mRNA correlation.Furthermore, the genes with mRNA overexpression showed co-expression features and tended to be clustered within the same genomic neighborhoods. Then, several genes were selected to carry out further analysis and demonstrated that shared transcription factors, a conserved bidirectional promoter, and competition for a limited pool of microRNAin the 3'UTR of mRNA may be the underlying mechanisms behind the co-expression of physically adjacent genes.Finally, the databases, Lentivirus shRNA, and qPCR were used to find that these adjacent genes with co-expression cooperatively participated in the same biological pathways associated with the pathogenesis and development of colorectal cancer.
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Mutant SOD1 causes amyotrophic lateral sclerosis (ALS) by a dominant gain of toxicity. Previous studies have demonstrated therapeutic potential of mutant SOD1-RNAi delivered by intrathecal (IT) injection of recombinant adeno-associated virus (rAAV). However, optimization of delivery is needed to overcome the high degree of variation in the transduction efficiency and therapeutic efficacy. Here, on the basis of our previously defined, efficient IT injection method, we investigated the influence of injection speed on transduction efficiency in the central nervous system (CNS). We demonstrate that slow IT injection results in higher transduction of spinal cord and dorsal root ganglia (DRG) while fast IT injection leads to higher transduction of brain and peripheral organs. To test how these effects influence the outcome of RNAi therapy, we used slow and fast IT injection to deliver rAAVrh10-GFP-amiR-SOD1, a rAAV vector that expresses GFP and an artificial miRNA targeting SOD1, in SOD1-G93A mice. Both slow and fast IT injection produced therapeutic efficacy but the slow injection trended slightly toward a better outcome than the fast injection. These results demonstrate that IT injection speed influences the predominance of gene delivery at different CNS sites and should be taken into consideration in future therapeutic trials involving IT injection.
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Esclerose Lateral Amiotrófica , Terapia Genética/métodos , Mutação/genética , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Animais , Peso Corporal/genética , Proteínas de Ligação ao Cálcio/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Injeções Espinhais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Medula Espinal/patologia , Transdução GenéticaRESUMO
The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here, we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in 60 to 90 % of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells, and endothelial cells. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad central nervous system (CNS), the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ~100-fold. Finally, the transduced areas were co-localized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases.
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Dependovirus/metabolismo , Vértebras Lombares/metabolismo , Recombinação Genética/genética , Transgenes , Animais , Inflamação/patologia , Injeções , Lidocaína/administração & dosagem , Lidocaína/farmacologia , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Camundongos , Especificidade de Órgãos , Punções , Transdução GenéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes motor neuron degeneration leading to progressive muscle atrophy, weakness, paralysis and death. The majority of ALS (>95%) shows intracellular aggregation of transactive response DNA binding protein (TDP-43) as a prominent pathological feature. TDP-43 is normally a nuclear protein. In ALS, TDP-43 accumulates and aggregates in the cytoplasm (thus forming TDP-43 proteinopathy) and is depleted from the nucleus in CNS cells, including motor neurons and glia. While TDP-43 aggregation can harm cells through a gain of toxicity, it can also cause a loss of TDP-43 function in conjunction with its nuclear depletion. TDP-43 regulates its own expression to maintain itself at a constant level. Perturbation of this level by either increasing or decreasing TDP-43 in animal models leads to neurodegeneration and ALS phenotypes. The evidence supports the hypothesis that TDP-43 dysfunction is a critical driver of neurodegeneration in the vast majority of ALS cases.
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RNA interference (RNAi) is a conserved cellular mechanism in most eukaryotes that can mediate specific gene silencing. Since its discovery in 1998, rapid progress has been made in understanding its basic mechanism and its application in research and drug discovery. In recent years, the application of RNAi in research, including research in neurodegeneration, has expanded rapidly such that it has become a regular tool for reverse genetics in cultured cells in many labs. However, an incomplete understanding of the RNAi mechanism and worries about its pitfalls still intimidate many others. Here, we present a streamlined and simple protocol for the design and implementation of an RNAi experiment in cultured cells, aiming to enable those who are inexperienced with RNAi to apply this powerful method in their research, particularly in the field of neurodegeneration.
Assuntos
Técnicas Genéticas , Interferência de RNA , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Doenças Neurodegenerativas/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa/genética , TransfecçãoRESUMO
TAR DNA binding protein 43 KD (TDP-43) is an essential gene that regulates gene transcription, mRNA splicing and stability. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal neurodegenerative diseases, TDP-43 is fragmented, generating multiple fragments that include the C-terminal fragment of â¼25 KD. The role of these fragments in the pathogenesis of ALS and FTD is not clear. Here we investigated the aggregation propensity in various polypeptide regions of TDP-43 in mammalian cells and the effect of these fragments on cultured neurons. By expressing the full length and various TDP-43 fragments in motor neuron-derived NSC-34 cells and primary neurons, we found that both N- and C-terminal fragments of TDP-43 are prone to aggregate and the C-terminal end of RRM2 region is required, though not sufficient, for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregation with the full-length TDP-43, consequently reducing the nuclear TDP-43. In addition, the TDP-43 fragments can impair neurite growth during neuronal differentiation. Importantly, overexpression of the full-length TDP-43 rescues the neurite growth phenotype whereas knockdown of the endogenous TDP-43 reproduces this phenotype. These results suggest that TDP-43 fragments, particularly the pathologically relevant C-terminal fragments, can impair neuronal differentiation by dominant-negatively interfering with the function of the full length TDP-43, thus playing a role in pathogenesis in ALS and FTD.