RESUMO
ABCC9 genetic polymorphisms are associated with increased risk for various human diseases including hippocampal sclerosis of aging. The main goals of this study were 1 > to detect the ABCC9 variants and define the specific 3' untranslated region (3'UTR) for each variant in human brain, and 2 > to determine whether a polymorphism (rs704180) associated with risk for hippocampal sclerosis of aging pathology is also associated with variation in ABCC9 transcript expression and/or splicing. Rapid amplification of ABCC9 cDNA ends (3'RACE) provided evidence of novel 3' UTR portions of ABCC9 in human brain. In silico and experimental studies were performed focusing on the single nucleotide polymorphism, rs704180. Analyses from multiple databases, focusing on rs704180 only, indicated that this risk allele is a local expression quantitative trait locus (eQTL). Analyses of RNA from human brains showed increased ABCC9 transcript levels in individuals with the risk genotype, corresponding with enrichment for a shorter 3' UTR which may be more stable than variants with the longer 3' UTR. MicroRNA transfection experiments yielded results compatible with the hypothesis that miR-30c causes down-regulation of SUR2 transcripts with the longer 3' UTR. Thus we report evidence of complex ABCC9 genetic regulation in brain, which may be of direct relevance to human disease. ABCC9 gene variants are associated with increased risk for hippocampal sclerosis of aging (HS-Aging--a prevalent brain disease with symptoms that mimic Alzheimer's disease). We describe novel ABCC9 variants in human brain, corresponding to altered 3'UTR length, which could lead to targeting by miR-30c. We also determined that the HS-Aging risk mutation is associated with variation in ABCC9 transcript expression.
Assuntos
Envelhecimento/patologia , Encefalopatias/genética , Hipocampo/patologia , Doenças Neurodegenerativas/genética , Receptores de Sulfonilureias/genética , Idoso de 80 Anos ou mais , Encefalopatias/patologia , Feminino , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Humanos , Masculino , MicroRNAs/genética , Doenças Neurodegenerativas/patologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Esclerose/genética , Esclerose/patologiaRESUMO
Genome-wide association studies are identifying novel Alzheimer's disease (AD) risk factors. Elucidating the mechanism underlying these polymorphisms is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we elucidate the mechanism of action of the AD-associated polymorphism rs3865444 in the promoter of CD33, a member of the sialic acid-binding Ig-superfamily of lectins (SIGLECs). Immunostaining established that CD33 is expressed in microglia in human brain. Consistent with this finding, CD33 mRNA expression correlated well with expression of the microglial genes CD11b and AIF-1 and was modestly increased with AD status and the rs3865444C AD-risk allele. Analysis of CD33 isoforms identified a common isoform lacking exon 2 (D2-CD33). The proportion of CD33 expressed as D2-CD33 correlated robustly with rs3865444 genotype. Because rs3865444 is in the CD33 promoter region, we sought the functional polymorphism by sequencing CD33 from the promoter through exon 4. We identified a single polymorphism that is coinherited with rs3865444, i.e., rs12459419 in exon 2. Minigene RNA splicing studies in BV2 microglial cells established that rs12459419 is a functional single nucleotide polymorphism (SNP) that modulates exon 2 splicing efficiency. Thus, our primary findings are that CD33 is a microglial mRNA and that rs3865444 is a proxy SNP for rs12459419 that modulates CD33 exon 2 splicing. Exon 2 encodes the CD33 IgV domain that typically mediates sialic acid binding in SIGLEC family members. In summary, these results suggest a novel model wherein SNP-modulated RNA splicing modulates CD33 function and, thereby, AD risk.
Assuntos
Processamento Alternativo/genética , Doença de Alzheimer/genética , Éxons/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Genótipo , Humanos , Imuno-Histoquímica , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de RiscoRESUMO
Hippocampal sclerosis of aging (HS-Aging) is a high-morbidity brain disease in the elderly but risk factors are largely unknown. We report the first genome-wide association study (GWAS) with HS-Aging pathology as an endophenotype. In collaboration with the Alzheimer's Disease Genetics Consortium, data were analyzed from large autopsy cohorts: (#1) National Alzheimer's Coordinating Center (NACC); (#2) Rush University Religious Orders Study and Memory and Aging Project; (#3) Group Health Research Institute Adult Changes in Thought study; (#4) University of California at Irvine 90+ Study; and (#5) University of Kentucky Alzheimer's Disease Center. Altogether, 363 HS-Aging cases and 2,303 controls, all pathologically confirmed, provided statistical power to test for risk alleles with large effect size. A two-tier study design included GWAS from cohorts #1-3 (Stage I) to identify promising SNP candidates, followed by focused evaluation of particular SNPs in cohorts #4-5 (Stage II). Polymorphism in the ATP-binding cassette, sub-family C member 9 (ABCC9) gene, also known as sulfonylurea receptor 2, was associated with HS-Aging pathology. In the meta-analyzed Stage I GWAS, ABCC9 polymorphisms yielded the lowest p values, and factoring in the Stage II results, the meta-analyzed risk SNP (rs704178:G) attained genome-wide statistical significance (p = 1.4 × 10(-9)), with odds ratio (OR) of 2.13 (recessive mode of inheritance). For SNPs previously linked to hippocampal sclerosis, meta-analyses of Stage I results show OR = 1.16 for rs5848 (GRN) and OR = 1.22 rs1990622 (TMEM106B), with the risk alleles as previously described. Sulfonylureas, a widely prescribed drug class used to treat diabetes, also modify human ABCC9 protein function. A subsample of patients from the NACC database (n = 624) were identified who were older than age 85 at death with known drug history. Controlling for important confounders such as diabetes itself, exposure to a sulfonylurea drug was associated with risk for HS-Aging pathology (p = 0.03). Thus, we describe a novel and targetable dementia risk factor.
Assuntos
Envelhecimento/genética , Envelhecimento/patologia , Hipocampo/patologia , Polimorfismo de Nucleotídeo Único , Receptores de Sulfonilureias/genética , Idoso de 80 Anos ou mais , Envelhecimento/efeitos dos fármacos , Estudos de Coortes , Bases de Dados como Assunto , Endofenótipos , Estudo de Associação Genômica Ampla , Hipocampo/efeitos dos fármacos , Humanos , Esclerose/genética , Esclerose/patologia , Compostos de Sulfonilureia/efeitos adversos , Compostos de Sulfonilureia/uso terapêuticoRESUMO
MicroRNAs (miRNAs) target mRNAs in human cells via complex mechanisms that are still incompletely understood. Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, 'RIP-Chip' experiments enable direct analyses of miRNA targets. RIP-Chip studies (and parallel assessments of total input mRNA) were performed in cultured H4 cells after transfection with miRNAs corresponding to the miR-15/107 gene group (miR-103, miR-107, miR-16 and miR-195), and five control miRNAs. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. Computational analyses queried for determinants of miRNA:mRNA binding. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3' portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3'-untranslated region targeting, and stable AGO association versus mRNA knockdown. Future studies should take this important miRNA-to-miRNA variability into account.
Assuntos
Regulação para Baixo , MicroRNAs/química , RNA Mensageiro/metabolismo , Proteínas Argonautas/isolamento & purificação , Sequência de Bases , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/químicaRESUMO
MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the "RIP-Chip" assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.
Assuntos
Imunoprecipitação/métodos , MicroRNAs/genética , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Anticorpos Monoclonais , Proteínas Argonautas , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/imunologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Substâncias Macromoleculares , Camundongos , MicroRNAs/química , RNA Mensageiro/química , Ribonucleoproteínas/química , TransfecçãoRESUMO
Granulin (GRN, or progranulin) is a protein involved in wound repair, inflammation, and neoplasia. GRN has also been directly implicated in frontotemporal dementia and may contribute to Alzheimer's disease pathogenesis. However, GRN regulation expression is poorly understood. A high-throughput experimental microRNA assay showed that GRN is the strongest target for miR-107 in human H4 neuroglioma cells. miR-107 has been implicated in Alzheimer's disease pathogenesis, and sequence elements in the open reading frame-rather than the 3' untranslated region-of GRN mRNA are recognized by miR-107 and are highly conserved among vertebrate species. To better understand the mechanism of this interaction, FLAG-tagged Argonaute constructs were used following miR-107 transfection. GRN mRNA interacts preferentially with Argonaute 2. In vitro and in vivo studies indicate that regulation of GRN by miR-107 may be functionally important. Glucose supplementation in cultured cells that leads to increased miR-107 levels also results in decreased GRN expression, including changes in cell compartmentation and decreased secretion of GRN protein. This effect was eliminated following miR-107 transfection. We also tested a mouse model where miR-107 has been shown to be down-regulated. In brain tissue subjacent to 1.0 mm depth controlled cortical impact, surviving hippocampal neurons show decreased miR-107 with augmentation of neuronal GRN expression. These findings indicate that miR-107 contributes to GRN expression regulation with implications for brain disorders.
Assuntos
Lesões Encefálicas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Proteínas Argonautas , Sequência de Bases , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Células Cultivadas , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Glucose/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Análise em Microsséries , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Progranulinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3'UTR of mRNAs,guided by the 5' "seed" portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a "RIP-Chip" experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide "seed" sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3'UTR that would be recognized by the 5' seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have 'seed' sequences in the mRNA open reading frame, but not the 3' UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5' seed in determining binding characteristics for some miRNAs; however, the "binding rules" are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting.
Assuntos
MicroRNAs/metabolismo , MicroRNAs/fisiologia , Interferência de RNA/fisiologia , RNA Mensageiro/metabolismo , Animais , Sequência de Bases/fisiologia , Sítios de Ligação/genética , Humanos , Imunoprecipitação/métodos , Camundongos , MicroRNAs/química , MicroRNAs/genética , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/química , RNA Mensageiro/genética , TransfecçãoRESUMO
MicroRNA (miRNA) gene expression profiling has provided important insights into plant and animal biology. However, there has not been ample published work about pitfalls associated with technical parameters in miRNA gene expression profiling. One source of pertinent information about technical variables in gene expression profiling is the separate and more well-established literature regarding mRNA expression profiling. However, many aspects of miRNA biochemistry are unique. For example, the cellular processing and compartmentation of miRNAs, the differential stability of specific miRNAs, and aspects of global miRNA expression regulation require specific consideration. Additional possible sources of systematic bias in miRNA expression studies include the differential impact of pre-analytical variables, substrate specificity of nucleic acid processing enzymes used in labeling and amplification, and issues regarding new miRNA discovery and annotation. We conclude that greater focus on technical parameters is required to bolster the validity, reliability, and cultural credibility of miRNA gene expression profiling studies.
Assuntos
Perfilação da Expressão Gênica , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.
Assuntos
Perfilação da Expressão Gênica , MicroRNAs/isolamento & purificação , Biologia Molecular/métodos , Tecido Nervoso/metabolismo , Idoso de 80 Anos ou mais , Animais , Astrócitos/metabolismo , Northern Blotting , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RatosAssuntos
Doenças do Sistema Nervoso Central/terapia , MicroRNAs , Terapia de Alvo Molecular , Traumatismos do Sistema Nervoso/terapia , Animais , Doenças do Sistema Nervoso Central/patologia , Regulação para Baixo/genética , Inativação Gênica/efeitos dos fármacos , Humanos , Hibridização In Situ , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/uso terapêutico , Terapia de Alvo Molecular/métodos , Ratos , Ativação Transcricional/efeitos dos fármacos , Traumatismos do Sistema Nervoso/patologia , Regulação para Cima/genéticaRESUMO
Hippocampal sclerosis of aging (HS-Aging) is a common high-morbidity neurodegenerative condition in elderly persons. To understand the risk factors for HS-Aging, we analyzed data from the Alzheimer's Disease Genetics Consortium and correlated the data with clinical and pathologic information from the National Alzheimer's Coordinating Center database. Overall, 268 research volunteers with HS-Aging and 2,957 controls were included; detailed neuropathologic data were available for all. The study focused on single-nucleotide polymorphisms previously associated with HS-Aging risk: rs5848 (GRN), rs1990622 (TMEM106B), and rs704180 (ABCC9). Analyses of a subsample that was not previously evaluated (51 HS-Aging cases and 561 controls) replicated the associations of previously identified HS-Aging risk alleles. To test for evidence of gene-gene interactions and genotype-phenotype relationships, pooled data were analyzed. The risk for HS-Aging diagnosis associated with these genetic polymorphisms was not secondary to an association with either Alzheimer disease or dementia with Lewy body neuropathologic changes. The presence of multiple risk genotypes was associated with a trend for additive risk for HS-Aging pathology. We conclude that multiple genes play important roles in HS-Aging, which is a distinctive neurodegenerative disease of aging.
Assuntos
Envelhecimento , Doença de Alzheimer , Hipocampo/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Sulfonilureias/genética , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Bases de Dados Factuais/estatística & dados numéricos , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Genômica/estatística & dados numéricos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Progranulinas , Fatores de Risco , Esclerose/etiologia , Esclerose/patologiaRESUMO
MicroRNAs (miRNAs) play key roles in gene expression regulation in both healthy and disease brains. To better understand those roles, it is necessary to characterize the miRNAs that are expressed in particular cell types under a range of conditions. In situ hybridization (ISH) can demonstrate cell- and lamina-specific patterns of miRNA expression that would be lost in tissue-level expression profiling. In the present study, ISH was performed with special focus on the human entorhinal cortex (EC) and transentorhinal cortex (TEC). The TEC is the area of the cerebral cortex that first develops neurofibrillary tangles in Alzheimer's disease (AD). However, the reason for TEC's special vulnerability to AD-type pathology is unknown. MiRNA ISH was performed on three human brains with well-characterized clinical and pathological parameters. Locked nucleic acid ISH probes were used referent to miR-107, miR-124, miR-125b, and miR-320. In order to correlate the ISH data with AD pathology, the ISH staining was compared with near-adjacent slides processed using Thioflavine stains. Not all neurons or cortical lamina stain with equal intensity for individual miRNAs. As with other areas of brain, the TEC and EC have characteristic miRNA expression patterns. MiRNA ISH is among the first methods to show special staining characteristics of cells and laminae of the human TEC.
RESUMO
The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played by these miRNAs in human diseases. Membership in this group is defined based on sequence similarity near the mature miRNAs' 5' end: all include the sequence AGCAGC. Phylogeny of this group of miRNAs is incomplete; thus, a definitive taxonomic classification (e.g., designation as a "superfamily") is currently not possible. While all vertebrates studied to date express miR-15a, miR-15b, miR-16, miR-103, and miR-107, mammals alone are known to express miR-195, miR-424, miR-497, miR-503, and miR-646. Multiple different miRNAs in the miR-15/107 group are expressed at moderate to high levels in human tissues. We present data on the expression of all known miR-15/107 group members in human cerebral cortical gray matter and white matter using new miRNA profiling microarrays. There is extensive overlap in the mRNAs targeted by miR-15/107 group members. We show new data from cultured H4 cancer cells that demonstrate similarities in mRNAs targeted by miR-16 and miR-103 and also support the importance of the mature miRNAs' 5' seed region in mRNA target recognition. In conclusion, the miR-15/107 group of miRNA genes is a fascinating topic of study for evolutionary biologists, miRNA biochemists, and clinically oriented translational researchers alike.
Assuntos
Regulação da Expressão Gênica , MicroRNAs , Animais , Proliferação de Células , Córtex Cerebral/metabolismo , Evolução Molecular , Cardiopatias/genética , Humanos , MicroRNAs/fisiologia , Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Doenças Neurodegenerativas/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
MicroRNAs (miRNAs) play fundamental roles in human brain neurochemistry. However, much remains to be learned in this fast-paced field. To understand how miRNAs contribute to normal biologic functions and disease states, it is critical to understand the miRNAs that are expressed in particular cell types under a range of conditions. Many tools have been developed to help describe the repertoire of miRNAs present at the tissue level in a given sample. However, tissue level miRNA profiling is inadequate to pinpoint the cellular and sub-cellular distribution of individual miRNAs. Such knowledge is especially important in the nervous system with its many cell types, microscopic heterogeneity with regard to functionally distinct cell groups, and extreme geometrical complexity in cellular shapes. We have found that in situ hybridization shows important cerebral cortical lamina-specific patterns of miRNA expression that would be lost on most tissue level expression studies, and these lamina-specific patterns can be directly relevant to human brain disease. Thus, in situ hybridization is an important experimental complement to tissue level miRNA expression profiling. Technical and theoretical aspects of this important technique are described, especially those pertinent to studying the human brain.
Assuntos
Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , Hibridização In Situ , MicroRNAs/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
MicroRNAs (miRNAs) are powerful regulators of gene expression. Although first discovered in worm larvae, miRNAs play fundamental biological roles-including in humans-well beyond development. MiRNAs participate in the regulation of metabolism (including lipid metabolism) for all animal species studied. A review of the fascinating and fast-growing literature on miRNA regulation of metabolism can be parsed into three main categories: (1) adipocyte biochemistry and cell fate determination; (2) regulation of metabolic biochemistry in invertebrates; and (3) regulation of metabolic biochemistry in mammals. Most research into the 'function' of a given miRNA in metabolic pathways has concentrated on a given miRNA acting upon a particular 'target' mRNA. Whereas in some biological contexts the effects of a given miRNA:mRNA pair may predominate, this might not be the case generally. In order to provide an example of how a single miRNA could regulate multiple 'target' mRNAs or even entire human metabolic pathways, we include a discussion of metabolic pathways that are predicted to be regulated by the miRNA paralogs, miR-103 and miR-107. These miRNAs, which exist in vertebrate genomes within introns of the pantothenate kinase (PANK) genes, are predicted by bioinformatics to affect multiple mRNA targets in pathways that involve cellular Acetyl-CoA and lipid levels. Significantly, PANK enzymes also affect these pathways, so the miRNA and 'host' gene may act synergistically. These predictions require experimental verification. In conclusion, a review of the literature on miRNA regulation of metabolism leads us believe that the future will provide researchers with many additional energizing revelations.