Ação regulatória de microRNAs durante a regeneração epimórfica em nadadeira caudal de zebrafish (Danio rerio)
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2020-02-19
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Universidade Estadual Paulista (Unesp)
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Regeneração é a capacidade de reconstruir partes do corpo e formar uma nova estrutura, idêntica morfológica e funcionalmente àquela perdida. De modo geral, a habilidade de regenerar tecidos, órgãos e membros é evolutivamente conservada nos vertebrados, ainda que envolva a ativação de processos celulares distintos, como desdiferenciação, reorganização e rediferenciação das células adjacentes ao tecido injuriado ou amputado. A regeneração denominada "epimórfica", comum em diversos teleósteos, anfíbios e répteis, caracteriza-se pela formação de um agregado de células desdiferenciadas (blastema) que posteriormente se rediferenciam, proliferam e migram para recompor a estrutura originalmente perdida. Durante a regeneração epimórfica, típica na nadadeira caudal do teleósteo zebrafish (Danio rerio), ocorre extensa desdiferenciação celular de células ósseas (osteoblastos) ativada por mecanismos genéticos ainda não completamente elucidados. Do mesmo modo, pouco se conhece acerca dos mecanismos epigenéticos mediados por microRNAs (miRNAs, pequenos RNAs regulatórios) atuantes no controle da desdiferenciação celular durante a regeneração. Neste contexto, o presente estudo teve por objetivo caracterizar a atividade de microRNAs na regulação de genes alvo importantes nos processos celulares relacionados à regeneração epimórfica da nadadeira caudal no modelo zebrafish. Para isso, realizamos a amputação da nadadeira caudal e coletamos tecidos de 80 zebrafish, subdivididos em 8 grupos experimentais: 0, 12, 24, 36, 48, 72, 96 e 120 horas pós-amputação (hpa). Essas amostras foram subdividas e utilizadas para o sequenciamento em larga escala de microRNAs (miRNA-seq) dos períodos 0, 24 , 48 e 72 hpa e para PCR quantitativo em Tempo Real (qPCR), após etapa prévia de predição in silico para seleção de miRNAs (miR-20a-3p, miR-203a-3p, miR-203a-3p, miR-19a-5p e let-7c-3p) e genes alvo candidatos expressos em células ósseas (sp7, runx2, bglap e spp1) potencialmente atuantes nas vias de interesse do estudo. Também realizamos análises in silico explorando bancos de dados públicos para avaliar se existe conservação evolutiva, nos diferentes grupos de vertebrados, das interações miRNA-alvo preditas. Os dados gerados por miRNA-seq revelaram mais de 600 microRNAs maduros expressos na região da amputação, sendo 37 miRNAs diferencialmente expressos nos 4 períodos pós-amputação analisados, e 14 miRNAs reconhecidos como relevantes nos processos de formação e reparo do tecido ósseo, com base na literatura. Além disso três miRNAs novos, ainda não reportados previamente no zebrafish, tiveram expressão detectada em nosso estudo. Com a análise do perfil de expressão por qPCR, identificamos o pico de expressão dos genes alvo próximo a 12hpa, sugerindo que este se constitui em um período crítico para a ocorrência de desdiferenciação de osteoblastos. A comparação temporal dos perfis de expressão pós-amputação também mostrou expressão negativamente correlacionada entre o gene sp7 e os quatro miRNAs selecionados, indicando uma potencial atividade coordenada na regulação deste e dos demais genes avaliados. As análises evolutivas revelaram que os sítios de interação do gene sp7 (na região 3'UTR do gene) aos miRNAs possuem mutações específicas em suas sequências de DNA nas diferentes espécies estudadas, mostrando que os processos moleculares de regeneração epimórfica não são conservados entre os grupos de vertebrados. Interessantemente, nas espécies de mamíferos, cujas sequências 3'UTR de sp7 são menores e variáveis, não foram detectados sítios de interação para os miRNAs estudados, o que está de acordo com a ausência de ativação da resposta de regeneração epimórfica mediada por blastema observada nesses animais. Juntos, esses resultados evidenciam que a regulação gênica mediada por microRNAs é um mecanismo importante para que haja regeneração epimórfica, particularmente durante a desdiferenciação de osteoblastos induzida pela modulação da atividade de sp7 que culmina na formação do blastema e restauração da estrutura danificada.
Regeneration is the ability to regain lost parts, making new structures morphologically and functionally identical to the lost one. Generally, this ability to regenerate tissues, organs and limbs is evolutionarily conserved on vertebrates, even if it involves the activation of distinct cellular processes, such as dedifferentiation, reorganization and redifferentiation of cells around to the injured or amputated tissue. The regeneration so-called “epimorphic”, found in teleost fish, amphibians and reptiles, is characterized by a blastema, a mass of dedifferentiated cells that will differentiate, proliferate and migrate to rebuild the lost structure. During epimorphic regeneration, typically found in the caudal fin of the teleost zebrafish (Danio rerio), there is an huge cell dedifferentiation of osteoblasts (bone cells) activated by genetic mechanism that have not yet been fully elucidated. Likewise, little is known about the epigenetic mechanisms mediated by microRNAs (miRNAs, small regulatory RNAs) acting in the control of cell dedifferentiation during regeneration. In this context, the present study aimed to characterize the activity of microRNAs in the regulation of important target genes in cellular processes related to epimorphic regeneration of the caudal fin in the zebrafish. To do so, we performed the amputation of the caudal fin and from 80 zebrafish and collected its tissue dividing it in 8 experimental groups: 0, 12, 24, 36, 48, 72, 96 and 90 hours post amputation (hpa). These samples were subdivided and used to perform a large-scale sequencing of microRNAs (miRNA-seq) of the periods 0, 24, 48 and 72 hpa and a real time quantitative PCR (qPCR), after a previous in silico prediction to select miRNAs (miR-20a-3p, miR-203a-3p, miR-203a-3p, miR-19a-5p e let-7c-3p) and target genes to be expressed in bone cells (sp7, runx2, bglap e spp1), potentially present on the investigated pathways of this study. We also performed in silico analysis exploring public databases to evaluate the possibility of evolutive conservation of the miRNA-target interactions predicted in different vertebrate groups. Our data from miRNA-seq shown more than 600 mature microRNAs expressed in the amputation site, with 37 of then differentially expressed in the 4 periods post amputation, and 14 miRNAs known as relevant on process of bone tissue repair and formation, based on literature. Also, three new miRNAs, yet not previously reported on zebrafish, had its expression detected on our study. The expression profile by qPCR have shown a peak of target genes expression close to the 12 hpa, suggesting that it is a critic period for the differentiation of osteoblasts. The comparison of post amputation’s temporal expression profile also shown a negative correlated expression between sp7 gene and the four selected miRNAs, evince a potential coordinated activity on the regulation of this and the other genes analysed. The evolutive analyses revealed that the interaction sites of sp7 gene (at the 3’UTR region) for miRNAs have specific mutations in their DNA sequences on all of the studied species, showing that the molecular process of epimorphic regeneration are not conserved between the vertebrate groups. Interestingly, on mammals species, where sp7 3’UTR sequences are smaller and variable, ware not detected interaction sites for the analyzed miRNAs, what agrees with the absence of response activation of epimórfica regeneration mediated by blastema formation in these animals. Altogether, our results evince that the genic regulation mediated by miRNAs is an important mechanism for the epimorphic regeneration to happens, specifically during the dedifferentiation of osteoblasts induced by the sp7’s activity modulation that ends in the blastema formation and restoration of the damaged structure.
Regeneration is the ability to regain lost parts, making new structures morphologically and functionally identical to the lost one. Generally, this ability to regenerate tissues, organs and limbs is evolutionarily conserved on vertebrates, even if it involves the activation of distinct cellular processes, such as dedifferentiation, reorganization and redifferentiation of cells around to the injured or amputated tissue. The regeneration so-called “epimorphic”, found in teleost fish, amphibians and reptiles, is characterized by a blastema, a mass of dedifferentiated cells that will differentiate, proliferate and migrate to rebuild the lost structure. During epimorphic regeneration, typically found in the caudal fin of the teleost zebrafish (Danio rerio), there is an huge cell dedifferentiation of osteoblasts (bone cells) activated by genetic mechanism that have not yet been fully elucidated. Likewise, little is known about the epigenetic mechanisms mediated by microRNAs (miRNAs, small regulatory RNAs) acting in the control of cell dedifferentiation during regeneration. In this context, the present study aimed to characterize the activity of microRNAs in the regulation of important target genes in cellular processes related to epimorphic regeneration of the caudal fin in the zebrafish. To do so, we performed the amputation of the caudal fin and from 80 zebrafish and collected its tissue dividing it in 8 experimental groups: 0, 12, 24, 36, 48, 72, 96 and 90 hours post amputation (hpa). These samples were subdivided and used to perform a large-scale sequencing of microRNAs (miRNA-seq) of the periods 0, 24, 48 and 72 hpa and a real time quantitative PCR (qPCR), after a previous in silico prediction to select miRNAs (miR-20a-3p, miR-203a-3p, miR-203a-3p, miR-19a-5p e let-7c-3p) and target genes to be expressed in bone cells (sp7, runx2, bglap e spp1), potentially present on the investigated pathways of this study. We also performed in silico analysis exploring public databases to evaluate the possibility of evolutive conservation of the miRNA-target interactions predicted in different vertebrate groups. Our data from miRNA-seq shown more than 600 mature microRNAs expressed in the amputation site, with 37 of then differentially expressed in the 4 periods post amputation, and 14 miRNAs known as relevant on process of bone tissue repair and formation, based on literature. Also, three new miRNAs, yet not previously reported on zebrafish, had its expression detected on our study. The expression profile by qPCR have shown a peak of target genes expression close to the 12 hpa, suggesting that it is a critic period for the differentiation of osteoblasts. The comparison of post amputation’s temporal expression profile also shown a negative correlated expression between sp7 gene and the four selected miRNAs, evince a potential coordinated activity on the regulation of this and the other genes analysed. The evolutive analyses revealed that the interaction sites of sp7 gene (at the 3’UTR region) for miRNAs have specific mutations in their DNA sequences on all of the studied species, showing that the molecular process of epimorphic regeneration are not conserved between the vertebrate groups. Interestingly, on mammals species, where sp7 3’UTR sequences are smaller and variable, ware not detected interaction sites for the analyzed miRNAs, what agrees with the absence of response activation of epimórfica regeneration mediated by blastema formation in these animals. Altogether, our results evince that the genic regulation mediated by miRNAs is an important mechanism for the epimorphic regeneration to happens, specifically during the dedifferentiation of osteoblasts induced by the sp7’s activity modulation that ends in the blastema formation and restoration of the damaged structure.
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Palavras-chave
Peixe-zebra, MicroRNAs, Reação em Cadeia da Polimerase, Genética