RESUMO
PU.1 is an Ets family transcription factor involved in the myelo-lymphoid differentiation. We have previously demonstrated that PU.1 is also expressed in the adipocyte lineage. However, the expression levels of PU.1 mRNA and protein in preadipocytes do not match the levels in mature adipocytes. PU.1 mRNA level is higher in preadipocytes, whereas its protein is expressed in the adipocytes but not in the preadipocytes. The underlying mechanism remains elusive. Here, we find that miR-155 knockdown or overexpression has no effect on the levels of PU.1 mRNA and protein in preadipocytes or adipocytes. MiR-155 regulates adipogenesis not through PU.1, but via C/EBPß which is another target of miR-155. We also checked the expression levels of PU.1 mRNA and antisense long non-coding RNA (AS lncRNA). Interestingly, compared with the level of PU.1 mRNA, the level of PU.1 AS lncRNA is much higher in preadipocytes, whereas it is opposite in the adipocytes. We further discover that PU.1 AS lncRNA binds to its mRNA forming an mRNA/AS lncRNA compound. The knockdown of PU.1 AS by siRNA inhibits adipogenesis and promotes PU.1 protein expression in both preadipocytes and adipocytes. Furthermore, the repression of PU.1 AS decreases the expression and secretion of adiponectin. We also find that the effect of retroviral-mediated PU.1 AS knockdown on adipogenesis is consistent with that of PU.1 AS knockdown by siRNA. Taken together, our results suggest that PU.1 AS lncRNA promotes adipogenesis through preventing PU.1 mRNA translation via binding to PU.1 mRNA to form mRNA/AS lncRNA duplex in preadipocytes.
Assuntos
Adipogenia/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Transativadores/metabolismo , Células 3T3-L1 , Adipogenia/genética , Adiponectina/genética , Adiponectina/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genéticaRESUMO
Natural antisense transcripts (NATs) exist ubiquitously as pivotal molecules to regulate coding gene expression. Sirtuin 1 (Sirt1) is a NAD-dependent deacetylase which is involved in myogenesis. However, whether Sirt1 transcribes NAT during C2C12 differentiation is still unknown. In this study, we identified a Sirt1 NAT which was designated as Sirt1 antisense long non-coding RNA (AS lncRNA) by sequencing and bioinformatic analysis. The level of Sirt1 AS lncRNA was greater in spleen but less in muscle tissue. The expression of both Sirt1 mRNA and Sirt1 AS lncRNA decreased during C2C12 myogenic differentiation, whereas the levels of miR-34a, which targets Sirt1, increased gradually. We further found that the half-life of Sirt1 AS lncRNA was 10h, but that of Sirt1 mRNA was 6h in C2C12 cells treated with 2 µg/ml Actinomycin D. Therefore, compared with Sirt1 mRNA, Sirt1 AS lncRNA was more stable. Overexpression of Sirt1 AS lncRNA increased the levels of Sirt1 protein, whereas overexpression of Sirt1 AS lncRNA mutant did not affect the level of Sirt1 protein in C2C12 cells. Moreover, downregulation of Sirt1 mRNA caused by miR-34a was counteracted by Sirt1 AS lncRNA in C2C12 cells. Taken together, we identified a novel NAT of Sirt1 which implicated in myogenesis through regulating Sirt1 expression.
Assuntos
MicroRNAs/genética , Desenvolvimento Muscular/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Sirtuína 1/genética , Animais , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Dactinomicina/farmacologia , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Meia-Vida , Camundongos , MicroRNAs/biossíntese , Dados de Sequência Molecular , Desenvolvimento Muscular/efeitos dos fármacos , Músculos/citologia , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Antissenso/biossíntese , RNA Longo não Codificante/biossíntese , RNA Mensageiro/biossíntese , Baço/citologiaRESUMO
The glioma-associated oncogene family zinc finger 3 gene (GLI3) mediates in all vertebrates hedgehog (Hh) signaling that plays an essential role in the induction and patterning of numerous cell types during invertebrate and vertebrate development. In this study, a total of 6 single nucleotide polymorphisms (SNPs: 1-6) were identified by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and DNA pool sequencing, including all 13 exons and 12 exon-intron boundaries within the bovine GLI3 gene. 16 haplotypes and 13 combined genotypes were revealed and the linkage disequilibrium was assessed in 708 individuals representing three main cattle breeds from China. The statistical analyses indicated that the SNP2, 3 and 4 are associated with the body weight at birth and 6 months in Nanyang cattle population (P<0.05). No significant association was detected between 11 combined genotypes and body weight at five different ages. Our results provide evidence that polymorphisms in the GLI3 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.