RESUMO
MicroRNA-449a (miR-449a) was significantly downregulated in 156 lung cancer tissues (p<0.001). We found that the low expression of miR-449a was highly correlated with cancer recurrence and survival of lung cancer patients. The transient introduction of miR-449a caused cell cycle arrest and cell senescence in A549 and 95D cells. Further studies revealed that E2F3 was a direct target of miR-449a in lung cancer cells. miR-449a also suppressed tumor formation in vivo in nude mice. These results suggest that miR-449a plays an important role in lung cancer tumorigenesis and that miR-449a might predict cancer recurrence and survival of lung cancer patients.
Assuntos
Fator de Transcrição E2F3/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Animais , Carcinogênese/genética , Pontos de Checagem do Ciclo Celular/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Senescência Celular/genética , Feminino , Genes Supressores de Tumor , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.
Assuntos
Fibra de Algodão , Giberelinas/biossíntese , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Genes de Plantas , Gossypium/genética , Microscopia Eletrônica de Varredura , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Óvulo Vegetal/metabolismo , Óvulo Vegetal/ultraestrutura , Plantas Geneticamente Modificadas , Homologia de Sequência de AminoácidosRESUMO
Five flavonoid structural genes, encoding chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and anthocyanidin reductase, were cloned from a brown-fiber cotton line (T586). The predicted proteins of these genes exhibit high sequence similarity with corresponding enzymes from various plants. RT-PCR analysis showed these genes are developmentally co-regulated and preferentially expressed in developing fibers of T586. Expression analyses and dimethylaminocinnaldehyde staining demonstrated that high transcript levels of these genes in developing fibers and presence of proanthocyanidins in mature fibers co-segregated with brown fiber in a recombination inbred line population. Our results indicated that the cloned flavonoid structural genes and proanthocyanidins were involved in the pigmentation in brown cotton fibers.
Assuntos
Flavonoides/metabolismo , Genes de Plantas , Gossypium/enzimologia , Proteínas de Plantas/metabolismo , Clonagem Molecular , Fibra de Algodão , Gossypium/genética , Gossypium/fisiologia , Filogenia , Pigmentação/genética , Proteínas de Plantas/genética , Proantocianidinas/metabolismoRESUMO
By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis.
