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Mol Genet Genomics ; 294(4): 1023-1036, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30972482


The miR-17-92 cluster has been involved in the cell cycle, apoptosis, and signaling. However, its transcriptional regulation has not been fully characterized. To elucidate the transcriptional regulation, the promoter of miR-17-92 was analyzed in detail in pig here. We found that, as an intronic miRNA, porcine miR-17-92 cluster was regulated by two independent promoters, an A/T-rich region directly upstream of the miR-17-92 coding sequence, and a G/C-rich region corresponding to the host gene promoter of the human miR-17-92 cluster. Several cis-regulatory elements were identified including sites for c-Myc, NFY, E2F3, and SP1, among which NFY and c-Myc sites were present in both A/T- and G/C-rich regions, while E2F3 and SP1 sites only existed in G/C-rich region. Sites for c-Myc, E2F3, and SP1 were positive for regulating transcription. NFY sites played bipartite roles, functioning as a repressor for the A/T-rich region, and as an activator for the G/C-rich region. Additionally, we found that levels of individual miRNAs in the cluster were not promoted completely in parallel with each other or with pri-miR-17-92 by the A/T-rich region, through using a self-made vector by modifying pGL3-basic in which firefly luciferase gene was replaced with an miR-17-92 cluster and a direct upstream A/T-rich region. The expression regulation of miR-17-92 is complicated and the results will contribute to further revealing the regulatory mechanisms under the expression of the miR-17-92 cluster.

MicroRNAs/genética , Elementos Reguladores de Transcrição , Animais , Composição de Bases , Linhagem Celular , Regulação da Expressão Gênica , Família Multigênica , Regiões Promotoras Genéticas , Suínos
Mol Genet Genomics ; 291(3): 1431-42, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26995495


Speckled 110 kDa (Sp110) plays an important role in infectious diseases, as revealed by studies in humans. However, little is known regarding porcine Sp110. To elucidate its potential role in porcine resistance to viral diseases, here, the complete coding sequence of porcine Sp110 gene and its 26 alternatively spliced isoforms were isolated using reverse transcription (RT)-polymerase chain reaction (PCR), and another seven splicing patterns were obtained using a minigene construct. Subcellular distribution of 11 representative isoforms was characterized in PK-15 cells transiently transfected with their respective GFP fusion constructs, and only isoforms (R and V) bearing all functional domains were localized in nucleus, indicating all the other isoforms lose normal functions of Sp110 owing to alternative splicing. Real-time quantitative PCR and competitive RT-PCR showed that both isoforms R and V had similar tissue expression profile, half-life and response to poly(I:C), a synthetic analog of viral double-stranded RNA, while the longer one (isoform R) was transcribed at a higher level. The results indicated that porcine Sp110 has a role in viral infection and that isoform R is the dominant active form. Overall the data provide potential resource for molecular breeding of pig resistant to diseases and contributes to breeding pigs resistant to viral infection.

Clonagem Molecular/métodos , Proteínas Nucleares/genética , Porco Miniatura/genética , Processamento Alternativo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Poli I-C , Polinucleotídeos/farmacologia , Suínos , Distribuição Tecidual
Yi Chuan ; 37(9): 926-31, 2015 09.
Artigo em Inglês | MEDLINE | ID: mdl-26399532


Paired immunoglobulin-like type 2 receptors (PILRs) are members of the immunoglobulin superfamily and composed of two subtypes, α and ß. PILRα plays an important role in the immune response against invading pathogens, but so far there is no report on porcine PILRα. In order to analyze the potential role of PILRα in porcine disease-resistant breeding, we first cloned the PILRA gene (V1-V3, GenBank accession Nos. KJ143679-81) into pigs, and identified its three splice variants. Each variant conceptually translates into proteins of 271 amino acids (aa), 254aa and 283aa, respectively. Furthermore, quantitative real-time PCR was used to construct expression profiles of each variant in tissues and that induced by Poly(I:C). All three variants had the highest expression levels in the spleen, followed by liver and lung tissues. While levels were low or undetectable in the heart, kidney, stomach, muscle, lymph, large intestine, small intestine and bladder. Poly(I:C) significantly induced the expression of splice variant 1 (V1) of porcine PILRA, but hardly affected the expression of V2 and V3. The results lay a foundation for further study on the role of PILRA in porcine breeding and disease resistance.

Clonagem Molecular , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Sequência de Aminoácidos , Animais , Cruzamento , Feminino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Processamento de RNA , Receptores Imunológicos/química , Receptores Imunológicos/fisiologia , Suínos