RESUMEN

PURPOSE: To explore the regulatory relationship between Chloride intracellular channel 1 (CLIC1) and Angiomotin (AMOT)-p130, and reveal the role of AMOT-p130 in gastric cancer (GC). METHODS: Immunohistochemistry was performed to analyze the expression of CLIC1 and AMOT-p130 in GC tissues and adjacent tissues. The expression of AMOT-p130 upon CLIC1 silencing was analyzed using RT-PCR, western blot, and immunofluorescence in GC cells. Transwell and wound-healing assays were performed to detect migration and invasion in GC cells. The changes in EMT-related proteins were detected using western blot. RESULTS: Our study found that high CLIC1 expression was significantly associated with low AMOT-p130 expression in GC tissues. Silencing CLIC1 expression in MGC-803 cells (MGC-803 CLIC1 KO) and AGS cells (AGS CLIC1 KO) decreased the invasive and migratory abilities of tumor cells, which were induced by the upregulation of AMOT-p130. Subsequently, we demonstrated that AMOT-p130 inhibits the invasive and migratory abilities of GC cells by inhibiting epithelial-mesenchymal transition. CONCLUSIONS: Our study suggests that AMOT-p130 could inhibit epithelial-mesenchymal transition in GC cells. CLIC1 may participate in the metastatic progression of GC by downregulating the expression of AMOT-p130.

Asunto(s)

Canales de Cloruro/metabolismo , Transición Epitelial-Mesenquimal , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Angiomotinas , Línea Celular Tumoral , Movimiento Celular , Canales de Cloruro/genética , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Cicatrización de Heridas

RESUMEN

The polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) is a newly identified protein that is specifically expressed in testis tissue and participates in spermatogenesis. In this study, we characterized a novel bovine GALNTL5 splice variant, designated as GALNTL5-AS, by using real-time polymerase chain reaction (RT-PCR) and clone sequencing methods. The novel GALNTL5 isoform was derived from the complete transcript, GALNTL5-complete, via alternative splicing (AS). The pattern of the splice variant was exon skipping. Bovine GALNTL5 transcripts were expressed in the testis, as demonstrated by RT-PCR. The expression levels of both transcripts were higher in adult testes than in calf testes (P < 0.05). In addition, prediction analysis showed that the GALNTL5-AS transcript only encoded 122 amino acids and lost its glycosyltransferase 1 and Gal/GalNAc-T motifs, which may result in a dysfunctional protein compared with the predominant transcript GALNTL5-complete. This study improves our understanding of the bovine GALNTL5 gene function during bull sperm formation.

Asunto(s)

Empalme Alternativo , N-Acetilgalactosaminiltransferasas/genética , Testículo/metabolismo , Secuencias de Aminoácidos , Animales , Bovinos , Exones , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Polymorphonuclear neutrophil (PMN) leukocytes are primary phagocytic cells of the bovine mammary gland and a first line of defense against invading pathogens during bovine mastitis infection. Cluster of differentiation 14 (CD14) is mainly expressed in macrophages and neutrophils and acts as a co-receptor that binds bacterial lipopolysaccharide (LPS) and recruits PMNs to CD14-LPS complexes in mammary epithelial cells. In this study, we identified a novel splice variant in PMNs, named CD14-SV, characterized by a deleted region from c.143-579 nt compared to the CD14 reference mRNA sequence. Moreover, a single nucleotide polymorphism (c.523 A>G) in exon 2 of CD14 was identified and found to modify the secondary structure and hydrophilicity of the CD14 protein. Association analysis also showed that the milk somatic cell score, an indicator of mastitis, of cows with the GG genotype was lower than that of cows with the AA and AG genotypes. Our findings suggest that the expression of CD14 in bovine blood PMNs is regulated by alternative splicing, and that CD14-SV is a candidate functional marker that may influence mastitis-resistance in dairy cows.

Asunto(s)

Bovinos/genética , Receptores de Lipopolisacáridos/genética , Mastitis Bovina/genética , Neutrófilos/metabolismo , ARN Mensajero/genética , Empalme Alternativo , Animales , Femenino , Variación Genética , Receptores de Lipopolisacáridos/metabolismo , Masculino , Mastitis Bovina/sangre , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo

RESUMEN

Phospholipase C zeta 1 (PLCζ1), which transcribes a key protein, has an important function in oocyte activation and embryo development because PLCζ1 can trigger a series of intracellular Ca2+ oscillations in mammals. In this study, a novel splice variant in the testis tissues of adult and fetal Chinese Holstein bulls was characterized by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis. The novel splice variant PLCζ1-sv1 was derived from the PLCζ1 complete transcript (PLCζ1-complete) by alternative splicing; the alternative splicing pattern exhibited alternative 5'-splice sites. The full-length transcript, PLCζ1-complete, is the main transcript found in fetal and adult cow testis tissue. Quantitative real-time PCR (qPCR) analysis demonstrated that the expression levels of the PLCζ1-complete transcript were significantly higher than those of the PLCζ1-sv1 splice variant in bovine testis tissues. PLCζ1 protein sequencing analysis showed that the amino acids at positions 453 to 457 were deleted in PLCζ1-sv1, thereby terminating transcription prematurely. In summary, this study provided information to elucidate the structure and function of the bovine PLCζ1 gene.

Asunto(s)

Empalme Alternativo/genética , Fosfolipasa C gamma/genética , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , China , Elementos de Facilitación Genéticos/genética , Exones/genética , Masculino , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Fosfolipasa C gamma/química , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética

RESUMEN

Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated.

Asunto(s)

Bovinos/genética , Fosfoenolpiruvato Carboxilasa/genética , Secuencia de Aminoácidos , Animales , China , Clonación Molecular , ADN Complementario , Exones , Femenino , Masculino , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Fosfoenolpiruvato Carboxilasa/metabolismo , Conformación Proteica , ARN/genética , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN

RESUMEN

Mastitis is an economically devastating disease affecting the dairy industry. Dairy cows with mastitis give reduced milk yield and produce milk that is unfit for consumption. The chemokine receptor CXCR1 is an excellent prospective genetic marker for mastitis resistance in cattle because it regulates neutrophil migration, killing, and survival during infection. We detected 4 single nucleotide polymorphisms (SNPs) of the CXCR1 gene in Chinese native cattle and analyzed their associations with milk traits. Screening for genetic variations in CXCR1 among 648 Chinese Holstein, Luxi Yellow, and Bohai Black cattle by created restriction site polymerase chain reaction (PCR), nested PCR, and DNA sequencing revealed 4 new SNPs with allelic frequencies ranging from 0.676 to 0.821, 0.706 to 0.803, 0.647 to 0.824, and 0.558 to 0.581. All four CXCR1 gene SNPs were located in exon II. Two SNPs, c.337A>G and c.365C>T, were nonsynonymous mutations [ATC (Ile) > GTC (Val) and GCC (Ala) > GTC (Val)], whereas two, c.291C>T and c.333C>T, were synonymous mutations [TTC (Gly) > TTT (Gly) and GGC (Phe) > GGT (Phe)]. Statistical analyses revealed the significant association of c.337A>G and c.365C>T with the somatic cell score, which suggests the possible role of these SNPs in the host response against mastitis. Our data suggest that combined genotypes CCAC/CCGC, CCAC/CTAT, and CCAT/CTAT (lowest somatic cell scores); CTAC/CTAT (highest protein rate); CCAC/CTGC (highest fat rate); and CCAT/CTAT (highest 305-day milk yield) can be used as possible candidates for marker-assisted selection in dairy cattle breeding programs.

Asunto(s)

Bovinos/genética , Lactancia/genética , Polimorfismo de Nucleótido Simple , Receptores de Interleucina-8A/genética , Animales , Animales Endogámicos , Exones , Femenino , Estudios de Asociación Genética , Mastitis Bovina/genética , Leche/metabolismo , Mutación , Carácter Cuantitativo Heredable

RESUMEN

XRCC1-399 allele polymorphisms have been reported to be associated with susceptibility to hepatocellular carcinoma (HCC), but the conclusions of the various studies have been inconsistent. We conducted a meta-analysis of available studies to determine whether XRCC1-399 alleles influence susceptibility to hepatocellular carcinoma. We searched English-language databases, including PubMed, Medline and Embase, using terms such as "hepatocellular carcinoma" (or "HCC"), "X-ray repair cross-complementing group 1" (or "XRCC1") and "genetic polymorphism" (or "SNP"), among others; we also searched Chinese-language databases, including CNKI, VIP, Wanfang Data, and CBM, using terms such as "ganai", "ganxibaoai", "ganzhongliu", "duotaixing", and "X-xian xiufu jiaocha hubu jiyin 1". Eight independent studies, including 1604 HCC cases and 2185 controls, were included. The pooled odds ratio for XRCC1-399 was 0.99 (95% confidence interval = 0.75-1.31). We conclude that XRCC1- 399 gene polymorphisms are unrelated to risk for HCC.

Asunto(s)

Carcinoma Hepatocelular/genética , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas/genética , Polimorfismo Genético , Intervalos de Confianza , Heterogeneidad Genética , Humanos , Oportunidad Relativa , Sesgo de Publicación , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X

RESUMEN

The mannan-binding lectin gene (MBL) participates as an opsonin in the innate immune system of mammals, and single nucleotide polymorphisms (SNPs) in MBL cause various immune dysfunctions. In this study, we detected SNPs in MBL2 at exon 1 using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing techniques in 825 Chinese Holstein cows. Four new SNPs with various allele frequencies were also found. The g.1164 G>A SNP was predicted to substitute arginine with glutamine at the N-terminus of the cysteine-rich domain. In the collagen-like domain, SNPs g.1197 C>A and g.1198 G>A changed proline to glutamine, whereas SNP g.1207 T>C was identified as a synonymous mutation. Correlation analysis showed that the g.1197 C>A marker was significantly correlated to somatic cell score (SCS), and the g.1164 G>A locus had significant effects on SCS, fat content, and protein content (P < 0.05), suggesting possible roles of these SNPs in the host response against mastitis. Nine haplotypes and nine haplotype pairs corresponding to the loci of the 4 novel SNPs were found in Chinese Holsteins. Haplotype pairs MM, MN, and BQ were correlated with the lowest SCS; MN with the highest protein yield; MM with the highest protein rate, and MN with the highest 305- day milk yield. Thus, MM, MN, and BQ are possible candidates for marker-assisted selection in dairy cattle breeding programs.

Asunto(s)

Bovinos/genética , Estudios de Asociación Genética , Lectina de Unión a Manosa/genética , Leche/metabolismo , Polimorfismo de Nucleótido Simple/genética , Carácter Cuantitativo Heredable , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , China , Exones/genética , Frecuencia de los Genes/genética , Haplotipos/genética , Heterocigoto , Análisis de los Mínimos Cuadrados , Lectina de Unión a Manosa/química , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple/genética , Análisis de Secuencia de ADN , Tinción con Nitrato de Plata

RESUMEN

The complement system helps in the direct lysis of invading pathogens and modulates phagocytic, humoral and cellular immune responses. Complement 4 is a critical component in complement activity and protection against many bacterial pathogens because it is essential to classical and lectin activation pathways. We used reverse transcription and PCR to investigate alternative splicing and expression of the complement component 4 (C4A) gene in Chinese Holstein cattle. The PCR products were cloned and sequenced. A novel splice variant involving intron 10 was identified, which we named C4A-AS. To examine how C4A gene activity is affected by bovine mastitis, six Chinese Holstein cattle were divided into healthy (non-mastitic) and Staphylococcus aureus-induced mastitic groups. Real-time quantitative PCR (qRT-PCR) revealed that the C4A-complete and C4A-AS transcripts are expressed at significantly different levels in healthy cows, while there were no significant differences in the mastitic group (P = 0.257). Expression of C4A-AS increased significantly when mastitis developed. We also examined the expression of C4A-complete and C4A-AS in several tissues (liver, heart, spleen, lung, kidney, tongue, and muscle). The two transcripts were expressed in all of these tissues but there were no significant differences in expression between healthy and mastitic cows. We therefore conclude that the C4A-complete transcript is the main transcript under normal physiological conditions, while C4A-AS is augmented when mastitis develops.

Asunto(s)

Empalme Alternativo/genética , Bovinos/genética , Bovinos/inmunología , Complemento C4a/genética , Industria Lechera , Mastitis Bovina/genética , Mastitis Bovina/inmunología , Animales , China , Femenino , Mastitis Bovina/microbiología , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Staphylococcus aureus/fisiología

RESUMEN

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

Asunto(s)

Lactoferrina/genética , Glándulas Mamarias Animales/inmunología , Mastitis Bovina/metabolismo , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/genética , Empalme Alternativo , Animales , Bovinos , Exones , Femenino , Expresión Génica , Lactoferrina/inmunología , Lactoferrina/metabolismo , Hígado/inmunología , Hígado/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/microbiología , Mastitis Bovina/inmunología , Mastitis Bovina/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inmunología , Bazo/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología

RESUMEN

Transferrin (Tf) is a ß-globulin protein that transports iron ions in mammalian cells. It contributes to innate immunity to microbial pathogens, primarily by limiting microbial access to iron. Thus, polymorphisms present in bovine Tf could potentially underlie inherited differences in mastitis resistance and milk production traits. We detected three novel single-nucleotide polymorphisms of the Tf gene in Chinese native cattle by screening for genetic variation of Tf in 751 individuals of three Chinese cattle breeds, namely China Holstein, Luxi Yellow and Bohai Black, using PCR-RFLP and DNA sequencing techniques. The three new SNPs, g.-1748G>A ss250608649, g.13942T>C ss250608650, and g.14037A>G ss250608651, had allele frequencies of 85.9, 86.3 and 92.5%, 64.5, 73.3 and 65.0%, and 67.6, 73.7 and 60.0%, respectively. SNP g.-1748G>A was located in the 5' flanking region of Tf. SNP g.14037A>G was located in intron 8 of Tf. SNP g.13942T>C, located in exon 8 of Tf, was a synonymous mutation (TTA > CTA), encoding a leucine (326 aa) in the Tf protein. Associations of the Tf SNPs with milk traits were also analyzed. Significant (P < 0.05) relationships among the Tf polymorphisms, somatic cell scores (SCS), and milk productive traits were observed. Cows with genotypes TT (g.13942T>C), GG (g.-1748G>A) and AG (g.14037A>G) had a lower SCS and higher protein levels and 305-day milk yield. Nineteen combinations of different haplotypes from the three SNPs were identified in Chinese Holstein cattle. The haplotype combination ATA/GCA, GCA/GCA and GCG/ GTA was dominant in cows with a lower SCS, a higher protein level and a higher 305-day milk yield, respectively. Moreover, the gene expression level of Tf was higher in mastitis-affected mammary tissues than in normal mammary tissues. These results suggest that the Tf gene affects milk production, as well as mastitis-resistance traits, in Chinese Holsteins.

Asunto(s)

Leche , Polimorfismo de Nucleótido Simple/genética , Transferrina/genética , Animales , Bovinos , Variación Genética/genética , Genotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción/genética , Análisis de Secuencia de ADN