RESUMO
We aimed to investigate the biological role of miR-367 in uveal melanoma cell growth and migration, and the underlying mechanism responsible. Quantitative real-time polymerase chain reaction was performed to evaluate miR-367 expression in uveal melanoma tissue samples and cell lines. A miR-367 mimic, miR-367 inhibitor, and negative control oligonucleotide were transfected into these cells to investigate the function of this microRNA. In addition, the role of PTEN in miR-367-mediated uveal melanoma cell growth and migration was evaluated. miR-367 was significantly upregulated in uveal melanoma cells and tissue samples (both P < 0.01). Its inhibition suppressed the proliferation, cell cycle transition, and migration of such cells, and increased levels had the opposite effect. PTEN was confirmed to be a target gene of miR-367. More importantly, co-transfection with a PTEN construct lacking the 3'-untranslated region mitigated miR-367 mimic-induced promotion of uveal melanoma cell proliferation and migration. In summary, miR-367 was found to be upregulated in this malignancy, and may promote uveal melanoma cell proliferation and migration, at least in part by regulating PTEN.
Assuntos
Movimento Celular , Proliferação de Células , Melanoma/metabolismo , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Uveais/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Melanócitos/metabolismo , Melanócitos/fisiologia , Melanoma/genética , Melanoma/patologia , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , Regulação para Cima , Neoplasias Uveais/genética , Neoplasias Uveais/patologiaRESUMO
Porcine ear size is an important characteristic for distinguishing among pig breeds. In a previous genome-wide association study of porcine ear size, LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3), high mobility group AT-hook 2 (HMGA2), and Wnt inhibitory factor 1 (WIF1) were implicated as important candidate genes for ear size. This study investigated the expression levels of four candidate genes for ear size in Erhualian and Large White pigs. Ten Erhualian pigs with large ears and eight Large White pigs with small ears at 60 days of age were examined. The mRNA expression levels of the four candidate genes were quantified by real-time polymerase chain reaction. WIF1 mRNA expression was significantly higher in Large White than in Erhualian pigs (P < 0.05), whereas the expression levels of the other three genes were not significantly different between the two breeds. The protein expression levels of the four genes were analyzed using western blot. WIF1 protein expression was significantly higher in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 protein expression was significantly higher in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 is the main causal gene for ear size in pigs.
Assuntos
Orelha/crescimento & desenvolvimento , Locos de Características Quantitativas , Suínos/genética , Animais , Animais Endogâmicos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs.
Assuntos
MAP Quinase Quinase Quinase 5/genética , Sus scrofa/genética , Sequência de Aminoácidos , Animais , Domínio Catalítico , Clonagem Molecular , Variações do Número de Cópias de DNA , Dosagem de Genes , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , MAP Quinase Quinase Quinase 5/química , MAP Quinase Quinase Quinase 5/metabolismo , Modelos Moleculares , Especificidade de Órgãos , Filogenia , Conformação Proteica em alfa-Hélice , Sus scrofa/metabolismoRESUMO
Thermotherapy has been proven to be effective for the treatment of various tumors, including glioma. We determined whether tumor necrosis factor-alpha (TNF-α) is involved in the regulation of the biological processes of glioma development. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the levels of TNF-α mRNA and heat shock factor-1 (HSF1) protein, respectively, in glioma cells. Radioimmunoassay was used to dynamically monitor the contents of TNF-α in the nutrient fluid of C6 cells after thermotherapy treatment. Crystal violet staining was used to determine glioma invasiveness. The most obvious increases in HSF1 protein and TNF-α mRNA in C6 cells were observed at 30 and 60 min after thermotherapy, respectively. In addition, the radioactivity of TNF-α in the culture fluid of the C6 cells reached a peak after 120 min of thermotherapy. In addition, glioma invasiveness decreased and the concentration of TNF-α reached a maximum after 120 min of thermotherapy. Our results show that the decrease in thermotherapy-mediated glioma invasiveness is due to the accelerated release of TNF-α, which could promote the release of HSF1 from neurospongioma cells.
Assuntos
Neoplasias Encefálicas/terapia , Regulação Neoplásica da Expressão Gênica , Glioma/terapia , Hipertermia Induzida , Fator de Necrose Tumoral alfa/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Fatores de Transcrição de Choque Térmico , Masculino , Invasividade Neoplásica , Transplante de Neoplasias , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Blumea balsamifera DC is a member of the Compositae family and is frequently used as traditional Chinese medicine. Blumea balsamifera is rich in monoterpenes, which possess a variety of pharmacological activities, such as antioxidant, anti-bacteria, and anti-viral activities. Farnesyl diphosphate synthase (FPS) is a key enzyme in the biosynthetic pathway of terpenes, playing an important regulatory role in plant growth, such as resistance and secondary metabolism. Based on the conserved oligo amino acid residues of published FPS genes from other higher plant species, a cDNA sequence, designated BbFPS, was isolated from B. balsamifera DC using polymerase chain reaction. The clones were an average of 1.6 kb and contained an open reading frame that predicted a polypeptide of 342 amino acids with 89.07% identity to FPS from other plants. The deduced amino acid sequence was dominated by hydrophobic regions and contained 2 highly conserved DDxxD motifs that are essential for proper functioning of FPS. Phylogenetic analysis indicated that FPS grouped with other composite families. Prediction of secondary structure and subcellular localization suggested that alpha helices made up 70% of the amino acids of the sequence.
Assuntos
Asteraceae/enzimologia , Asteraceae/genética , Genes de Plantas , Geraniltranstransferase/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Evolução Molecular , Geraniltranstransferase/química , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.
Assuntos
Animais , Humanos , Masculino , Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , S-Adenosilmetionina/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Fator C de Crescimento do Endotélio Vascular/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Carcinogênese/efeitos dos fármacos , Metilação de DNA/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos/efeitos dos fármacos , Imuno-Histoquímica , Camundongos Nus , Oncogenes/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/análise , Neoplasias Gástricas/metabolismo , Fator C de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.
Assuntos
Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , S-Adenosilmetionina/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Metilação de DNA/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Camundongos Nus , Oncogenes/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/metabolismo , Fator C de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Blumea balsamifera is a commercially important medicinal herb in China and other parts of Asia. It is used to produce borneol. This plant grows in the wild, but resources have diminished greatly in recent years. We examined the genetic diversity of this species to help develop conservation strategies; 35 plants from five provinces were analyzed using AFLPs. Eight AFLP primer combinations generated 1367 fragments, giving a mean of 172 fragments per primer combination. Polymorphism in the germplasm analysis was found for 1360 (99.48%) of the fragments, of which 264 (19.27%) fragments were unique (accession specific) and 423 (25.33%) of the fragments were rare (present in less than 10% of the accessions). The polymorphic fragments were used to group the accessions in a UPGMA phenogram. Most grouping was geographical. In general, accessions coming from Guizhou and Guangxi showed higher diversities as these accessions were scattered in different groups. The genetic distance estimated by Jaccard similarity coefficient index showed low variability among genotypes (coefficient value ranged from 0.60 to 0.95). More attention should be given to the study and conservation of the biodiversity of this economically important genus.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Asteraceae/genética , Variação Genética , Marcadores Genéticos , Humanos , Medicina Tradicional ChinesaRESUMO
Neochrysocharis formosa (Westwood) (Hymenoptera: Eulophidae), one of the dominant natural enemies of agromyzid leafminers, is a synovigenic parasitoid. We compared the longevity, oogenesis, and nutrient levels of female wasps provided with 10% solutions of five naturally occurring sugars. All five sugars significantly increased the longevity of female wasps, which was 6.5-9.3-fold higher than that of parasitoids provided with water only. We found no significant difference in longevity of female wasps fed on glucose versus fructose or trehalose versus melezitose, but longevity of wasps fed on glucose or fructose was significantly longer than those fed on trehalose or melezitose. Also, we examined the oosorption capability of wasps fed on the five sugars. Some mature eggs were present in the ovaries of newly emerged females, but these were fully reabsorbed within 72 h when wasps were starved. Once wasps were fed with any of the sugars, the number of mature eggs increased at first and then decreased due to oosorption. The longevity and oogenesis dynamics of female wasps fed on five sugars were related with their function of hydrolysis and digestion. As female wasps have no lipogenesis capability, by acquiring exogenous sugars for oogenesis, they can either maintain or exceed the original level of capital nutrients held on adult emergence because none of the wasps' glycogen need be metabolized to burn as sugar.