RESUMO
PURPOSE: To investigate the effect of microRNA-543 (miR-543) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of triple-negative breast cancer (TNBC) cells, and the associated mechanism. METHODS: Human breast cancer cells (MDA-MB-231, HCC1937, and MCF-7, ZR-75-1) and normal human breast epithelial cell line (MCF10A) were transfected with miR-543 mimics or inhibitor using lipofectamine 2000. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the mRNA and protein expression levels of miR-543, actin-like protein 6A (ACTL6A), vimentin, Snail, and E-cadherin in breast cancer cells/tissue. Cell counting kit-8 (CCK-8), wound-healing, and Transwell assays were used to measure the effect of miR-543 on TNBC cell proliferation, invasion, and migration. Overall survival was determined using data from Gene Expression Omnibus (GEO) and Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis and luciferase reporter gene assay were used to determine the regulatory effect of miR-543 on ACTL6A. RESULTS: The level of expression of miR-543 was significantly lower in breast cancer cells/tissue than in normal human breast epithelial cell/tissue (p < 0.05). MicroRNA-543 expression level was significantly reduced in TNBC cells/tissue, relative to the other breast cancer cells/normal breast tissue (p < 0.05). MicroRNA-543 significantly suppressed tumor growth and the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells, in mouse xenograft model (p < 0.05). CONCLUSIONS: miR-543 influences the biological behavior of TNBC cells by directly targeting ACTL6A gene. miR-543 could serve as a novel diagnostic and therapeutic target for TNBC.
Assuntos
Actinas/fisiologia , Movimento Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Transição Epitelial-Mesenquimal , MicroRNAs/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Humanos , Camundongos , Invasividade Neoplásica , Células Tumorais CultivadasRESUMO
Hurood cheese (HC) and Jueke (Jk) are 2 traditional fermented dairy products produced from raw milk (RM) in the Inner Mongolia region of China. They have a long history of production and consumption. The microbial compositions of RM, HC, and Jk vary greatly, and are influenced by their geographical origins and unique processing methods. In this study, 2 batches of RM, HC, and Jk samples were collected (April and August 2015) from the Zhenglan Banner, a region located in the southern part of Inner Mongolian belonging to the Xilingol league prefecture. The bacterial and fungal diversities of the samples were determined by 16S rRNA and 18S rRNA gene sequence analysis, respectively. A total of 112 bacterial and 30 fungal sequences were identified, with Firmicutes and Ascomycota being the predominant phyla for bacteria and fungi, respectively. Lactococcus and Lactobacillus were identified as the main bacterial genera, whereas Kluyveromyces was the predominant fungus identified in the 3 dairy products. Different bacterial and fungal compositions were observed in RM, HC, and Jk samples collected at different times. These results suggested that time of production may be an important factor influencing the microbial diversity present in RM, HC, and Jk.
Assuntos
Produtos Fermentados do Leite/microbiologia , DNA Bacteriano/genética , Leite/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , Queijo/microbiologia , China , Microbiologia de Alimentos , Fungos/genética , Fungos/isolamento & purificação , Leite/química , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
In this study, expression levels of miRNAs (miRNAs), miR-375 and miR-7, were detected in different tissues of cattle to determine whether adenohypophysis-prefer or exclusively expressed miRNAs, and target genes could be predicted by TargetScan, RNA22, and other software. Target genes related to pituitary function or reproductive traits were identified using a dual-luciferase assay. miR-375 and miR-7 were expressed differently in various tissues. miR-375 and miR-7 showed higher expression in the adenohypophysis, and there was a significant difference compared with expression in other tissues (P < 0.01). The binding sites for miR-7 were the mRNAs of bone morphogenetic protein receptor type II (BMPR2), prostaglandin F2 receptor negative regulator, gonadotropin-releasing hormone receptor, follicle-stimulating hormoneß, somatostatin receptor 1, and interleukin-1ß by bioinformatic analysis; similarly, the mRNAs of BMPR2 and leptin contained binding sites for miR-375, suggesting that these genes are affected by miR-7 or miR-375. Dual-luciferase reporter assays showed that miR-7 regulated prostaglandin F2 receptor negative regulator expression, while miR-375 regulated BMPR2 expression. The mutated plasmid and miRNA mimics were used to co-transfect NIH3T3 cells; luciferase reporter assays showed that the inhibition of luciferase activity in the wild-type cells dramatically decreased from 75 to 26% with a 3-5-nucleotide mismatch mutation into the seed region of miR-7. miR-375 had nearly lost the ability to inhibit luciferase activity, suggesting that GTCTTCC is the site of interaction between miR-7 and the prostaglandin F2 receptor negative regulator sequence and that GAACAAA is the site of interaction between miR-375 and the BMPR2 sequence.
Assuntos
MicroRNAs/genética , Adeno-Hipófise/metabolismo , RNA Mensageiro/genética , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , MicroRNAs/química , Conformação de Ácido Nucleico , Especificidade de Órgãos/genética , Interferência de RNA , RNA Mensageiro/químicaRESUMO
The results of previous case-control studies examining the relationship between the interleukin (IL)-6 gene -174G>C polymorphism and lung cancer are controversial. In this study, we evaluated the relationship between the IL-6 gene -174G>C polymorphism and lung cancer. We selected 5 case-control studies related to the IL-6 gene -174G>C polymorphism and lung cancer by searching the PubMed, EMBase, Chinese Biomedical Literature Database, and Wanfang database. We utilized the Q-test and I2 test to determine heterogeneity between each study. To merge the odds ratios (OR) and 95% confidence intervals (CI), we utilized the fixed effects model and random effect model for analyses. The present study included 2801 patients with lung cancer and 3234 cancer-free control subjects. The meta-analysis revealed no association between the IL-6 gene -174G>C polymorphism and lung cancer in either genotype or allele distribution [CC+GC vs GG: OR = 1.04, 95%CI (0.86-1.26), P = 0.70; GG+GC vs CC: OR = 0.93, 95%CI (0.82-1.05), P = 0. 23; CC vs GG: OR = 1.08, 95%CI (0.95-1.23), P = 0.23; C allele vs D allele: OR = 1.03, 95%CI (0.96-1.11), P = 0.44]. We concluded that the IL-6 gene -174G>C polymorphism was not associated with lung cancer.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Interleucina-6/genética , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único/genética , Humanos , Viés de Publicação , Fatores de RiscoRESUMO
Twelve microsatellite loci were developed from Haliotis ovina by the magnetic bead hybridization method. Genetic variability was assessed using 30 individuals from 3 wild populations. The number of alleles per locus ranged from 2 to 5, and the polymorphism information content ranged from 0.1228 to 0.6542. Observed and expected heterozygosities ranged from 0.0000 to 0.7778 and 0.1288 to 0.6310, respectively. These loci should provide useful information for genetic studies such as genetic diversity, pedigree analysis, construction of genetic linkage maps, and marker-assisted selection breeding in H. ovina.
Assuntos
DNA/genética , Gastrópodes/genética , Loci Gênicos , Repetições de Microssatélites/genética , Polimorfismo Genético , Animais , Marcadores GenéticosRESUMO
Hailey-Hailey disease (HHD) is an autosomal dominant disorder in which the ATP2C1 gene has been implicated. Many mutations of this gene have been detected in HHD patients. To analyze such mutations in HHD and summarize all those identified in Chinese patients with this disease, we examined four familial and two sporadic cases and searched for case reports and papers by using the Chinese Biological Medicine Database and PubMed. HHD diagnoses were made based on clinical features and histopathological findings. Polymerase chain reaction and direct sequencing of the ATP2C1 gene were performed using blood samples from HHD patients, unaffected family members, and 120 healthy individuals. Three mutations were identified, including the recurrent mutation c.2126C>T (p.Thr709Met), and two novel missense mutations, c.2235_2236insC (p.Pro745fs*756) and c.689G>A (p.Gly230Asp). Considering our data, 81 different mutations have now been reported in Chinese patients with HHD. In cases of misannotation or duplication, previously published mutations were renamed according to a complementary DNA reference sequence. These mutations are scattered throughout the ATP2C1 gene, with no evident hotspots or clustering. It is of note that some reported "novel" mutations were in fact found to be recurrent. Our findings expand the range of known ATP2C1 sequence variants in this disease.
Assuntos
ATPases Transportadoras de Cálcio/genética , Predisposição Genética para Doença , Mutação , Pênfigo Familiar Benigno/genética , Adulto , Povo Asiático , Sequência de Bases , Estudos de Casos e Controles , Criança , Feminino , Expressão Gênica , Genes Dominantes , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Pênfigo Familiar Benigno/diagnóstico , Pênfigo Familiar Benigno/etnologia , Pênfigo Familiar Benigno/patologia , Análise de Sequência de DNA , Terminologia como AssuntoRESUMO
Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.
Assuntos
Animais , Feminino , Humanos , Masculino , Camundongos , Expressão Gênica/fisiologia , Fragmentos de Imunoglobulinas/biossíntese , Molécula 1 de Adesão Intercelular/imunologia , Redobramento de Proteína , Renaturação Proteica , Anticorpos de Cadeia Única/biossíntese , Complexo Antígeno-Anticorpo , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/biossíntese , Adesão Celular , Cromatografia , Diálise , Ensaio de Imunoadsorção Enzimática , Pavilhão Auricular/efeitos dos fármacos , Escherichia coli/genética , Vetores Genéticos , Fragmentos de Imunoglobulinas/farmacologia , Corpos de Inclusão/metabolismo , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Plasmídeos , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/farmacologia , Xilenos/farmacologiaRESUMO
Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19 × 10(-8) M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35 × 10(-7) M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.
Assuntos
Expressão Gênica/fisiologia , Fragmentos de Imunoglobulinas/biossíntese , Molécula 1 de Adesão Intercelular/imunologia , Redobramento de Proteína , Renaturação Proteica , Anticorpos de Cadeia Única/biossíntese , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/biossíntese , Complexo Antígeno-Anticorpo , Adesão Celular , Cromatografia , Diálise , Pavilhão Auricular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Vetores Genéticos , Humanos , Fragmentos de Imunoglobulinas/farmacologia , Corpos de Inclusão/metabolismo , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Plasmídeos , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/farmacologia , Xilenos/farmacologiaRESUMO
This study aimed to provide additional anatomical information for axillary lymph node dissection (ALND) through in vivo anatomy studies of intercostobrachial nerve (ICBN) preservation in order to provide theoretical and practical experience for clinicians. A total of 156 patients with breast cancer underwent ALND at the Department of Gynecology of Baotou Tumor Hospital between June 2009 and March 2010. The origin, destination, main source, length, branch type, and direction of ICBN in axilla were observed, as well as its relationship with adjacent major blood vessels and nerves within the axilla. There were 120 cases of single trunk, 23 cases of double trunks, 9 cases of multiple trunks, and 4 cases without trunks in 156 patients with ICBN preservation. The transverse diameter at the origin of the ICBN was 1.89 ± 0.44 mm with a length of 94.45 ± 12.08 mm; the distances were 77.19 ± 21.04 mm, 29.34 ± 6.73 mm, 90.04 ± 13.13 mm, and 28.63 ± 13.01 mm from origin to the inferior margin at the midpoint of the clavicle, inferior margin of the axillary vein, the bottom of axilla, and branch point, respectively. The identification, dissection, and preservation of ICBN was simple and easy in a modified radical mastectomy for breast cancer and breast-conserving surgery, which only took 10-20 min, but effectively reduced the incidence of post-mastectomy pain syndrome and significantly improved the quality of life for patients after surgery.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Nervos Intercostais/patologia , Excisão de Linfonodo , Tratamentos com Preservação do Órgão , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Nervos Intercostais/cirurgia , Pessoa de Meia-IdadeRESUMO
We investigated 2 Chinese families with dyschromatosis symmetrica hereditaria (DSH) and search for mutations in the adenosine deaminase acting on RNA1 (ADAR1) gene in these 2 pedigrees. We performed a mutation analysis of the ADAR1 gene in 2 Chinese families with DSH and reviewed all articles published regarding ADAR1 mutations reported since 2003 by using PubMed. By direct sequencing, a 2-nucleotide AG deletion, 2099-2100delAG, was found in family 1, and a CâT mutation was identified at nucleotide 1420 that changed codon 474 from arginine to a translational termination codon in family 2. Two different pathogenic mutations were identified, c.2099-2100delAG and c.1420C>T, the former being a novel mutation, and the latter previously reported in 3 other families with DSH. To date, a total of 110 mutations in the ADAR1 gene have been reported, and 10 of them were recurrent; the mutations R474X, R1083C, R1096X, and R1155W might be the DSH-related hotspots.
Assuntos
Adenosina Desaminase/genética , Mutação , Transtornos da Pigmentação/congênito , Adolescente , Adulto , Criança , China , Feminino , Genes Dominantes , Humanos , Masculino , Linhagem , Transtornos da Pigmentação/diagnóstico , Transtornos da Pigmentação/genética , Proteínas de Ligação a RNARESUMO
The luteinizing hormone receptor (LHR) plays a key role in testosterone production through its interaction with the gonadotropins, LH and chorionic gonadotropin. We examined the LHR splicing pattern in bovine Leydig cells; LH-induced expression of eight cloned splicing variants was detected by real-time PCR. Luteinizing hormone applied to cultured Leydig cells resulted in expression of full-length LHR and the A and B isoforms, as well as secretion of testosterone, which first increased, then declined, and then increased further, with increased LH levels. The secretion of testosterone progressively increased with increasing LH, but the expression levels of LHR (FL, A, and B) did not increase correspondingly. We conclude that the LHR splicing pattern is complex in bovine Leydig cells, and that expression of full-length LHR and isoforms A and B changes when induced with LH.
Assuntos
Células Intersticiais do Testículo/metabolismo , Receptores do LH/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Análise de Variância , Animais , Bovinos , Células Cultivadas , Éxons , Expressão Gênica , Hormônio Luteinizante/fisiologia , Masculino , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores do LH/genética , Testosterona/metabolismoRESUMO
Follicle-stimulating hormone (FSH) plays an essential role in mammalian spermatogenesis and follicular development. In a previous study, we demonstrated that some bulls carry numerous linked mutations in the FSH beta-subunit (FSHB) gene, and that these bulls have poor-quality semen, low fertility, and slightly lower serum FSH concentration compared to those without such mutations. Here, we identified the different FSHB mRNA transcripts in such individuals and analyzed the evolutionary pattern of the FSHB open reading frame (ORF) in different species. Two different lengths of FSHB mRNA transcripts corresponding to two different polyadenylation sites in the 3'-UTR were detected in wild-type bull pituitary glands, and four different mRNA transcripts resulting from the different polyadenylation sites and linked mutations were identified in mutation-bearing bull pituitaries. All transcripts had almost the same putative FSHB precursor molecule. When the ORF sequences of wild-type and mutation-bearing genes were compared with those of other tetrapod species, the leopard frog had the lowest level of homology (57.8 and 58.1%) and the buffalo had the highest level (95.9 and 96.7%), respectively. These results indicated that the bovine FSHB gene transcribes at least two classes of mRNA in the wild-type and four classes of mRNA in the mutation-bearing individuals, which provides a new insight into the bovine FSHB evolutionary pattern. In addition, these findings lay a foundation for further study of gene expression regulation and the effects of mutations on male fertility traits in cattle.