RESUMO
PURPOSE: To investigate the potential candidate microRNA (miRNA) biomarkers for the clinical diagnosis, classification, and prognosis of gastric cancer (GC). METHODS: We use bioinformatics overlapping subclasses analysis to find the tumor grade and lymphatic metastasis-related GC specific miRNAs from the Cancer Genome Atlas (TCGA) database. Then, we further investigated these GC specific miRNAs distributions in different GC clinical features and their correlations overall survival on the basis of GC patients' information and their related RNA sequencing profile from TCGA. Finally, we randomly selected some of key miRNAs use qRT-PCR to confirm the reliability and validity. RESULTS: 22 GC specific key miRNAs were identified (Fold-change >2, P < 0.05), 11 of them were discriminatively expressed with tumor size, grade, TNM stage and lymphatic metastasis (P < 0.05). In addition, nine miRNAs (miR-196b-5p, miR-135b-5p, miR-183-5p, miR-182-5p, miR-133a-3p, miR-486-5p, miR-144-5p, miR-129-5p and miR-145-5p) were found to be significantly associated with overall survival (log-rank P < 0.05). Finally, four key miRNAs (miR-183-5p, miR-486-5p, miR-30c-2-3p and miR-133a-3p) were randomly selected to validation and their expression levels in 53 newly diagnosed GC patients by qRT-PCR. Results showed that the fold-changes between TCGA and qRT-PCR were 100 % in agreement. We also found miR-183-5p and miR-486-5p were significantly correlated with tumor TNM stage (P < 0.05), and miR-30c-2-3p and miR-133a-3p were associated with tumor differentiation degree and lymph-node metastasis (P < 0.05). These verified miRNAs clinically relevant, and the bioinformatics analysis results were almost the same. CONCLUSION: These key miRNAs may functions as potential candidate biomarkers for the clinical diagnosis, classification and prognosis for GC.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/secundário , Biomarcadores Tumorais/genética , MicroRNAs/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Idoso , Estudos de Casos e Controles , Biologia Computacional , Progressão da Doença , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Taxa de SobrevidaRESUMO
Fusarium wilt (also known as Panama disease) is one of the most destructive banana diseases, and greatly hampers the global production of bananas. Consequently, it has been very detrimental to the Chinese banana industry. An infected plant is one of the major causes of the spread of Fusarium wilt to nearby regions. It is essential to develop an efficient and environmentally sustainable disease control method to restrict the spread of Fusarium wilt. We isolated Trichoderma spp from the rhizosphere soil, roots, and pseudostems of banana plants that showed Fusarium wilt symptoms in the infected areas. Their cellulase activities were measured by endoglucanase activity, ß-glucosidase activity, and filter paper activity assays. Safety analyses of the Trichoderma isolates were conducted by inoculating them into banana plantlets. The antagonistic effects of the Trichoderma spp on the Fusarium pathogen Foc tropical Race 4 (Foc TR4) were tested by the dual culture technique. Four isolates that had high cellulase activity, no observable pathogenicity to banana plants, and high antagonistic capability were identified. The isolates were used to biodegrade diseased banana plants infected with GFP-tagged Foc TR4, and the compost was tested for biological control of the infectious agent; the results showed that the fermentation suppressed the incidence of wilt and killed the pathogen. This study indicates that Trichoderma isolates have the potential to eliminate the transmission of Foc TR4, and may be developed into an environmentally sustainable treatment for controlling Fusarium wilt in banana plants.
Assuntos
Fermentação , Fusarium/fisiologia , Musa/microbiologia , Doenças das Plantas/microbiologia , Trichoderma/fisiologia , Bioensaio , Proteínas de Fluorescência Verde/metabolismo , Filogenia , Folhas de Planta/microbiologia , Caules de Planta/microbiologia , Trichoderma/isolamento & purificaçãoRESUMO
SNX-2112 is a potential molecular targeted therapeutic drug against esophageal cancer (EC). However, its exact mechanism of action remains to be explained. The aim of this study was to investigate the effect of SNX-2112 on excision repair cross- complementing 1 (ERCC1), epidermal growth factor receptor (EGFR), and p53, to elucidate the mechanism of action of SNX-2112 on EC. Fresh tumor sections were surgically obtained from 65 patients with EC, and the expression of ERCC1, EGFR, and p53 was determined by immunohistochemical staining. Furthermore, the effect of SNX-2112 (0.2 µM) on the proliferation of EC-9706 cells and the expression of ERCC1, EGFR, and p53 in these cells were analyzed by a cell proliferation assay and western blot, respectively. We observed a significant decrease and increase in ERCC1 (P = 0.001) and p53 (P = 0.043) expression, respectively, and no significant difference in EGFR (P = 0.59) expression, with the TNM stage of EC, which suggested that ERCC1 and p53 could be potential markers for the TNM stage of EC. We also observed a significant increase in ERCC1 expression, and decrease in p53 and EGFR expression, in EC-9706 cells treated with SNX-2112 (P < 0.05), indicating the regulation of EC by SNX-2112. Furthermore, SNX-2112 treatment induced a significant decrease in the proliferation of EC-9706, which confirmed the function of SNX- 2112. In summary, SNX-2112 inhibits the proliferation of EC cells by regulating the expression of ERCC1, EGFR, and p53.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
5-Fluorouracil (5-FU) is widely used in chemotherapy for treatment of colorectal cancer. Leucine-rich repeat-containing G protein-coupled receptor (LGR) is known to participate in the occurrence and development of breast cancer by regulating the rebirth of tumor vessels. This study aimed to explore the proliferation and apoptosis of HCT116 colorectal cancer cells treated with 5-FU and related molecular mechanisms. 5-FU (20 µg/mL) was used to treat cultured HCT116 cells. An MTT test, flow cytometry, and colony formation assays were used to examine the proliferation and apoptosis of HCT116 cells. Western blotting was applied to detect the expression of the LGR4 protein in HCT116 cells. Small interference RNA or over-expression techniques were used to manipulate LGR4 expression in HCT116 cells and describe the proliferation and apoptosis of HCT116 treated with 5-FU. A dosage of 20 µg/mL 5-FU resulted in a significant decrease in the proliferation and apoptosis of HCT116 cells and significantly decreased expression levels of LGR4. The specific gene silence or over-expression of LGR4 in HCT116 cells increased and decreased the levels of apoptosis in HCT116, respectively. 5-FU induces apoptosis of colorectal cancer cells and inhibits proliferation by suppressing LGR4 proteins.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Fluoruracila/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Apoptose , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
This study investigated CapG gene expression in prostate cancer cell lines; in addition, we explored the effects of CapG suppression on DU145 cell growth, and the underlying mechanism with which CapG affects DU145 cell growth and invasiveness. The expression of CapG and 18 related genes in DU145 cells was analyzed by flow cytometry, quantitative polymerase chain reaction (qPCR), CCK8 assay, western blot, and the trans-well assay. DU145 cells were transfected with designed small interfering RNA (siRNA). CapG expression was quantified by qPCR and western blot. DU145 cell proliferation and invasiveness was analyzed using the CCK8, flow cytometric, and trans-well assays. CapG, TMPRSS1, EGFR, ETS-1, ERBB2, AKT, Cyclin D1, P21, Bcl-2, and Bak1 gene and Bcl-2, Cyclin D1, and CapG protein expressions were significantly lower in the siRNA group compared to the negative control group (P < 0.05). The proliferation of CapG siRNA DU145 cells was lower than that of the two control groups, 48 h after transfection. The cell inhibition rate was 24.5, 35.4, and 16,5% at 24, 48, and 72 h, respectively. The growth curve indicated that CapG siRNA DU145 cells showed a significantly slower proliferation rate (P < 0.05). The trans-well assay showed a significant decrease in the migratory and invasive capacities of DU145 cells in the siRNA group (P < 0.05). The suppression of CapG expression caused a significant decrease in the proliferation, invasiveness, and metastasis of DU145 cells. The mechanism with which CapG, with other oncogenes, influences cancer cell cycle remains to be elucidated.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
This study aims to explore the possible associations between a genetic variation in the miR-34b binding site in the 3'-untranslated region (UTR) of the methylenetetrahydrofolate reductase (MTHFR) gene (rs55763075) with male infertility in a Chinese population. Genotype distributions of the rs55763075 single nucleotide polymorphism were investigated by polymerase chain reaction and direct sequencing in a Chinese cohort that included 464 infertile men with idiopathic azoospermia or oligospermia and 458 controls with normal fertility. Overall, no significant differences in the distributions of the genotypes of the MTHFR rs55763075 polymorphism were detected between the infertility and control groups. A statistically significant increased risk of male infertility was found for carriers of the rs55763075 AA genotype when compared with homozygous carriers of the rs55763075 GG genotype in the azoospermia subgroup (OR = 1.721; 95% CI = 1.055-2.807; P = 0.031). Furthermore, we found that rs55763075 was associated with folate and homocysteine levels in patients with idiopathic azoospermia. Our results indicated that the MTHFR 3'-UTR rs55763075 polymorphism might modify the susceptibility to male infertility with idiopathic azoospermia.
Assuntos
Regiões 3' não Traduzidas/genética , Infertilidade Masculina/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Ácido Fólico/sangue , Genótipo , Homocisteína/sangue , Humanos , Infertilidade Masculina/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.
Assuntos
Proteínas Fúngicas/genética , Proteínas Luminescentes/genética , Magnaporthe/genética , Proteínas Recombinantes de Fusão/genética , Fusão Gênica Artificial/métodos , Clonagem Molecular/métodos , DNA Complementar/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Amplificação de Genes , Expressão Gênica , Genes Fúngicos , Vetores Genéticos/química , Vetores Genéticos/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/química , Oryza/microbiologia , Plasmídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteína Vermelha FluorescenteRESUMO
The aim of this study was to construct overexpression vectors and selecting strains of the Magnaporthe oryzae effectors BAS1 and BAS4. Primer pairs of BAS1, BAS4, and mCherry were designed based on their known nucleotide sequences. The coding sequences of BAS1 and BAS4 were amplified, and the pXY201 plasmid was selected as a template to amplify the mCherry sequence. Fragments of BAS1 and mCherry, and BAS4 and mCherry were ligated into the pCAMBIA1302 vector. The recombinant pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into E. coli DH5α competent cells. Transformants were screened by PCR, and plasmids from the positive transformants were extracted by enzymatic digestion to obtain pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry. The pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into protoplasts of rice blast strains and the transformed strains were screened by PCR using primer pairs against the hygromycin gene. The result showed that the PCR products corresponded with the theoretical sizes. RT-PCR was used to analyze the expression of BAS1 and BAS4 in five transformed strains of BAS1 and BAS4, and the result showed that the higher expression level of the two genes was occurred in five transformant strains comparing to wild-type strain A3467-40 (the strain containing BAS1 and BAS4), but there was no difference among the five overexpression strains. The sporulation and spore germination of transformed strains was higher than in wild type strain, and there was no difference in the germination time. Construction of overexpression vectors and strains of M. oryzae effectors BAS1 and BAS4 provide reference material for other new effectors.
Assuntos
Proteínas Fúngicas/genética , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Magnaporthe/genética , Plasmídeos/metabolismo , Transativadores/genética , Clonagem Molecular , Primers do DNA/síntese química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Proteínas Luminescentes/metabolismo , Magnaporthe/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Plasmídeos/química , Engenharia de Proteínas , Protoplastos/microbiologia , Protoplastos/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismo , Transformação Bacteriana , Proteína Vermelha FluorescenteRESUMO
Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population.
Assuntos
DNA Fúngico/isolamento & purificação , Fungos/classificação , Fungos/genética , Parasitos/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Animais , FenótipoRESUMO
We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.