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1.
Anticancer Res ; 39(10): 5361-5367, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570430

RESUMO

BACKGROUND/AIM: The mechanism responsible for B-cell translocation gene 1 (BTG1) down-regulation in breast carcinoma remains unknown. We examined the BTG1 expression status in breast carcinoma cells and investigated the mechanism underlying the observed alterations. MATERIALS AND METHODS: Four breast carcinoma cell lines (SK-BR-3, MDA-MB-231, T-47D, and MCF-7), and one normal mammary epithelial cell line (MCF-10A) were analyzed. BTG1 expression was examined using quantitative reverse transcription polymerase chain reaction (PCR) and western blot. Methylation status of the BTG1 promoter was analyzed using methylation-specific PCR (MSP). To investigate the effect of methylation on BTG1, the cells were treated with a demethylating agent. RESULTS: The carcinoma cells expressed significantly lower levels of BTG1 mRNA and protein than normal cells. The BTG1 promoter was highly methylated in the carcinoma cells. 5-aza-2-deoxycytidine significantly restored BTG1 expression. CONCLUSION: Down-regulation of BTG1 expression through epigenetic repression is involved in mammary carcinogenesis. BTG1 is a potential diagnostic marker and therapeutic target for breast carcinoma.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA/genética , Regulação para Baixo/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , RNA Mensageiro/genética
2.
Anticancer Res ; 39(10): 5715-5720, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570472

RESUMO

BACKGROUND/AIM: The PRKCI gene encodes Protein kinase C iota. The overexpression of protein kinase C iota is associated with poor outcomes in patients with gastric and other cancers, but the role of the PRKCI gene in gastric cancer is not fully understood. Thus, we evaluated the clinical significance of PRKCI gene expression in gastric cancer. MATERIALS AND METHODS: PRKCI mRNA expression levels in cancerous tissues and adjacent normal mucosa from 398 patients with gastric cancer were measured. Relationships between PRKCI gene expression and clinicopathological characteristics and outcomes were examined. RESULTS: Overall survival was lower in patients with a high expression of PRKCI than in those with low expression (p=0.016). No other relationships were observed. A high PRKCI expression was found to be an independent prognostic factor (p=0.036, HR=1.44, 95%CI=1.02-2.02). CONCLUSION: PRKCI gene expression in cancerous tissue might be a useful prognostic factor in patients with gastric cancer after gastrectomy.


Assuntos
Expressão Gênica/genética , Isoenzimas/genética , Proteína Quinase C/genética , Neoplasias Gástricas/genética , Gastrectomia/métodos , Mucosa Gástrica/patologia , Humanos , Prognóstico , RNA Mensageiro/genética , Neoplasias Gástricas/patologia
3.
Zhonghua Yi Xue Za Zhi ; 99(35): 2761-2767, 2019 Sep 17.
Artigo em Chinês | MEDLINE | ID: mdl-31550799

RESUMO

Objective: To investigate the mechanisms of lncRNA on the occurrence and development of NOA by constructing ceRNA regulation network of lncRNA, miRNA and mRNA. Methods: Samples of adult human testis were obtained from NOA patients and OA patients with normal spermatogenesis (controls), recruited from the Reproductive Medicine Center of Nanfang Hospital from June 2017 to June 2018. Differentially expressed lncRNAs and mRNAs in testicular tissues from patients with NOA were identified by microarray analysis in previous association study. In this study, differentially expressed lncRNAs and mRNA were used to construct the ceRNA regulatory network in NOA and clarify the interaction relationship among lncRNA, miRNA and mRNA. GeneMANIA database was used to construct Protein-Protein Interaction (PPI) of the mRNAs in ceRNA regulatory network. WebGestalt toolkit was employed to perform gene function and pathway enrichment analyses of those coding genes. Finally, qRT-PCR and dual luciferase reporter system were employed for further experimental validation. Results: The ceRNA regulatory network of lncRNA, miRNA and mRNA consists of 21 nodes and 26 edges, of which 4 lncRNAs, 13 miRNAs and 4 mRNAs. 19 proteins were found to interact with the mRNA coding proteins in ceRNA regulatory network by PPI analysis. Gene oncology and KEGG pathway enrichment analyses indicate these coding genes were significantly enriched in pentose metabolic process and pentose phosphate pathway. Furthermore, lncRNA ANXA2P3 was found binding with miR-613 and miR-206 to inhibit mRNA TKT expression. Conclusion: lncRNAs exert an important role in the occurrence and development of NOA via ceRNA regulatory network, which could be used as new biomarkers for NOA treatment.


Assuntos
Azoospermia/genética , Redes Reguladoras de Genes , RNA Longo não Codificante/genética , Humanos , Masculino , MicroRNAs/genética , RNA Mensageiro/genética
4.
Acta Virol ; 63(3): 286-291, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507194

RESUMO

Schmallenberg virus (SBV), a neurotropic member of the genus Orthobunyavirus, infects ruminants and causes neurological lesions and fetal malformations including cerebellar hypoplasia, hydranencephaly, and porencephaly. The aim of this study is to establish intracerebral (i.c.) infection of SBV in newborn BALB/c mice and to investigate some of the transcription factors in brain. For this aim, brain samples of newborn BALB/c mice which were infected with SBV i.c. were analyzed by plaque titration and real-time RT-PCR for T-bet, Gata3, RoRγt, Foxp3 and Eomes mRNA levels. Study results showed that SBV can replicate in BALB/c mice brain and cause death of newborn mice with generation of infectious viral particles. Analyses of transcription factor mRNA levels indicated up-regulation of T-bet, Gata3, RoRγt, Foxp3 and down-regulation of Eomes. In this report, we introduce preliminary data of T cell transcription factors affected by SBV infection of BALB/c mice. Keywords: Eomes; Foxp3; Gata3; RoRγt; Schmallenberg virus; T-bet.


Assuntos
Encéfalo , Infecções por Bunyaviridae , Regulação da Expressão Gênica , Orthobunyavirus , Fatores de Transcrição , Animais , Animais Recém-Nascidos , Encéfalo/virologia , Infecções por Bunyaviridae/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ruminantes , Fatores de Transcrição/genética , Replicação Viral
5.
Anticancer Res ; 39(9): 4637-4642, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519561

RESUMO

AIM: The aim of this study was to characterize the role of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) in human lung cancer cell lines. MATERIALS AND METHODS: AMMECR1 gene expression was evaluated in four lung cell lines, with A549 then selected for further in-depth examination. To characterize the role of AMMECR1, silencing was achieved utilizing lentivirus-mediated RNA interference, and confirmed by quantitative real-time polymerase chain reaction and western blotting. The impact of AMMECR1 silencing on cellular proliferation was assessed using Celigo-based and MTT assays. Apoptosis was determined using the annexin V-allophycocyanin single staining method. Cell-cycle arrest was assessed by flow cytometry. Finally, colony formation was assessed using Giemsa staining. RESULTS: In A549 cells, AMMECR1 silencing was found to significantly suppress cell proliferation, reduce colony formation, promote apoptosis, and arrest cells in the S and G2/M phases. CONCLUSION: AMMECR1 plays a critical role in cell proliferation, cell-cycle progression, and apoptosis of human lung cancer cells, and may serve as a potential therapeutic target for non-small-cell lung cancer.


Assuntos
Apoptose/genética , Ciclo Celular/genética , Neoplasias Pulmonares/genética , Proteínas/genética , Células A549 , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas/metabolismo , RNA Mensageiro/genética
6.
Anticancer Res ; 39(9): 4729-4736, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519572

RESUMO

BACKGROUND/AIM: Amphiregulin (AREG) and epiregulin (EREG) mRNA expression levels are predictors of response to anti-EGFR antibody therapy. Left-sided colon cancer is more sensitive to anti-EGFR antibodies than right-sided, although the mechanism is unclear. The aim of this study was to determine the relationship between AREG, EREG mRNA expression levels and tumor location as well as the efficacy of anti-EGFR antibody agents. MATERIALS AND METHODS: Real-time PCR was used to assess AREG and EREG mRNA expression in metastatic colorectal cancer (CRC) samples from 153 patients. RESULTS: Among KRASwt samples, high AREG expression (AREGHigh) was significantly more common in left-sided tumors than in right-sided. Among patients who received anti-EGFR antibody, response rates were significantly higher in AREGHigh than in AREGLow In the left-sided tumor group, overall survival was significantly longer in patients with high EREG levels than with low levels, whereas the right-sided tumor group showed no survival difference between them. CONCLUSION: AREG and EREG mRNA expression levels in left-sided CRC were higher than in right-sided tumors. This may help explain why left-sided CRC is more responsive to anti-EGFR antibodies.


Assuntos
Anfirregulina/genética , Neoplasias Colorretais/genética , Epirregulina/genética , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Especificidade de Órgãos/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética
7.
Anticancer Res ; 39(9): 4749-4755, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519575

RESUMO

BACKGROUND: Cisplatin is a platinum compound capable of inducing apoptosis of cancer cells. However, cancer cells can become cisplatin-resistant. A recent study showed that a pregnane X receptor (PXR) antagonist, leflunomide, can enhance the antitumor activity of cisplatin and overcome such resistance. This study determined whether PXR antagonists ketoconazole and phenethyl isothiocyanate (PEITC) enhance the antitumor activity of platinum compounds and by which mechanism(s) of action. MATERIALS AND METHODS: Caspase-3 activity, intracellular platinum level, and expression of ATP-binding cassette subfamily C member 2 (ABCC2; previously named multidrug resistance-associated protein 2) were assessed in HepG2 human hepatocellular carcinoma cells exposed to carboplatin or cisplatin with and without PXR antagonist. RESULTS: In combination with platinum compounds, PEITC increased the intracellular platinum level, while ketoconazole induced higher caspase-3 activity. Additionally, PEITC suppressed ABCC2 protein expression. CONCLUSION: These results suggested that ketoconazole and PEITC enhance the antitumor activity of platinum compounds by different and complex mechanisms.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Receptor de Pregnano X/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Biomarcadores , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , RNA Mensageiro/genética
8.
Tumour Biol ; 41(8): 1010428319869101, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31423948

RESUMO

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Antígeno CD24/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Galinha , Membrana Corioalantoide/citologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/genética , Células Tumorais Cultivadas
9.
Anticancer Res ; 39(8): 4073-4077, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366490

RESUMO

BACKGROUND/AIM: ANRIL is a long noncoding RNA located on INK4 locus, which encodes p15 and p16 that cause G1 phase arrest in the cell cycle. ANRIL positively regulates proliferation of several kinds of cancer cells such as lung and gastric cancers. This study, examined the effect of ANRIL in head and neck squamous cell carcinoma cells. MATERIALS AND METHODS: Cells were transfected with siRNA oligonucleotides targeting ANRIL. Transfected cells were subjected to cell-cycle and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. RESULTS: Depletion of ANRIL increased p15 mRNA in FaDu cells, and p15 and p16 mRNA in CAL27 cells and inhibited proliferation of these cells. Cell cycle analysis showed that depletion of ANRIL caused arrest at the G1 phase of the cell cycle. CONCLUSION: ANRIL promotes G1 phase progression by repressing p15 and p16, and thus promotes FaDu and CAL27 cell proliferation.


Assuntos
Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , RNA Longo não Codificante/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
10.
Anticancer Res ; 39(8): 4129-4136, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366497

RESUMO

BACKGROUND/AIM: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP). MATERIALS AND METHODS: The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively. RESULTS: Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU. CONCLUSION: Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição Sp1/genética , Timidina Fosforilase/genética , Sítios de Ligação/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Decitabina/metabolismo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , Timidina Fosforilase/química
11.
Life Sci ; 234: 116747, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408661

RESUMO

AIMS: The present study was aimed to investigate the neuroprotective effect of HSP70 against neuroinflammation in a rotenone-induced Parkinson's disease model. MATERIALS AND METHODS: In the present study, SH-SY5Y cells were treated with HSP70 (5-20 mg/L) for 72 h. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), levels of oxidative markers, mitochondrial fragmentation, apoptosis, and mRNA and protein expressions of signal transducer and activator of transcription (STAT)-3 and nuclear factor-kappa B (NF-κB) were assessed. KEY FINDINGS: Cells treated with 5, 10, 15, and 20 mg/L of HSP70 exhibited increased, by 61.7%, 70.3%, 84.6%, and 96.7%, respectively, in cell viability. ROS and lipid peroxidation levels decreased following treatment with HSP70, and reductions in glutathione (GSH), catalase, glutathione peroxidase (Gpx), and superoxide dismutase (SOD) levels were reversed following treatment with HSP70. Additionally, MMP levels were reduced by 29.7, 46.4, 79.5, and 125.2 relative units following treatment with 5-20 mg/L of HSP70, respectively. HSP70 treatment also decreased levels of fragmented mitochondria and apoptosis, and mRNA and protein expressions of NF-κB and STAT3 were reduced by >25%. SIGNIFICANCE: Taken together, these findings indicate that supplementation with HSP70s recovered cell viability and MMP and reduced levels of ROS, apoptosis, and mitochondrial fragmentation. Additionally, supplementation with HSP70 significantly reduced the expressions of STAT3 and NF-κB.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/farmacologia , Inflamação/tratamento farmacológico , NF-kappa B/genética , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson Secundária/tratamento farmacológico , Fator de Transcrição STAT3/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Humanos , Inflamação/genética , Doença de Parkinson Secundária/genética , RNA Mensageiro/genética , Rotenona
12.
Mol Biol (Mosk) ; 53(4): 692-704, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397443

RESUMO

miRNAs regulate the expression of many genes and are involved in the development of diseases. We studied miRNAs that interact partly or fully complementarily with the 5'UTR, CDS and 3'UTR of mRNAs of target genes. The MirTarget program used in this study allows for the discovery of miRNA binding sites (BS) in the entire nucleotide sequence of the mRNA and for determining the characteristics of the interactions of miRNAs with mRNAs. We identified five pairs of fully complementary BS for miR-127-5p and miR-127-3p, miR-136-5p and miR-136-3p, miR-431-5p and miR-431-3p, miR-432-5p and miR-432-3p, and miR-433-5p and miR-433-3p in the CDS of the human and animal mRNA of RTL1 gene. The fully complementary BS for miR-6720-5p, miR-6720-3p were identified in the CDS of the FOXF2 gene; BS for miR-3187-5p, miR-3187-3p were found in the CDS of the PLPPR3 gene; BS for miR-4665-5p, miR-4665-3p were found in the 5'UTR of the KIAA2026 gene; BS for miR-135a-5p, miR-135a-3p were found in the 3'UTR of the GLYCTK gene; BS for miR-7106-5p, miR-7106-3p were found in the 3'UTR of the CCDC42B gene. The miRNA-5p and miRNA-3p associated with the RTL1 gene have BS in the mRNAs of 32 target human genes. The miRNA-5p and miRNA-3p associated with the FOXF2, PLPPR3, KIAA2026, GLYCTK and CCDC42B genes have BS in the mRNAs of 27 target genes, involved in development of several diseases. Nucleotide sequences of miRNA-5p and miRNA-3p and BS are conserved over tens of millions of years of divergence of the studied animal species. Binding characteristics of miR-3120-3p and miR-3120-5p, miR-196b-3p and miR-196b-5p, miR-125a-3p and miR-125a-3p, let-7e-3p and let-7e-5p, miR-99b-3p in fully complementary BS of non-coding DMN3OS, HOXA10-AS, SPACA6P-AS genes have been established.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação/genética , Humanos
13.
Gene ; 720: 144056, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31437466

RESUMO

Repeated implantation failure (RIF) was mainly due to poor endometrium receptivity. Long noncoding RNAs (lncRNAs) could regulate endometrium receptivity and act in competing endogenous RNA (ceRNA) theory. However, the regulatory mechanism of the lncRNA-miRNA-mRNA network in repeated implantation failure (RIF) is unclear. We obtained RIF-related expression profiles of lncRNAs, mRNAs, and miRNAs using mid-secretory endometrial tissue samples from 5 women with RIF and 5 controls by RNA-sequencing. Co-expression analysis revealed that three functional modules were enriched in immune response/inflammation process; two functional modules were enriched in metabolic/ biosynthetic process, and one functional module were enriched in cell cycle pathway. By adding the miRNA data, ceRNA regulatory relationship of each module was reconstructed. The ceRNA network of the whole differentially expressed RNAs revealed 10 hub lncRNAs. Among them, TRG-AS1, SIMM25, and NEAT1 were involved in the module1, module2, and module3, respectively; LNC00511 and SLC26A4-AS1 in the module4; H19 in the module5. The real-time polymerase chain reaction (RT-PCR) results of 15 randomly selected RNAs were consistent with our sequencing data. These can be used as novel potential biomarkers for RIF. Furthermore, they might be involved in endometrium receptivity by acting as ceRNA.


Assuntos
Biomarcadores/metabolismo , Endométrio/metabolismo , Redes Reguladoras de Genes , Genoma Humano , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Adulto , Estudos de Casos e Controles , Implantação do Embrião , Endométrio/patologia , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
14.
Gene ; 719: 144075, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31449843

RESUMO

The human L-DOPA decarboxylase (DDC) is a gene that has been in the center of research attention in many laboratories the last decades, due to its major implication in various disorders, including many types of cancer. In the current work, we used in-house developed RACE and high-throughput sequencing approaches, in order to detect and identify novel DDC transcripts. Bioinformatic analysis revealed new alternative splicing events that support the existence of novel DDC transcripts. As a result, a total of 14 DDC splice variants were identified and their expression profile was investigated in a wide panel of human cancer cell lines. From all 14 novel DDC transcripts that were identified, 9 transcripts are predicted to encode new protein isoforms, while the remaining 5 are nonsense-mediated mRNA decay (NMD) candidates. Our results demonstrate that the human DDC gene undergoes complex processing leading to the figuration of multiple mRNA isoforms in cancer cells.


Assuntos
Processamento Alternativo , Descarboxilases de Aminoácido-L-Aromático/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de Bases , Linhagem Celular Tumoral , Éxons , Perfilação da Expressão Gênica , Humanos , RNA Mensageiro/genética
15.
Medicine (Baltimore) ; 98(35): e16576, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464892

RESUMO

OBJECTIVES: G protein-coupled receptor 137 (GPR137) was reported to be associated with several cancers, but its role in bladder cancer has not been reported. The purpose of this study was to evaluate clinical significance of GPR137 in bladder cancer. METHODS: The expressions of GPR137 in pathological tissues and corresponding normal tissues from bladder cancer patients were detected via quantitative real time polymerase chain reaction (qRT-PCR). Western blot was performed to detect GPR137 expression in bladder cancer tissues and adjacent normal tissues. Chi-Squared test analyzed the relationship between GPR137 expression and clinical features of bladder cancer patients. Additionally, Kaplan-Meier method was adopted in estimating overall survival of bladder cancer patients. Prognostic value of GPR137 was evaluated through Cox regression analysis. RESULTS: The expression of GPR137 mRNA and protein in pathological tissues was significantly higher than that in adjacent normal tissues (P < .001). Moreover, similar result was found for bladder cancer patients and healthy controls (P < .001). And GPR137 expression was associated with tumor size (P = .006) and TNM stage (P = .012). The results of Kaplan-Meier analysis suggested that patients with high expression of GPR137 had shorter overall survival time than those with low expression (Log rank test, P = .001). Cox regression analysis indicated that GPR137 could act as an independent biomarker for bladder cancer prognosis (HR = 1.850, 95% CI = 1.272-2.689, P = .001). CONCLUSION: Abnormal expression of GPR137 is associated with bladder cancer and GPR137 is a potential biomarker for the therapy and prognosis of bladder cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Regulação para Cima , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Acoplados a Proteínas-G/genética , Análise de Sobrevida , Carga Tumoral , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
16.
Nat Commun ; 10(1): 2907, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266958

RESUMO

Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.


Assuntos
Anticorpos/análise , Núcleo Celular/genética , Genômica/métodos , Análise de Célula Única/métodos , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Córtex Pré-Frontal/química , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Neoplasma ; 20192019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31307199

RESUMO

Gastric cancer (GC) is a malignant tumor that affects individuals worldwide, and miRNA and mRNA are closely connected to this disease. However, it is still unclear how these molecules affect GC and whether their effects are associated with circRNA in GC patients. Therefore, we obtained the miRNA, mRNA and circRNA expression profiles of GC patients from the GEO database. For comparison, shared miRNAs and mRNAs from the results of microarrays were annotated by gene ontology (GO) and pathway analysis. We also identified mRNAs that were targeted by miRNA through TargetScan 7.2 and circRNAs that were targeted by miRNA through CircInteractome. A comprehensive analysis of the microarray results revealed 72 shared miRNAs, and the expression profiles of 6 miRNAs were significantly different between the tumor and control groups (the absolute value of fold change>2, P<0.05). Hsa-miR-1, hsa-miR-142-3p, hsa-miR-95, hsa-miR-133a and hsa-miR-181d were upregulated in GC, whereas hsa-miR-375 was downregulated. The analysis results also revealed 1201 shared mRNAs and 27 mRNAs, respectively, by microarray and TargetScan. Pathway analysis demonstrated that the Glypican pathway, Proteoglycan syndecan-mediated signalling events, Glypican 1 network and PAR1-mediated thrombin signalling events play important roles. GO analysis revealed significant enrichment in the three terms cellular component, molecular function and biological process, suggesting that organelles, enzyme binding, RNA-binding and nitrogen metabolism may have a strong relationship in GC. The increase in PAX6 in GC may be related to hsa-miR-375. Three circRNAs, hsa_circ_0001658, hsa_circ_0004928 and hsa_circ_0000376, were then found to be significantly differentially expressed between GC and normal tissues (the absolute value of fold change>2, P<0.05). In conclusion, the circ0001658/circ0004928/circ0000376-miR-375-PAX6 axis may represent a new regulatory network that should be further investigated, and the results of this study provide a better understanding of GC.


Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs , RNA Mensageiro , Neoplasias Gástricas , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética , RNA Mensageiro/genética , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/fisiopatologia
18.
Zool Res ; 40(4): 317-323, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31310065

RESUMO

Leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional hepatokine, is involved in many pathological conditions. However, its role in atherosclerosis remains undefined. In this study, we administered vehicle or LECT2 to male Apoe-/- mice fed a Western diet for 15 weeks. Atherosclerotic lesions were visualized and quantified with Oil-red O and hematoxylin staining. The mRNA expression levels of MCP-1, MMP-1, IL-8, IL-1ß, and TNF-α were analyzed by quantitative real-time polymerase chain reaction. Serum TNF-α, IL-1ß, IL-8, MCP-1, and MMP-1 concentrations were measured by enzyme-linked immunosorbent assay. CD68, CD31, and α-SMA, markers of macrophages, endothelial cells, and smooth muscle cells, respectively, were detected by immunostaining. Results showed that LECT2 reduced total cholesterol and low-density lipoprotein concentrations in serum and inhibited the development of atherosclerotic lesions, accompanied by reductions in inflammatory cytokines and lower MCP-1, MMP-1, TNF-α, IL-8, and IL-1ß mRNA abundance. Furthermore, LECT2 decreased CD68, but increased α-SMA in atherosclerotic lesions, suggesting an increase in smooth muscle cells and reduction in macrophages. In summary, LECT2 inhibited the development of atherosclerosis in mice, accompanied by reduced serum total cholesterol concentration and lower inflammatory responses.


Assuntos
Aterosclerose/prevenção & controle , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Animais , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória
19.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
Nat Commun ; 10(1): 2947, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270320

RESUMO

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.


Assuntos
Epitopos/metabolismo , Sondas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/metabolismo , Coloração e Rotulagem , Peixe-Zebra/embriologia
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