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1.
Gene ; 722: 144101, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31479714

RESUMO

The catadromous species, eels, invariably exposed to variable Ca2+ concentrations circumstance i.e., lagoon or ocean. They need to maintain Ca2+ homeostasis by exchanging Ca2+ under different culture conditions. To understand the effects of environmental Ca2+ to fish, three types of genes coding for voltage-dependent L-type calcium channels (cacnb1, 2, 3) were cloned by screening an A. marmorata cDNA library. Tissue distribution analysis of Western blot showed that Cacnb1, 2, 3 had a significantly high expression in gill; while mRNA results showed the expressions of cacnb1 and cacnb3 were predominated in skin tissue but only cacnb2 was expressed in intestine. Serum osmolality and Ca2+ concentrations of A.marmorata were increased in a high calcium environment while reduced in a low calcium environment within 7 days; however, they were not significantly different among Ca2+ treatments after the eels were acclimated for 7 days. We also examined the influence of ambient Ca2+ levels on cacnbs expression of eels. With the increasing of exposure time, mRNA and protein expressions of cacnb1 were up-regulated in high level of Ca2+ (10 mM) and down-regulated in deficient Ca2+ (0 mM) compared to the control Ca2+ (2 mM). However, the opposite results were observed in cacnb2 and cacnb3. Notably, the cacnb2 expression was not significant different among Ca2+ treatments on day 7. Our study provided the insightful evidence that cacnbs play important roles in maintaining Ca2+ homeostasis of fish.


Assuntos
Anguilla/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/fisiologia , Aclimatação , Anguilla/sangue , Anguilla/genética , Animais , Cálcio/sangue , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Clonagem Molecular , Brânquias/metabolismo , Concentração Osmolar , RNA Mensageiro/metabolismo , Distribuição Tecidual
2.
Gene ; 722: 144058, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31494240

RESUMO

PURPOSE: Adipose-derived mesenchymal stem cells (MSCs) are attractive biological agents in regenerative medicine. To optimize cell therapies, it is necessary to determine the most effective delivery method for MSCs. Therefore, we evaluated the biological properties of MSCs after exposure to various temperatures to define optimal storage conditions prior to therapeutic delivery of MSCs. DESIGN: Prospective observational study. METHODS AND MATERIALS: Adherent and non-adherent MSCs were incubated at multiple temperatures (i.e., 4, 23 and 37 °C) in Lactated Ringers (LR) solution lacking essential cell growth ingredients, or in culture media which is optimized for cell growth. Cells were assessed either after the temperature changes (4 h) or after recovery (24 h). Metabolic activity of MSCs, cell number and expression of representative mRNA biomarkers were evaluated to assess the biological effects of temperature. We monitored changes in mRNAs expression related to cytoprotective- or stress-related responses (e.g., FOS, JUN, ATF1, ATF4, EGR1, EGR2, MYC), proliferation (e.g., HIST2H4, CCNB2), and extracellular matrix production (ECM; e.g., COL3A1, COL1A1) by quantitative real time reverse-transcriptase polymerase chain reaction (RT-qPCR) analysis. RESULTS: Our study demonstrates that storing MSCs in Lactated Ringers (LR) solution for 4 h decreases cell number and metabolic activity. The number of viable MSCs decreased significantly when cultured at physiological temperature (37 °C) and severe hypothermia (4 °C), while cells grown at ambient temperature (23 °C) exhibited the least detrimental effects. There were no appreciable biological differences in mRNA markers for proliferation or ECM deposition at any of the temperatures. However, biomarkers related to cytoprotective- or stress-responses were selectively elevated depending on temperature or media type (i.e., LR versus standard media). CONCLUSION: The biological impact of nutrient-free media and temperature changes after 4 h exposure persists after a 24 h recovery period. Hence, storage temperature and media conditions should be optimized to improve effective dosing of MSCs.


Assuntos
Tecido Adiposo/citologia , Temperatura Baixa , Células-Tronco Mesenquimais/citologia , Sobrevivência Celular , Meios de Cultura , Humanos , Células-Tronco Mesenquimais/metabolismo , Nutrientes , RNA Mensageiro/metabolismo , Temperatura Ambiente
3.
Gene ; 722: 144105, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31521702

RESUMO

BACKGROUND: Caulophyllum robustum Maxim (CRM) is a medicinal compound of the Northeast and is commonly used in China for the treatment of rheumatic pain and rheumatoid arthritis (RA). A preliminary study found that CRM has good anti-inflammatory, analgesic and immunosuppressive effects. However, the specific links and targets for its function remain unclear. Our study aimed to provide a mechanism for the action of Caulophyllum robustum Maxim extraction (CRME) against RA and to establish a method for studying disease treatment using Chinese medicine. METHODS: The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method was used to detect the toxicity of CRME in L929 cells, and the concentration ranges of the blank, model, and CRME drug groups were determined. Differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) were identified between the three groups. Gene Ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways of the differentially expressed genes. Expression of Hist1h2bj, Hist1h2ba, Zfp36, Ccl3, Cxcl2 and Egr1 in the blank, model and drug groups was detected by quantitative real-time PCR (qRT-PCR), and the role of CRME on the above factors was determined to ensure consistency with the chip data. RESULTS: A total of 329 significantly upregulated genes and 141 downregulated genes were identified between the blank and model groups. A total of 218 significantly upregulated genes and 191 downregulated genes were identified between the CRME drug group and model group. CRME has a significant role in multiple pathways involved in the occurrence and development of RA. Additionally, Hist1h2bj, Hist1h2ba, Zfp36, Ccl3, Cxcl2, and Egr1 were observed in modules of the lncRNA-mRNA weighted co-expression network, consistent with the chip data. CONCLUSIONS: CRME has regulatory effects on inflammatory factors, the histone family, chemokines and their ligands that are related to RA-related cytokines, the RA pathway, the TNF signaling pathway, the Toll receptor-like signaling pathway, the chemokine signaling pathways and other pathways are related to the course of RA.


Assuntos
Artrite Reumatoide/genética , Caulophyllum , Medicamentos de Ervas Chinesas/farmacologia , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Animais , Artrite Reumatoide/metabolismo , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento de Interação de Proteínas
4.
Biomed Khim ; 65(5): 385-387, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666410

RESUMO

The neurotransmitter systems of the brain are exposed to dysregulation during alcohol withdrawal. This contributes to the development of the pathological craving for alcohol in which corticotropin-releasing hormone receptors are may be involved. During the period of alcohol withdrawal, the level of CRFR2 mRNA in the ventral tegmental area of the brain on the seventh day of abstinence was significantly increased in comparison with the control group. This supports existing concepts on possible participation in the modulation of dopaminergic and GABA-neural neurons in the ventral tegmental area the brain.


Assuntos
Alcoolismo , Encéfalo/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Síndrome de Abstinência a Substâncias , Animais , Encéfalo/efeitos dos fármacos , Etanol , RNA Mensageiro , Ratos
5.
Braz J Med Biol Res ; 52(10): e8343, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618295

RESUMO

The objective was to study the effect of mechanical intestinal obstruction in rats on the phenotype of interstitial cells of Cajal (ICC). Healthy Wistar rats were randomly divided into sham-operation group (C), one day obstruction group (M1), two days obstruction group (M2), and three days obstruction group (M3), with 10 rats in each group. The expression of SCF mRNA and c-Kit protein in intestinal tissue was investigated by RT-PCR and immunohistochemistry. Compared with the sham-operation group, the relative expression of SCF mRNA and the expression of c-Kit protein in intestinal tissue were significantly decreased in both obstruction groups. Levels decreased gradually with the prolongation of obstruction time, and significantly decreased on the 3rd day after obstruction (P<0.05). Immunohistochemical staining of the small intestine showed that the number of ICC in the sham-operation group was the highest, and they were gradually decreased with the extension of obstruction time in the M1 to M3 groups. There was a significant difference between groups (P<0.05). Intestinal obstruction caused a decrease in the concentrations of SCF mRNA and c-Kit protein in ICC. With the prolongation of intestinal obstruction, the number of ICCs gradually decreased.


Assuntos
Células Intersticiais de Cajal/metabolismo , Obstrução Intestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Mensageiro/metabolismo , Fator de Células-Tronco/metabolismo , Animais , Modelos Animais de Doenças , Imuno-Histoquímica , Células Intersticiais de Cajal/patologia , Obstrução Intestinal/patologia , Masculino , Fenótipo , Ratos , Ratos Wistar
6.
Medicine (Baltimore) ; 98(43): e17578, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31651862

RESUMO

BACKGROUND: To evaluate the methylation levels of human telomerase reverse transcriptase (hTERT) promoter three CpG island (CGIs) regions and its prognostic impact in Chinese patients with acral and mucosal melanoma. METHODS: Bioinformatics software was used to analyze hTERT gene promoter. Fresh frozen tissues were taken from 14 patients with melanoma (6 acral melanoma and 8 mucosal melanoma) and 14 pigmented nevus as control subjects (14 acral pigmented nevus). Bisulfite sequencing PCR (BSP) combined TA clone sequencing was used to assess the methylation levels of hTERT promoter CGIs regions. The relative expression level of hTERT mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: CGIs-1 (-1392--1098 bp), CGIs-2 (-945--669 bp), and CGIs-3 (-445--48 bp) were selected for our study. Our results indicated that the methylation levels of hTERT promotor CGIs regions in melanoma were greater than pigmented nevus (CGIs-1: 69.3 ±â€Š18.7% vs 46.8 ±â€Š20.4%, t = 3.048 P = .005; CGIs-2: 73.8 ±â€Š14.7% vs 55.6 ±â€Š16.0%, t = 3.120 P = .004; CGIs-3: 5.8 ±â€Š2.2% vs 2.2 ±â€Š1.3%, t = 5.164 P < .001). The relative expression level of hTERT in melanoma was greater than in pigmented nevus (50.39 ±â€Š9.16 vs 26.10 ±â€Š7.25, t = 7.778, P < .001). Linear regression analysis showed that the methylation level of CGIs-2 in melanoma was positively correlated with the relative expression level of hTERT mRNA (R = .490, F = 13.478, P = .003). Combined with the analysis of clinicopathological features, the methylation level of CGIs-2 in melanoma with lymph node metastasis was greater than in melanoma without lymph node metastasis, and the methylation level of CGIs-2 increased with TNM staging. CONCLUSION: CGIs-2 methylation level was associated with the relative expression level of hTERT mRNA, lymph node metastasis and TNM staging, suggesting that CGIs-2 hypermethylation might be used to evaluate the prognosis in Chinese patients with acral and mucosal melanoma.


Assuntos
Ilhas de CpG/genética , Metilação de DNA/genética , Melanoma/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Telomerase/metabolismo , Idoso , Grupo com Ancestrais do Continente Asiático/genética , China , Biologia Computacional , Feminino , Humanos , Modelos Lineares , Metástase Linfática , Masculino , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , RNA Mensageiro/metabolismo
7.
Medicine (Baltimore) ; 98(39): e17216, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31574831

RESUMO

The present study aimed to explore the expression of latent transforming growth factor ß binding protein 2 (LTBP2) in patients with hepatocellular carcinoma (HCC) and their correlation to clinicopathologial features.Serum levels of LTBP2 in 60 patients with HCC, 35 patients with hepatocellular benign tumors, 60 patients with precancerous lesions of HCC, and 60 healthy volunteers were determined by enzyme-linked immunosorbent assay. The expression levels of LTBP2 at messenger RNA (mRNA) and protein levels in 60 cases of HCC and adjacent tissues were detected by quantitative real-time polymerase chain reaction and immunohisochemistry. Statistical analysis was used to analyze the relationship between LTBP2 and clinical characteristics of patients with HCC.The mRNA and protein levels of LTBP2 were significantly upregulated in HCC tissues compared to adjacent tissues. Additionally, higher serum LTBP2 level was also observed in HCC patients relative to normal controls. Further investigation demonstrated that LTBP2 expression was associated with malignant degree of tumor, tumor progression, tumor differentiation, tumor size, tumor stage and hepatitis virus infection, and has prognostic implications in HCC patients.LTBP2 might be served as a potential biomarker in diagnosis and treatment of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Neoplasias Hepáticas/genética , Adulto , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/sangue , Feminino , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/metabolismo
8.
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
9.
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
10.
N Engl J Med ; 381(17): 1644-1652, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31597037

RESUMO

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).


Assuntos
Proteínas de Membrana Transportadoras/genética , Mutagênese Insercional , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , Oligonucleotídeos Antissenso/uso terapêutico , Medicina de Precisão , Doenças Raras/tratamento farmacológico , Biópsia , Criança , Desenvolvimento Infantil , Descoberta de Drogas , Drogas em Investigação/uso terapêutico , Eletroencefalografia , Feminino , Humanos , Testes Neuropsicológicos , RNA Mensageiro , Convulsões/diagnóstico , Convulsões/tratamento farmacológico , Pele/patologia , Sequenciamento Completo do Genoma
11.
Postepy Biochem ; 65(3): 173-182, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643164

RESUMO

Endoribonuclease III Dicer plays a crucial role in the biogenesis of small regulatory RNAs, such as microRNAs (miRNAs) and small inter­fering RNAs (siRNAs). However, this is not the only role that Dicer plays in cells. For example, it has been shown that Dicer is involved in processing of diverse classes of RNA, including tRNA and snoRNA, cleavage of repeat-element-derived RNAs, and maintenance of genome integrity. Dicer has also been found to participate in the chromosome fragmentation during apoptosis or in the inflammatory processes. More­over, a recent discovery of Dicer-binding passive sites in mRNAs and long non-coding RNAs, and its putative nucleic acid chaperone activity, has pointed out a novel regulatory role of the enzyme. Here we focus on human Dicer and review its structure and function including recent findings on miRNA-independent roles and their impact on cell biology.


Assuntos
Ribonuclease III/química , Ribonuclease III/metabolismo , Fragmentação do DNA , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/metabolismo
12.
Psychiatr Danub ; 31(3): 347-354, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31596828

RESUMO

BACKGROUND: The role of sirtuins as a pathogenetic element of some mental disorders is becoming increasingly more common. They participate in many cellular processes, such as ageing, transcription, apoptosis, inflammatory processes, post-translational modification of proteins, gene transcription silencing, activation of DNA repair mechanisms, and regulation of many metabolic processes. The aim of this paper is to verify the statistical hypothesis assuming the difference in expression at the level of mRNA in genes for sirtuins 1-7 between patients with recurrent depressive disorders (rDD) and patients from the control group, and the hypothesis assuming the relation between the expression at the level of mRNA for these genes and clinical variables in the course of recurrent depressive disorders. SUBJECTS AND METHODS: A total of 198 individuals took part in the study (rDD gropup, N=99; control group, N=99). RESULTS: SIR-1 and SIR-6 expression at the mRNA level was significantly higher among the people with rDD as compared to the subjects from the control group. A reversed relationship was observed for SIR-2, SIR-3, SIR-4 and SIR-5. Statistically significant correlations were observed only in the case of SIR-1 and the number of depression episodes (negative relationship), as well as SIR-5 and the severity of depression measured by the Hamilton Depression Rating Scale (positive relationship). CONCLUSIONS: Expression at the mRNA level for selected sirtuins is a factor that significantly differentiates people with depressive episodes from healthy ones. SIR-1 and SIR-6 expression at the mRNA level was significantly higher among the people with depression as compared to the subjects from the control group. A reversed relationship (also statistically significant) was observed for SIR-2, SIR-3, SIR-4 and SIR-5.


Assuntos
Transtorno Depressivo Maior/genética , Regulação da Expressão Gênica , Sirtuínas/genética , Doença Crônica , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genética
13.
Zhonghua Xue Ye Xue Za Zhi ; 40(9): 759-763, 2019 Sep 14.
Artigo em Chinês | MEDLINE | ID: mdl-31648479

RESUMO

Objective: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. Methods: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. Results: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) . Conclusion: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.


Assuntos
Receptor de Morte Celular Programada 1/genética , Linfócitos T , Antígenos CD19 , Humanos , RNA Mensageiro , Receptores de Antígenos de Linfócitos T
14.
Medicine (Baltimore) ; 98(40): e17126, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577702

RESUMO

BACKGROUND: The aim of this study was to investigate the role of n-acetyl cysteine (NAC) in the lipopolysaccharide (LPS)-mediated induction of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) synthesis by human periodontal ligament fibroblast cells (hPDLFs). In addition, we aimed to determine the involvement of the nuclear factor-kappa B (NF-κB) pathway in any changes in IL-1ß and TNF-α expression observed in response to LPS and NAC. METHODS: HPDLFs were obtained by primary culture. The culture medium used in this experiment was Dulbecco's Modified Eagle Medium (DMEM low-glucose). Cells were stimulated with various concentrations of NAC or LPS. Cell proliferation was measured at various time-points with the cell Counting Kit 8 (CCK-8) assay. mRNA levels of IL-1ß and TNF-α were determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Protein levels of IL-1ß and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). Protein and mRNA expression levels of NF-κB were measured by western blot and RT-qPCR. RESULTS: The results showed that LPS treatment in hPDLFs induced mRNA and protein expression of IL-1ß, TNF-α, and NF-κB. However, these effects were eliminated by pretreatment with NAC. Pretreatment with both NAC (1 mmol/L) and BAY11-7082 (10 µmol/L) significantly inhibited the NF-κB activity induced by LPS. CONCLUSION: NAC inhibits the LPS-mediated synthesis of tumor TNF-α and IL-1ß in hPDLFs, through the NF-κB pathway.


Assuntos
Acetilcisteína/farmacologia , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Humanos , Interleucina-1beta/biossíntese , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Ligamento Periodontal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
15.
Medicine (Baltimore) ; 98(40): e17471, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577778

RESUMO

Hand, foot, and mouth disease (HFMD) spreads rapidly and has been recognized as a public health problem in recent years in China. Unfortunately, there is no effective vaccine or antiviral drug currently for EV71 infection. In this study, we aim to identify biomarker which are associated with for severity of EV71 infection cases using high-throughput RNA sequencing approach.RNA sequencing of samples from severe HFMD (S) patients group (n = 10) and control HFMD (C) patients group (n = 10) were performed and the results were verified by qPCR. mRNA with the highest expression level was selected to be validated in an independent cohort comprising of 45 severe EV71 infected patients and 45 control by qPCR assay.Seventeen significant differentially expressed genes were identified. Scavenger receptor class A, member 3 (SCARA3) was one of the significantly upregulated genes with the highest expression level and was selected for validation. The mean relative expression levels in severe HFMD and control HFMD patients were 10.1-fold and 5.0-fold, respectively, P value <.001.We found that SCARA3 is associated with severity of HFMD, and it may be a potential prognostic marker to predict the HFMD progression in EV71 infected patients.


Assuntos
Doença de Mão, Pé e Boca/diagnóstico , Doença de Mão, Pé e Boca/genética , Proteínas de Choque Térmico/genética , Receptores Depuradores Classe A/genética , Biomarcadores/metabolismo , Estudos de Casos e Controles , Pré-Escolar , Feminino , Doença de Mão, Pé e Boca/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Lactente , Masculino , RNA Mensageiro/metabolismo , Receptores Depuradores Classe A/metabolismo , Índice de Gravidade de Doença
16.
Chem Biol Interact ; 314: 108822, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31580832

RESUMO

Aldehyde dehydrogenase (ALDH) activity is not only a valuable marker for cancer cells with stem-like features, but also plays a vital role in drug resistance and disease progression in many tumors including melanoma. However, the precise role of ALDH activity in patient prognosis remains unclear. In this study, using the Cancer Genome Atlas (TCGA) RNA-sequencing expression data, we analyzed gene expression of ALDH isozymes in melanoma tumors to define the expression patterns and the prognostic and predictive values of these enzymes. We found that ALDH1A1 and ALDH1A3 had both higher and broader expression ranges in melanoma patients, and that ALDH1A3 expression correlated with better overall survival in metastatic melanoma. Further, stratification of the TCGA cohorts by the mutational subtypes of melanoma specifically revealed that expression of ALDH1A3 correlated with better prognosis in metastatic BRAF-mutant melanoma while expression of ALDH1A1 correlated with better prognosis in BRAF wild-type melanoma. Gene set enrichment analysis (GSEA) of these cohorts identified upregulation in oxidative phosphorylation, adipogenesis, and fatty acid metabolism signaling in ALDH1Alo patients, suggesting BRAF/MEK inhibitor resistance in that subset of patients. On the other hand, GSEA of ALDH1A3hi cohorts revealed upregulation in glycolysis, hypoxia and angiogenesis, suggesting BRAF/MEK inhibitor sensitivity in that subset of patients. Gene expression analysis using pre-treatment tumor samples supports high ALDH1A3 expression before BRAF/MEK inhibitor treatment as predictive of better treatment response in BRAF-mutant melanoma patients. Our study provides evidence that high ALDH1A3 mRNA expression is not only a prognostic marker but also a predictive marker for BRAF/MEK inhibitor treatment response in BRAF-mutant metastatic melanoma patients.


Assuntos
Aldeído Desidrogenase/genética , Aldeído Oxirredutases/genética , Melanoma/patologia , RNA Mensageiro/metabolismo , Idoso , Aldeído Desidrogenase/metabolismo , Aldeído Oxirredutases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Melanoma/metabolismo , Melanoma/mortalidade , Pessoa de Meia-Idade , Mutação , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética
17.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(5): 1476-1481, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31607301

RESUMO

OBJECTIVE: To explore the mechanisms of angiogenesis in chronic myeloid leukemia (CML) through detecting the levels of angiogenesis-related factors secreted from K562 cells after overexpression and interference of HIF-1α gene in K562 cells. METHODS: The K562 cells were transfected by lentiviruses carried and interfered HIF-1α gene, then the transtected K562 cells with carried and interfered with HIF-1α gene were enrolled in overexpression and interference groups respectively, at the same time the K562 cells transfected by the empty virus were enrolled in control group. The cells were harvested after culture for 72 hours under normoxid condition. The transfection efficient in 3 groups was detected by fluorescence microscopy; the mRNA expression of HIF-1α gene and angiogenesis-related factors was detected by RT-PCR; the concentration of angiogenesis-related factors in the caltured supernatant was detected by ELISA. RESULTS: The optimal MOI of K562 cells transfected with lentivirus was 10 and the transfection efficiency was about 50%. The positive rate of transfection after screening by puromycin was more than 90%. The mRNA expression of ANG-I, ANG-II, TGF-α and VEGF in the interference group was lower than that in the over-expression group, and the TGF-ß1 mRNA expression in the interference group was higher than in the over-expression group. The mRNA expression of ANG-I and VEGF in the interference group was lower than that in the control group. TGF-αdid not could be detected, and the culture supernatant concentration of ANG-I and TNF-α in the interference group was lower than in the over-expression group, while the VEGF concentration in the interference group was higher than that in the over-expression group. All of the above-mentioned differences were statistically significant (P<0.05). CONCLUSION: The positive K562 cells transfected with leutivirus have been harvested by screening with puromycin. The HIF-1α mRNA positively regulates the mRNA expression of ANG-1, ANG-2, TGF-α, VEGF in K562 cells, promotes the antocrine ability of ANG-1 and TNF-α, moreover not stimulates the autocrine of TGF-α, the up-regulation of HIF-1α expression can inhibit the expression TGF-ß1 in K562 cells and the autocrine of TGF-ß1.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Humanos , Células K562 , RNA Mensageiro , Fator A de Crescimento do Endotélio Vascular
18.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(10): 1033-1037, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31642440

RESUMO

OBJECTIVE: To study the expression levels of glial cell line-derived neurotrophic factor family receptor α-1 (GFRα1) and enhancer of zeste homolog 2 (EZH2) in the intestinal tissue of children with Hirschsprung's disease (HSCR), as well as the role of EZH2 in the regulation of GFRα1 gene expression and the pathogenesis of HSCR. METHODS: The samples of colon tissue with spasm from 24 children with HSCR after radical treatment of HSCR were selected as the experimental group, and the samples of necrotized colon tissue from 18 children with neonatal necrotizing enterocolitis after surgical resection were selected as the control group. Real-time PCR and Western blot were used to measure the expression levels of GFRα1 and EZH2 in colon tissue in both groups. Human neuroblastoma SH-SY5Y cells were divided into an EZH2 over-expression group and a negative control group. The cells in the EZH2 over-expression group were transfected with pCMV6-EZH2 plasmid, and those in the negative control group were transfected with pCMV6 plasmid. The expression levels of EZH2 and GFRα1 were measured after transfection. RESULTS: Compared with the control group, the experimental group had significant reductions in the mRNA and protein expression levels of GFRα1 and EZH2 in colon tissue (P<0.05), and the protein expression of EZH2 was positively correlated with that of GFRα1 (r=0.606, P=0.002). Compared with the negative control group, the EZH2 over-expression group had significant increases in the expression levels of EZH2 and GFRα1 after SH-SY5Y cells were transfected with EZH2 over-expression plasmid (P<0.05). CONCLUSIONS: Low expression of EZH2 in the colon tissue of children with HSCR may be one of the causes of inadequate expression of GFRα1 and onset of HSCR.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Doença de Hirschsprung , Criança , Colo , Doença de Hirschsprung/genética , Humanos , Recém-Nascido , RNA Mensageiro
19.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(8): 950-956, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31511216

RESUMO

OBJECTIVE: To investigate the effects of different doses of propofol on myelin basic protein (MBP) synthesis and myelination of oligodendrocytes in neonatal SD rats. METHODS: A total of 57 neonatal SD rats (7 days old) were randomly divided into control group (n=13), vehicle (fat emulsion) group (n=5), and 25, 50 and 100 mg/kg propofol groups (n=13 in each group). Eight hours after a single intraperitoneal injection of propofol or the vehicle, the rats were examined for expressions of mbp mRNA, caspase-3 mRNA, cleaved caspase-3 and MBP in the brain tissues using qPCR and Western blotting. Immunofluorescence assay was used to detect the apoptosis of the oligodendrocytes at 8 h after the injection and the myelination of the corpus callosum and internal capsule at 24 h. RESULTS: Compared with the control group, the neonatal rats with propofol injections showed significantly down-regulated expressions of mbp mRNA and MBP protein in the brain tissue (P < 0.05). Propofol dose-dependently increased the transcription level of caspase-3 and the protein levels of cleaved caspase-3 at 8 h after the injection (P < 0.05). Propofol injection significantly increased the apoptosis of the oligodendrocytes, and the effect was significantly stronger in 50 and 100 mg/kg groups than in 25 mg/kg group (P < 0.05). At 24 h after propofol injection, myelin formation was significantly decreased in the corpus callosum of the neonatal rats in 100 mg/kg propofol group and in the internal capsule in 50 and 100 mg/kg groups (P < 0.05). CONCLUSIONS: In neonatal SD rats, propofol can dose-dependently promote oligodendrocyte apoptosis, decrease MBP expressions in the brain, and suppress myelin formation in the corpus callosum and the internal capsule.


Assuntos
Oligodendroglia , Animais , Proteína Básica da Mielina , Propofol , RNA Mensageiro , Ratos , Ratos Sprague-Dawley
20.
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
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