RESUMO
Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro. Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication.
Assuntos
Vasos Coronários/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/metabolismo , Tunicamicina/farmacologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacosRESUMO
The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.
Assuntos
Manchas de Sangue , RNA Mensageiro/genética , RNA/genética , 5-Aminolevulinato Sintetase/sangue , 5-Aminolevulinato Sintetase/genética , Biomarcadores/sangue , Primers do DNA , Eletroforese Capilar , Feminino , Genética Forense , Humanos , Metaloproteinase 7 da Matriz/sangue , Metaloproteinase 7 da Matriz/genética , Menstruação , Reação em Cadeia da PolimeraseRESUMO
Stress-induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up-regulated 1 (Axud1), a novel pro-apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress-induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti-apoptotic Bcl-2 was decreased, whereas pro-apoptotic Bax and Cleaved caspase-3 (Cc3) were increased in a time-dependent manner. The Wnt/ß-catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/ß-catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE-induced cardiomyocytes apoptosis and also inactivated the Wnt/ß-catenin signaling. XAV-939, an inhibitor of Wnt/ß-catenin signaling, partially reversed the pro-apoptotic effect of NE. In conclusion, Axud1 accelerated stress-induced cardiomyocytes apoptosis through activation of Wnt/ß-catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/ß-catenin signaling might be promising in relation to RS-induced myocardial injury.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Miócitos Cardíacos/metabolismo , Estresse Psicológico/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt , beta Catenina/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imobilização , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Norepinefrina/antagonistas & inibidores , Norepinefrina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , beta Catenina/metabolismoRESUMO
DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1ß or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1ß or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dano ao DNA/genética , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , NF-kappa B/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/fisiologia , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
The mutation of short tandem repeat (STR) loci is affected by several factors, such as sex, age, and DNA architectures. Previous studies have shown a different profile of mutation rates at autosomal STR loci among populations. It is important to provide population data and reveal underlying factors influencing the evaluation of STR mutation rates. In this study, we performed a comprehensive analysis on the mutation of 19 autosomal STR loci through 124,773 parent-child allelic transfers from 5846 paternity testing cases. A total of 197 mutations were observed including 187 single-step mutations. The observed mutation rates ranged from 0.15 × 10-3 (TH01) to 4.57 × 10-3 (FGA), and the average mutation rate across all the 19 loci was 1.58 × 10-3. Furthermore, the average mutation rate of STR loci increases with the paternal conception ages and remains relatively stable in different maternal age groups, which suggest the profile of paternal conception ages as a potential factor influencing the evaluation of STR mutation rates and the ratio of paternal versus maternal mutation rate in populations. Multidimensional scaling analysis (MDS) shows a difference in the profile of mutation rates at 13 CODIS STR loci among ethnical groups. Based on our data, our results support that short alleles are biased towards expansion mutation and longer alleles favor contraction mutation. In conclusion, our results provide useful information for further investigation on STR mutation in forensic genetics and population genetics.
Assuntos
Etnicidade/genética , Genética Populacional , Repetições de Microssatélites , Taxa de Mutação , China , Feminino , Loci Gênicos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Paternidade , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. MATERIAL AND METHODS To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. RESULTS The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. CONCLUSIONS p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2.
Assuntos
Miosinas Cardíacas/metabolismo , Estenose Coronária/metabolismo , Vasoespasmo Coronário/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Biomarcadores/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Células Cultivadas , Angiografia Coronária/métodos , Vasos Coronários/metabolismo , Diagnóstico , Humanos , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Vasoconstritores/uso terapêuticoRESUMO
Viral myocarditis (VMC) is a life-threatening disease that leads to heart failure or cardiac arrhythmia. A large number of researches have revealed that mircroRNAs (miRNAs) participate in the pathological processes of VMC. We previously reported that miR-1 repressed the expression of gap junction protein α1 (GJA1) in VMC. In this study, miR-19b was found to be significantly upregulated using the microarray analysis in a mouse model of VMC, and overexpression of miR-19b led to irregular beating pattern in human cardiomyocytes derived from the induced pluripotent stem cells (hiPSCs-CMs). The upregulation of miR-19b was associated with decreased GJA1 in vivo. Furthermore, a miR-19b inhibitor increased, while its mimics suppressed the expression of GJA1 in HL-1 cells. When GJA1 was overexpressed, the miR-19b mimics-mediated irregular beating was reversed in hiPSCs-CMs. In addition, the effect of miR-19b on GJA1 was enhanced by miR-1 in a dose-dependent manner. These data suggest miR-19b contributes to irregular beating through regulation of GJA1 by cooperating with miR-1. Based on the present and our previous studies, it could be indicated that miR-19b and miR-1 might be critically involved in cardiac arrhythmia associated with VMC.
Assuntos
Conexina 43/genética , Regulação para Baixo , MicroRNAs/genética , Miocardite/virologia , Viroses/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Miocardite/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Precise estimation of postmortem interval (PMI) is crucial in some criminal cases. This study aims to find some optimal markers for PMI estimation and build a mathematical model that could be used in various temperature conditions. Different mRNA and microRNA markers in rat brain samples were detected using real-time fluorescent quantitative PCR at 12 time points within 144 h postmortem and at temperatures of 4, 15, 25, and 35 °C. Samples from 36 other rats were used to verify the animal mathematical model. Brain-specific mir-9 and mir-125b are effective endogenous control markers that are not affected by PMI up to 144 h postmortem under these temperatures, whereas the commonly used U6 is not a suitable endogenous control in this study. Among all the candidate markers, ΔCt (ß-actin) has the best correlation coefficient with PMI and was used to build a new model using R software which can simultaneously manage both PMI and temperature parameters. This animal mathematical model is verified using samples from 36 other rats and shows increased accuracy for higher temperatures and longer PMI. In this study, ß-actin was found to be an optimal marker to estimate PMI and some other markers were found to be suitable to act as endogenous controls. Additionally, we have used R code software to build a model of PMI estimation that could be used in various temperature conditions.
Assuntos
Encéfalo/patologia , Modelos Teóricos , Mudanças Depois da Morte , Estabilidade de RNA , Temperatura , Actinas/genética , Actinas/metabolismo , Animais , Encéfalo/metabolismo , Marcadores Genéticos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Animais , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Software , Manejo de EspécimesRESUMO
INTRODUCTION: Breast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI). METHODS: Immunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively. RESULTS: JMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo. CONCLUSION: We demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.
Assuntos
Neoplasias da Mama/genética , Histona Desmetilases com o Domínio Jumonji/genética , Transcrição Gênica/genética , Proteínas rho de Ligação ao GTP/biossíntese , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Fatores de Transcrição E2F/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Células HEK293 , Histona Desacetilases/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/biossíntese , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Transplante de Neoplasias , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Transplante Heterólogo , Cicatrização/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Specificity protein 1(Sp1) is a ubiquitous transcription factor and is highly expressed in breast cancer. However, its expression pattern and role in breast cancer progression remain unclear. The purpose of this study is to examine the expression pattern of Sp1 and determine its role in breast cancer progression. Immunohistochemistry (IHC) was performed on breast cancer tissues to reveal the expression pattern of Sp1. Spearman rank correlation was used for clinical statistics. Gene and protein expressions were monitored by IHC analysis, quantitative polymerase chain reaction, and Western blot. Wound-healing and Transwell assays were conducted to assess the role of Sp1 in breast cancer. Co-immunoprecipitation, deletion mutagenesis, chromatin immunoprecipitation, and dual luciferase reporter gene assays were used for investigation of the regulatory network. Sp1 expression was downregulated in late stage breast cancer and in highly invasive breast cancer cell lines. Expression of Sp1 was negatively correlated with TNM staging (P = 0.002) and metastasis status (P = 0.023). Overexpression of Sp1 inhibited breast cancer cell migratory and invasive abilities, whereas knockdown of GTP-binding RAS-like 3 (DIRAS3, also known as ARHI, NOEY2) attenuated the inhibitory effects. Moreover, re-expression of DIRAS3 abolished Sp1 knockdown-mediated cell migration and invasion. Jumonji domain containing 2A (JMJD2A) inhibited Sp1 autoregulation and explains Sp1 expression pattern in breast cancer. Sp1 negatively regulated breast cancer metastasis by transcriptional activation of DIRAS3. Inhibition of Sp1 autoregulation by JMJD2A contributed to Sp1 expression pattern in breast cancer. Our findings provided evidence that targeted therapy against Sp1 might be useful in early stage breast cancer. However, in late stages, development of Sp1 activator may be more promising for breast cancer treatments.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fator de Transcrição Sp1/genética , Proteínas rho de Ligação ao GTP/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
OBJECTIVE: To explore the relationship between the expression of EIIIA+ fibronectin in incised wound of rat's skin and injury time. METHODS: The wounding model was established by cutting the dorsal skin of 48 adult SD rats. The rats were sacrificed at the pre-set injury time as immediately, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h. The skin samples were taken at the margin of wound. The expression of the EIIIA? fibronectin was detected by immunohistochemistry and Western blotting and the relationship be- tween its expression and injury time was observed. Results The expression of EIIIA+ fibronectin was not observed immediately. The basal cell of skin began to show positive expression 0.5 h after injury. With the extension of injury time, positive staining became stronger. The value of relative optical density was gradually increased with prolonged injury time by the Western blotting analysis. CONCLUSION: The expression of EIIIA+ fibronectin could be used for estimation of injury time in the early stage of skin injury.
Assuntos
Fibronectinas/metabolismo , Proteínas/metabolismo , Pele/metabolismo , Animais , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Pele/lesões , Coloração e Rotulagem , Fatores de TempoRESUMO
OBJECTIVE: To observe the changes of relative expression of myocardial various RNAs in rats died of different causes and their relationship with PMI. METHODS: The rat models were established in which the rats were sacrificed by broken neck, asphyxia, and hemorrhagic shock. Total RNAs were extracted from myocardium. The quantitative real time PCR was used to calculate threshold cycle values of RNAs including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, inducible nitric oxide synthase (iNOS), hypoxia-inducible factor-1 (HIF-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and U6 small nuclear RNA (U6 snRNA) and to study the changes of the relative expressions of various indexes with PMI. RESULTS: U6 snRNA with stable expression level could be used as appropriate internal control. In the early PMI, the relative expression of GAPDH, HIF-1, iNOS, TNF-alpha, and IL-6 more characteristically increased in groups of asphyxia and hemorrhagic shock than in group of broken neck, but the quantity of beta-actin decreased in all groups. In the late PMI, all the relative expressions significantly declined in correlation with the degradation of RNA. CONCLUSION: The characteristic changes of each RNA expression can be used as references to estimate PMI in deaths by different causes.
Assuntos
Citocinas/metabolismo , Enzimas/metabolismo , Miocárdio/metabolismo , RNA/metabolismo , Actinas , Animais , Causas de Morte , Modelos Animais de Doenças , Gliceraldeído-3-Fosfato Desidrogenases , Óxido Nítrico Sintase Tipo II , RNA Nuclear Pequeno , Ratos , Choque Hemorrágico , Fator de Necrose Tumoral alfaRESUMO
Jumonji Domain Containing 2A (JMJD2A) may be a cancer-associated gene involved in human breast cancer. With a view to investigating expression of JMJD2A in human breast cancer and benign lesion tissues as well as relationship between JMJD2A and tumor related proteins, histological and immunohistochemical analysis, Western blot and quantitative real-time PCR in infiltrating duct carcinoma and fibroadenoma for JMJD2A and immunohistochemical analysis and quantitative real-time PCR in infiltrating duct carcinoma for tumor related proteins (ARHI, p53, ER, PR and CerbB-2) were performed. Histological examination validated the clinical diagnosis. The JMJD2A positive rate of infiltrating duct carcinoma was significantly higher than fibroadenoma by immunohistochemical analysis. The mean optical density of JMJD2A in infiltrating duct carcinoma was higher than fibroadenoma by western blot. JMJD2A mRNA level in infiltrating duct carcinoma was higher than fibroadenoma by quantitative real-time PCR. Spearman correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from immunohistochemical results respectively. Pearson correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from quantitative real-time PCR results respectively. Expression of JMJD2A in infiltrating duct carcinoma was higher, and associated with ARHI, p53 and ER. The results may take JMJD2A as a potential diagnostic and therapeutic target in human breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Fibroadenoma/metabolismo , Histona Desmetilases com o Domínio Jumonji/biossíntese , Receptores de Estrogênio/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteínas rho de Ligação ao GTP/biossíntese , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Receptor ErbB-2/biossíntese , Receptores de Progesterona/biossínteseRESUMO
MicroRNAs (miRNAs) are increasingly reported to have important roles in diverse biological and pathological processes. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the miR-1 function in the heart are only beginning to emerge. In this study, we investigated miR-1 expression and its potential role in the mouse model of viral myocarditis (VMC). The expression levels of miR-1 and its target gene Connexin 43 (Cx43) were measured by real-time PCR and western blotting, respectively. The miR-1 expression levels were significantly increased in cardiac myocytes from VMC mice in comparison with control samples (relative expression: 10 ± 2.5 vs. 31 ± 7.6, P < 0.05). Among the target genes of miR-1, the expression Cx43 protein was significantly reduced in such mice while there was no significant difference in the its mRNA levels. Our results revealed an inverse correlation between miR-1 levels and Cx43 protein expression in VMC samples. Using a bioinformatics-based approach, we found two identical potential binding sites were found in mouse miR-1 and Cx43 3'- untranslated region, this confirms a possible regulatory role of miR-1. In cultured, miRNA transfected myocardial cells, we show overexpression of miR-1 accompanied by a decrease in Cx43 protein's expression. There was only a slight (not statistically significant) drop in Cx43 mRNA levels. Our results indicate that miR-1 is involved in VMC via post-transcriptional repression of Cx43, and might constitute potentially valuable data for the development of a new approach in the treatment of this disease.
Assuntos
Conexina 43/metabolismo , Infecções por Coxsackievirus/genética , MicroRNAs/genética , Miocardite/genética , Miocardite/virologia , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Conexina 43/genética , Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Miocardite/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
OBJECTIVE: To explore the relationship between beta-actin mRNA degradation in SD rat's brain, heart and kidney and early postmortem interval (PMI) in order to find new markers for estimating early PMI. METHODS: Rats were sacrificed and kept in the place at a temperature of 20 degrees C. The total RNA were extracted from the brain, heart and kidney at different PMI points. Real time RT-PCR was applied to determine beta-actin mRNA levels in total RNA and the results were given in the form of Ct values. Linear relationships between PMI and Ct values were obtained and the functions of linear regression were established. RESULTS: The great decrease of beta-actin mRNA level were observed in the three organs. The degradation rate was obviously higher in 24 hours after death in the heart and kidney. However, there were no significant changes in the brain. The changes of Ct values and PMI showed a good linear relationship. CONCLUSION: beta-actin mRNA in rat's brain, heart and kidney degrades obviously after death and can be used for estimating early PMI by its degradation rules.
Assuntos
Actinas/metabolismo , Encéfalo/metabolismo , Rim/metabolismo , Miocárdio/metabolismo , Mudanças Depois da Morte , RNA Mensageiro/metabolismo , Actinas/genética , Animais , Medicina Legal/métodos , Masculino , Estabilidade de RNA , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
OBJECTIVE: To study the effects of triptolide-medicated serum on secretory function of adrenocortical cells isolated from rats. METHODS: Thirty SD rats were randomly divided into control group, prednisone group, and low-, medium- and high-dose triptolide groups. Rats were administered with normal saline, prednisone and low-, medium- and high-dose triptolide respectively by gastrogavage to prepare sera containing drugs. Primary adrenocortical cells were isolated from normal male rats and cultured with sera containing drug for 48 hours. Expression of proliferating cell nuclear antigen (PCNA) was observed by immunohistochemical method and number of PCNA-positive cells was counted. Ultrastructure of adrenocortical cells was observed under a transmission electron microscope. Content of corticosterone in supernatant of adrenocortical cell culture was detected by enzyme-linked immunosorbent assay, and real-time fluorescence quantitative polymerase chain reaction (PCR) was employed to investigate the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA. RESULTS: As compared with the control group, content of corticosterone in supernatant of adrenocortical cell culture and expression of 3beta-HSD mRNA were significantly increased in the triptolide-treated groups, and the numbers of PCNA-positive cells were increased in the medium- and high-dose triptolide groups, however, they were decreased in the prednisone group. CONCLUSION: Triptolide-medicated serum can increase the secretion of corticosterone in rat adrenocortical cells in vitro.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Diterpenos/farmacologia , Fenantrenos/farmacologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Animais , Linhagem Celular , Corticosterona/metabolismo , Compostos de Epóxi/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , SoroRESUMO
Status epilepticus (SE) is a neurological disorder associated with high morbidity and mortality rates, and is often difficult to treat. Moreover, the underlying mechanism of SE remains unknown. The lithium-pilocarpine model is a validated animal model that can reproduce the main clinical and neuropathological features of SE. In the present study, this SE model was utilized and SE was successfully established in rats, as determined by the corresponding epileptic electroencephalogram. Histology, immunohistochemistry, western blot analysis and co-immunoprecipitation were used to detect the phosphorylation (p-) of AKT substrate of 40 kDa (PRAS40), the combination of p-PRAS40 and 14-3-3 protein and the activation of the PI3K/mTOR signaling pathway in SE. In addition, the present study analyzed the dynamics of the expression of autophagy-associated factors in the hippocampus after SE induction, and the influence of suppressing the p- of PRAS40 on the autophagy process was detected in the pathogenesis of SE. The results indicated that increased p-PRAS40 expression could activate the mTOR pathway to decrease the level of autophagy. However, inhibition of the mTOR signaling pathway promoted autophagy flux. These results may provide further understanding of p-PRAS40 functions in SE.
RESUMO
Diabetic cardiomyopathy (DCM) is a severe cardiovascular complication of diabetes mellitus (DM). Detecting DCM during the early stages of the disease remains a challenge, as the molecular mechanisms underlying earlystage DCM are not clearly understood. Circular RNA (circRNA), a type of noncoding RNA, has been confirmed to be associated with numerous diseases. However, it is still unclear how circRNAs are involved in earlystage DCM. In the present study, heart tissues harvested from BKSdb/db knockout mice were identified through highthroughput RNA sequencing technology. A total of 58 significantly differentially expressed circRNAs were identified in the db/db sample. Among these, six upregulated circRNAs and seven downregulated circRNAs were detected by reverse transcriptionquantitative PCR and analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Furthermore, based on the predicted binding site with microRNAs (miRNAs) involved in DCM, five circRNAs (mmu_circ_0000652, mmu_circ_0000547, mmu_circ_0001058, mmu_circ_0000680 and novel_circ_0004285) were shown to serve as competing endogenous (ce)RNAs. The corresponding miRNAs and mRNAs of the ceRNAs were also verified, and two promising circRNAmiRNAmRNA regulatory networks were determined. Finally, internal ribosome entry site prediction combined with open reading frame prediction indicated that it was highly possible that mmu_circ_0001160 encoded a protein. In the present study, a comprehensive analysis of the circRNA expression profile during the early phase of DCM was performed, and two promising circRNAmiRNAmRNA regulatory networks were identified. These results lay the foundation for unravelling the underlying pathogenesis of DCM, and highlight potential biomarkers and therapeutic targets for the treatment of DCM at an early stage.