RESUMO
Transgenic animals have been established for studying gene function, improving animals' production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken ß-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.
Assuntos
Metilação de DNA , Regiões Promotoras Genéticas , Transgenes/genética , Actinas/genética , Actinas/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Feminino , Expressão Gênica , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Homozigoto , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Transgênicos , Análise de Sequência de DNA , DNA Metiltransferase 3BRESUMO
Ovarian cancer (OC) causes more deaths than any other gynecological cancer. Many cellular pathways have been elucidated to be associated with OC development and progression. Specifically, the insulin-like growth factor 1 receptor/insulin receptor substrate 1 (IGF1R/IRS1) pathway participates in OC development. Moreover, accumulating evidence has shown that microRNA deregulation contributes to tumor initiation and progression. Here, our study aimed to investigate the molecular functions and regulatory mechanisms of miR-150, specifically, in OC. We found that the expression of miR-150-5p/3p and their precursor, mir-150, was downregulated in OC tissues; lower mir-150 levels were associated with poor OC patient outcomes. Ectopic mir-150 expression inhibited OC cell growth and metastasis in vitro and in vivo. Furthermore, both IRS1 and IGF1R were confirmed as direct targets of miR-150-5p/3p, and the miR-150-IGF1R/IRS1 axis exerted antitumor effects via the PI3K/AKT/mTOR pathway. Forkhead box protein 3 (FoxP3) positively regulated the expression of miR-150-5p/3p by binding to the mir-150 promoter. In turn, the PI3K/AKT/mTOR pathway downregulated FoxP3 and miR-150-5p/3p. Taken together, these findings indicate that a complex FoxP3-miR-150-IGF1R/IRS1-PI3K/AKT/mTOR feedback loop regulates OC pathogenesis, providing a novel mechanism for miR-150 as a tumor suppressor miRNA in OC.
Assuntos
Movimento Celular , Proliferação de Células , Fatores de Transcrição Forkhead/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , MicroRNAs/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Carga TumoralRESUMO
By collecting daily data on measles cases, air pollutants, and meteorological data from 2005 to 2009 in Chengguan District of Lanzhou City, semi-parametric generalized additive model (GAM) was used to quantitatively study the impact of air pollutants and meteorological factors on daily measles cases. The results showed that air pollutants and meteorological factors had effect on the number of daily measles cases, and there was a certain lag effect. Except for SO2 and relative humidity, other factors showed statistically significant associations with daily measles cases: NO2 lag 6 days, PM10 and maximum temperature lag 5 days, minimum temperature and average temperature and average air pressure lag 4 days, visibility, and wind speed lag 3 days had the greatest impact on the number of daily measles cases. Under the optimum lag conditions, the number of daily measles cases increased by 15.1%, 17.6%, 7.0%, 116.6%, 98.6%, 85.7%, and 14.4% with the increase of 1 IQR in SO2, NO2, PM10, maximum temperature, minimum temperature, average temperature, and wind speed; with the increase of 1 IQR in average pressure, relative humidity, visibility, and daily measles cases decreased by 12.8%, 9.7%, and 13.1%, respectively. And different factors showed different seasonal effects. The effects of SO2 and temperature factors on daily measles cases were greater in spring and winter, but PM10 in summer.
Assuntos
Poluentes Atmosféricos/análise , Poluição do Ar/análise , Sarampo , China , Cidades , Humanos , Conceitos Meteorológicos , Material Particulado/análiseRESUMO
Cancer immune surveillance is an important host protection process that inhibits carcinogenesis and maintains cellular homeostasis. The major histocompatibility complex class I-related molecules A and B (MICA and MICB) are NKG2D ligands that play important roles in tumor immune surveillance. In the present study, by a combined bioinformatics prediction and experimental approach, we identify BCL11B 3'-UTR as a putative MICA and MICB ceRNA. We demonstrate in several human cell lines of different origins that the knockdown of BCL11B downregulates surface expression of MICA and MICB. Furthermore, we demonstrate miRNA dependency of BCL11B-mediated MICA and MICB regulation in Dicer knockdown HCT116 cells. In addition, MICA/B-targeting miRNAs (miR-17, miR-93, miR-20a, miR-20b, miR-106a, and miR-106b) repressed the expression of BCL11B by targeting its 3'-UTR. Moreover, we showed that the BCL11B knockdown-mediated downregulation of MICA/B resulted in reduced NK cell elimination in vitro and in vivo through reduced recognition of NKG2D. Of particular significance, BCL11B displays tumor-suppressive properties. The expression of BCL11B is downregulated in colon cancer tissues and associated with a reduced median survival of colon cancer patients. Taken together, our study revealed a new mechanism of BCL11B that prevents immune evasion of cancerous cells by upregulation of the NKG2D ligands MICA and MICB in a ceRNA manner.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunidade/genética , Antígenos de Histocompatibilidade Menor/metabolismo , RNA/genética , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Técnicas de Silenciamento de Genes , Células HCT116 , Células HT29 , Humanos , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer-related death worldwide. Increasing evidence indicates that the deregulation of long noncoding RNAs (lncRNAs) contributes to tumor initiation and progression; however, little is known about the biological role of cancer susceptibility candidate 9 (CASC9) in CRC. METHODS: Novel lncRNAs potentially involved in CRC tumorigenesis were identified from datasets downloaded from The Cancer LncRNome Atlas and The Atlas of Noncoding RNAs in Cancer. The CRC cell lines HCT-116, HCT-116 p53-/-, SW620, SW480, HT-29, LoVo, LS-174T, and RKO were used. Colony-formation, MTS, cell-cycle, apoptosis, and in-vivo tumorigenesis assays were used to determine the role of CASC9 in CRC cell growth in vitro and in vivo. Potential interaction between CASC9 and cleavage and polyadenylation specificity factor subunit 3 (CPSF3) was evaluated using RNA immunoprecipitation and RNA-protein pull-down assays. RNA-sequencing was performed to analyze gene expression following CASC9 knockdown. RT-qPCR, western blotting, and mRNA decay assays were performed to study the mechanisms involved. RESULTS: CASC9 was frequently upregulated in CRC, which was correlated with advanced TNM stage, and higher CASC9 levels were associated with poor patient outcomes. Knockdown of CASC9 inhibited growth and promoted apoptosis in CRC cells, whereas ectopic CASC9 expression promoted cell growth in vitro and in vivo. We demonstrated that CPSF3 is a CASC9-interacting protein, and knockdown of CPSF3 mimicked the effects of CASC9 knockdown in CRC cells. Furthermore, we found that CASC9 exerts its oncogenic activity by modulating TGFß2 mRNA stability and upregulating the levels of TGFß2 and TERT, resulting in an increase in phosphorylated SMAD3 and activation of TGF-ß signaling, and enhanced TERT complex function in CRC cells. Finally, CPSF3 was significantly upregulated in CRC tissues as compared with adjacent or non-adjacent normal colon tissues, and CASC9, CPSF3, and TGFß2 levels in human CRC tissues were positively correlated. CONCLUSIONS: CASC9 is a promising prognostic predictor for patients with CRC and the CASC9-CPSF3-TGFß2 axis is a potential therapeutic target for CRC treatment.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Biomarcadores Tumorais , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Interferência de RNA , Carga TumoralRESUMO
Although increasing evidence indicated that deregulation of microRNAs (miRNAs) contributed to tumor initiation and progression, but little is known about the biological role of miR-340 in ovarian cancer (OC). In this study, we found that miR-340 expression was downregulated in OC tissues compared with its expression in normal ovarian epithelium and endometrium, and treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) or trichostatin A (TSA) increased miR-340 expression in OC cells. In addition, ectopic miR-340 expression inhibited OC cell growth and metastasis in vitro and in vivo. Four and a half LIM domains protein 2 (FHL2) was confirmed as a direct target of miR-340 and silencing FHL2 mimicked the effects of miR-340 in OC cells. Further mechanistic study showed that miR-340 inhibited the Wnt/ß-catenin pathway by targeting FHL2, as well as downstream cell cycle and epithelial-to-mesenchymal transition (EMT) signals in OC cells. Moreover, the greatest association between miR-340 and FHL2 was found in 481 ovarian serous cystadenocarcinoma tissues via pan-cancer analysis. Finally, we revealed that lower miR-340 or higher FHL2 was associated with poor OC patient outcomes. Our findings indicate that the miR-340-FHL2 axis regulates Wnt/ß-catenin signaling and is involved in tumorigenesis in OC. Therefore, manipulating the expression of miR-340 or its target genes is a potential strategy in OC therapy.
Assuntos
Proliferação de Células , Proteínas com Homeodomínio LIM/metabolismo , MicroRNAs/metabolismo , Proteínas Musculares/metabolismo , Neoplasias Ovarianas/patologia , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Movimento Celular , Cistadenocarcinoma/metabolismo , Cistadenocarcinoma/patologia , Regulação para Baixo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Nus , MicroRNAs/química , MicroRNAs/genética , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Neoplasias Ovarianas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Via de Sinalização WntRESUMO
DNA methylation plays a very important role in the regulation of gene expression. Under general situations, methylation in a gene promoter region is frequently accompanied by transcriptional suppression, and those genes that are highly methylated display the phenomenon of low expression. In contrast, those genes whose methylation level is low display the phenomenon of active expression. In this study, we conducted DNA methylation analysis on the CpG sites within the promoter regions of five adipose tissue-specific transcriptional factors-Adiponectin, Chemerin, Leptin, Smaf-1, and Vaspin-and examined their messenger RNA (mRNA) expression levels in different mouse tissues. We also performed analyses on the correlation between the DNA methylation levels of these genes and their mRNA expression levels in these tissues. The correlation coefficient for Leptin was the highest, and it displayed a high expression in an adipose tissue-specific manner. Thus, we cloned the regulatory region of Leptin gene and incorporated its promoter into the eukaryotic expression vector pEGFP-N1 and constructed a recombinant plasmid named pEGFP-N1-(p-Lep). This recombinant plasmid was first verified by DNA sequencing and then transfected into mouse pre-adipocytes via electroporation. Measurement of the activity of luciferase (reporter) indicated that p-Lep was capable of driving the expression of the reporter gene. This study has paved a solid basis for subsequent studies on generating transgenic animals.