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Objective: This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Materials and methods: Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. Results: XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory responsein HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. Conclusion: LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.
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Retinopatia Diabética , MicroRNAs , Neovascularização Patológica , RNA Longo não Codificante , Fator A de Crescimento do Endotélio Vascular , RNA Longo não Codificante/genética , Retinopatia Diabética/genética , Retinopatia Diabética/sangue , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Ratos , Humanos , Masculino , Neovascularização Patológica/genética , Ratos Sprague-Dawley , Feminino , Movimento Celular/genética , Proliferação de Células/genética , Pessoa de Meia-Idade , Diabetes Mellitus ExperimentalRESUMO
ABSTRACT Objective: This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Materials and methods: Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. Results: XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory response in HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. Conclusions: LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.
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Abstract Objective This study aims to explore the regulatory mechanism of long noncoding RNA X inactive specific transcript (lncRNA XIST) in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were obtained from freshly extracted third molars and identified by flow cytometry. Methodology Odontogenic differentiation was induced in mineralized culture medium, and hDPSCs were infected with shRNA lentivirus targeting XIST or fused in sarcoma (FUS), followed by detection of alkaline phoshpatase (ALP) activity, alizarin red staining of mineralized nodules, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) quantification of XIST expression, and Western blot analysis of FUS, ZBTB16, and odontogenic differentiation markers (DSPP and DMP1). IF-FISH was performed to detect the cellular localization of XIST and FUS. RIP assay validated the XIST and FUS binding. ZBTB16 mRNA stability was tested after actinomycin D treatment. hDPSCs were infected with oe-ZBTB16 lentivirus and further treated with sh-XIST for a combined experiment. Results LncRNA XIST was highly expressed in hDPSCs with odontogenic differentiation. Downregulation of XIST or FUS weakened the ALP activity of hDPSCs, reduced mineralized nodules, diminished DSPP and DMP1 expressions. XIST binds to FUS to stabilize ZBTB16 mRNA and promote ZBTB16 expression. ZBTB16 overexpression partially reversed the inhibitory effect of XIST silencing on odontogenic differentiation of hDPSCs. Conclusion In conclusion, XIST stabilizes ZBTB16 mRNA and promotes ZBTB16 expression by binding to FUS, thereby facilitating the odontogenic differentiation of hDPSCs.
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Long non-coding RNAs (lncRNAs) are non-coding RNAs that contain more than 200 nucleotides but do not code for proteins. In tumorigenesis, lncRNAs can have both oncogenic and tumor-suppressive properties. X inactive-specific transcript (XIST) is a known lncRNA that has been implicated in X chromosome silencing in female cells. Dysregulation of XIST is associated with an increased risk of various cancers. Therefore, XIST can be a beneficial prognostic biomarker for human malignancies. In this review, we attempt to summarize the emerging roles of XIST in human cancers.
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MicroRNAs , Neoplasias , RNA Longo não Codificante , Humanos , Feminino , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro , Neoplasias/genética , CarcinogêneseRESUMO
The long non-coding RNA X inactivate-specific transcript (lncRNA XIST) has been verified as an oncogenic gene in non-small cell lung cancer (NSCLC) whose regulatory role is largely unknown. The important tumor suppressors, microRNAs: miR-449a and miR-16 are regulated by lncRNA XIST in NSCLC, these miRNAs share numerous common targets and experimental evidence suggests that they synergistically regulate the cell-fate regulation of NSCLC. LncRNA XIST is known to sponge miR-449a and miR-34a, however, the regulatory network connecting all these non-coding RNAs is still unknown. Here we propose a Boolean regulatory network for the G1/S cell cycle checkpoint in NSCLC contemplating the involvement of these non-coding RNAs. Model verification was conducted by comparison with experimental knowledge from NSCLC showing good agreement. The results suggest that miR-449a regulates miR-16 and p21 activity by targeting HDAC1, c-Myc, and the lncRNA XIST. Furthermore, our circuit perturbation simulations show that five circuits are involved in cell fate determination between senescence and apoptosis. The model thus allows pinpointing the direct cell fate mechanisms of NSCLC. Therefore, our results support that lncRNA XIST is an attractive target of drug development in tumor growth and aggressive proliferation of NSCLC, and promising results can be achieved through tumor suppressor miRNAs.
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PURPOSE: Diffuse intrinsic pontine gliomas (DIPGs) are the most fatal primary brainstem tumors in pediatric patients. The identification of new molecular features, mediating their formation and progression, as non-coding RNAs (ncRNAs), would be of great importance for the development of effective treatments. METHODS: We analyzed the DIPGs transcriptome with the HTA2.0 array and it was compared with pediatric non-brainstem astrocytoma expression profiles (GSE72269). RESULTS: More than 50% of the differentially expressed transcripts were ncRNAs and based on this, we proposed a DIPGs ncRNA signature. LncRNAs XIST and XIST-210, and the HBII-52 and HBII-85 snoRNA clusters were markedly downregulated in DIPGs. qPCR assays demonstrated XIST downregulation in all non-brainstem astrocytomas, in a gender, age, and brain location-independent manner, as well as in DIPGs affecting boys; however, DIPGs affecting girls showed both downregulation and upregulation of XIST. Girls' with longer survival positively correlated with XIST expression. CONCLUSIONS: The involvement of ncRNAs in DIPGs is imminent and their expression profile is useful to differentiate them from non-neoplastic tissues and non-brain stem astrocytomas, which suggests their potential use as DIPG biomarkers. In fact, XIST and XIST-210 are potential DIPG prognostic biomarkers.
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Biomarcadores Tumorais/metabolismo , Neoplasias do Tronco Encefálico/diagnóstico , Glioma Pontino Intrínseco Difuso/diagnóstico , RNA não Traduzido/metabolismo , Transcriptoma , Adolescente , Fatores Etários , Processamento Alternativo , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias do Tronco Encefálico/diagnóstico por imagem , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/mortalidade , Criança , Pré-Escolar , Bases de Dados Genéticas , Glioma Pontino Intrínseco Difuso/diagnóstico por imagem , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/mortalidade , Regulação para Baixo , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , RNA Nucleolar Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Regulação para CimaRESUMO
BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1). METHODS: The wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay. RESULTS: The expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function. CONCLUSION: XIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.
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Queimaduras/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , RNA Longo não Codificante/metabolismo , Western Blotting , Queimaduras/genética , Movimento Celular , Proliferação de Células , Matriz Extracelular/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
XIST, in association with the shorter ncRNA RepA, are essential for the initiation of X chromosome inactivation (XCI) in mice. The molecular mechanisms controlling XIST and RepA expression are well characterized in that specie. However, little is known in livestock. We aimed to characterize the DNA methylation status along the 5' portion of XIST and to characterize its transcriptional profile during early development in cattle. Three genomic regions of XIST named here as promoter, RepA and DMR1 had their DNA methylation status characterized in gametes and embryos. Expression profile of XIST was evaluated, including sense and antisense transcription. Oocytes showed higher levels of methylation than spermatozoa that was demethylated. DMR1 was hypermethylated throughout oogenesis. At the 8-16-cell embryo stage DMR1 was completed demethylated. Interestingly, RepA gain methylation during oocyte maturation and was demethylated at the blastocyst stage, later than DMR1. These results suggest that DMR1 and RepA are transient differentially methylated regions in cattle. XIST RNA was detected in matured oocytes and in single cells from the 2-cell to the morula stage, confirming the presence of maternal and embryonic transcripts. Sense and antisense transcripts were detected along the XIST in blastocyst. In silico analysis identified 63 novel transcript candidates at bovine XIST locus from both the plus and minus strands. Taking together these results improve our understanding of the molecular mechanisms involved in XCI initiation in cattle. This information may be useful for the improvement of assisted reproductive technologies in livestock considering that in vitro conditions may impair epigenetic reprogramming.
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Biomarcadores/análise , Metilação de DNA , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Oogênese/genética , RNA Longo não Codificante/genética , Animais , Bovinos , Embrião de Mamíferos/citologia , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Técnicas In Vitro , Oócitos/citologia , Oócitos/metabolismo , Regiões Promotoras Genéticas , Análise de Célula ÚnicaRESUMO
Background: Long noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1). Methods: The wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay. Results: The expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function. Conclusion: XIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.
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We report on a 16-year-old boy with a maternally inherited ~ 18.3 Mb Xq13.2-q21.31 duplication delimited by aCGH. As previously described in patients with similar duplications, his clinical features included intellectual disability, developmental delay, speech delay, generalized hypotonia, infantile feeding difficulties, self-injurious behavior, short stature and endocrine problems. As additional findings, he presented recurrent seizures and pubertal gynecomastia. His mother was phenotypically normal and had completely skewed inactivation of the duplicated X chromosome, as most female carriers of such duplications. Five previously reported patients with partial Xq duplications presented duplication breakpoints similar to those of our patient. One of them, a fetus with multiple congenital abnormalities, had the same cytogenetic duplication breakpoint. Three of the reported patients shared many features with our proband but the other had some clinical features of the Prader-Willi syndrome. It was suggested that ATRX overexpression could be involved in the major clinical features of patients with partial Xq duplications. We propose that this gene could also be involved with the obesity of the patient with the Prader-Willi-like phenotype. Additionally, we suggest that the PCDH11X gene could be a candidate for our patient's recurrent seizures. In males, the Xq13-q21 duplication should be considered in the differential diagnosis of Prader-Willi syndrome, as previously suggested, and neuromuscular diseases, particularly mitochondriopathies.
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DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.
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Humanos , Metilação de DNA/genética , Repressão Epigenética/genética , Genoma Humano , Genoma/genética , Impressão Genômica/genética , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , /genética , Metilação de DNA/efeitos dos fármacos , Técnicas de Inativação de Genes , Genoma Humano/efeitos dos fármacos , Hibridização in Situ Fluorescente/métodos , Análise em Microsséries , Polimorfismo de Nucleotídeo Único , Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Fatores epigenéticos são responsáveis por controlar a expressão de genes e a diferenciação durante a vida celular. Entre eles, a metilação do DNA é o mais estudado. Cada tipo celular possui um padrão epigenético altamente especializado. Após a fecundação natural ou a clonagem por transferência nuclear (SCNT), um padrão epigenético preexistente deve ser apagado e restabelecido para garantir o correto desenvolvimento embrionário. Porém, nos clones, essa reprogramação é ineficiente, mas é possível que o uso de substâncias desmetilantes de DNA utilizadas durante o cultivo celular de células doadoras de núcleo possa desmetilar o DNA, aumentando a eficiência da técnica. (AU)
Epigenetic factors are responsible for controlling gene expression and differentiation through the cell life. Among them, DNA methylation is the most studied. Each cell type possesses a specific and highly specialized epigenetic pattern. After natural fertilization or cloning by somatic cell nuclear transfer (SCNT), a pre-existing epigenetic pattern must be erased and reestablished to ensure correct embryo development. However, in cloning this reprogramming is inefficient and it is possible that the use of a DNA demethylating substance can improve the efficiency of the technique. (AU)
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Epigênese Genética/genética , Metilação de DNA/genética , Clonagem de Organismos , Desenvolvimento Embrionário/genéticaRESUMO
Fatores epigenéticos são responsáveis por controlar a expressão de genes e a diferenciação durante a vida celular. Entre eles, a metilação do DNA é o mais estudado. Cada tipo celular possui um padrão epigenético altamente especializado. Após a fecundação natural ou a clonagem por transferência nuclear (SCNT), um padrão epigenético preexistente deve ser apagado e restabelecido para garantir o correto desenvolvimento embrionário. Porém, nos clones, essa reprogramação é ineficiente, mas é possível que o uso de substâncias desmetilantes de DNA utilizadas durante o cultivo celular de células doadoras de núcleo possa desmetilar o DNA, aumentando a eficiência da técnica.
Epigenetic factors are responsible for controlling gene expression and differentiation through the cell life. Among them, DNA methylation is the most studied. Each cell type possesses a specific and highly specialized epigenetic pattern. After natural fertilization or cloning by somatic cell nuclear transfer (SCNT), a pre-existing epigenetic pattern must be erased and reestablished to ensure correct embryo development. However, in cloning this reprogramming is inefficient and it is possible that the use of a DNA demethylating substance can improve the efficiency of the technique.