RESUMEN

Formaldehyde, a common indoor air pollutant, is significantly toxic to the respiratory system, whereas its mechanism is unclear. CircRNAs exert critical functions via sponging microRNAs (miRNAs). To evaluate the effect of long-term formaldehyde exposure on rno_circRNA_006061 expression profiles, the downstream targets and signaling pathways associated with rno_circRNA_006061 were predicted and validated using bioinformatics methods and dual-luciferase reporter assay. Previously, our circRNA microarray showed that rno_circRNA_006061 was up-regulated in the formaldehyde-exposed lung tissue. Subsequently, bioinformatics analysis predicted that rno_circRNA_006061 bound to rno-miR-128-3p and co-regulated the p38/ATF3 signaling pathway. Meanwhile, the expressions of rno_circRNA_006061, rno-miR-128-3p and p38 were correlated with the lung histomorphopathological injury assessment. Furthermore, TUNEL and Bax/Bcl-2 ratio results revealed that up-regulated rno_circRNA_00606 induced by formaldehyde stimulated apoptosis in the lung. After the knockdown of rno_circRNA_006061, the expression of rno-miR-128-3p increased and the expression of p38 decreased slightly, which partially restored formaldehyde-induced apoptosis in alveolar epithelial cells. In conclusion, our study hinted that the rno_circRNA_006061 might enhance p38/ATF3 pathway expression via sponging the rno-miR-128-3p, thus significantly promoting apoptosis in lung tissues, which may provide potential new targets for preventing and treating lung injury by formaldehyde inhalation.

Asunto(s)

MicroARNs , ARN Circular , ARN Circular/genética , MicroARNs/metabolismo , Transducción de Señal , Pulmón/metabolismo , Apoptosis

RESUMEN

Oral squamous cell carcinoma (OSCC) accounts for about 90% of oral cancers. Expression of the long noncoding RNA (lncRNA) maternally expressed 3 (MEG3) has previously been reported to be downregulated in OSCC, and its overexpression can inhibit proliferation, migration, and invasion and promote apoptosis of OSCC cells. However, the mechanism underlying MEG3 downregulation in OSCC has not been well characterized. Here we report that low expression of MEG3 is caused by H3K27me3 modification of the MEG3 gene locus, and this is associated with the poor prognosis of OSCC. Overexpression of MEG3 inhibited the proliferation and invasion of OSCC cells. We observed that MEG3 was modified by m6A and bound to YTHDC1. Enhancer-controlled genes positively regulated by MEG3 were functionally enriched for the 'negative regulation of Wnt signaling pathway' term, as determined using metascape. GATA3 was predicted to be a transcription factor for these genes, and was demonstrated to bind to MEG3. Knockdown of GATA3 countered the effects on proliferation, invasion, and increased transcription of HIC1 and PRICKLE1 induced by MEG3 overexpression. In conclusion, our data suggest that MEG3 is downregulated in OSCC due to trimethylation of H3K27 at the MEG3 gene locus. The inhibitory effect of MEG3 on proliferation and invasion of OSCC cells was dependent on the binding of GATA3.

Asunto(s)

Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , ARN Largo no Codificante , Humanos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proliferación Celular/genética , Línea Celular Tumoral , Neoplasias de la Boca/metabolismo , Factor de Transcripción GATA3/genética

RESUMEN

Direct cell reprogramming, also called transdifferentiation, is valuable for cell fate studies and regenerative medicine. Current approaches to transdifferentiation are usually achieved by directly targeting the nuclear functions, such as manipulating the lineage-specific transcriptional factors, microRNAs, and epigenetic modifications. Here, a robust method to convert fibroblasts to neurons through targeting the cytoskeleton followed by exposure to lineage-specification surroundings is reported. Treatment of human foreskin fibroblasts with a single molecule inhibitor of the actomyosin contraction, can disrupt the cytoskeleton, promote cell softening and nuclear export of YAP/TAZ, and induce a neuron-like state. These neuron-like cells can be further converted into mature neurons, while single-cell RNA-seq shows the homogeneity of these cells during the induction process. Finally, transcriptomic analysis shows that cytoskeletal disruption collapses the original lineage expression profile and evokes an intermediate state. These findings shed a light on the underestimated role of the cytoskeleton in maintaining cell identity and provide a paradigm for lineage conversion through the regulation of mechanical properties.

Asunto(s)

Transdiferenciación Celular , Fibroblastos , Diferenciación Celular , Reprogramación Celular , Fibroblastos/fisiología , Humanos , Neuronas

RESUMEN

Fibrosis can occur in almost all tissues and organs and affects normal physiological function, which may have serious consequences, such as organ failure. However, there are currently no effective, broad-spectrum drugs suitable for clinical application. Revealing the process of fibrosis is an important prerequisite for the development of new therapeutic targets and drugs. Studies have shown that the limiting of myofibroblast activation or the promoting of their elimination can ameliorate fibrosis. However, it has not been reported whether a direct decrease in cell contraction can inhibit fibrosis in vivo. Here, we have shown that (-)-blebbistatin (Ble), a non-muscle myosin Ⅱ inhibitor, displayed significant inhibition of liver fibrosis in different chronic injury mouse models in vivo. We found that Ble reduced the stiffness of fibrotic tissues from the early stage, which reduced the extent of myofibroblast activation induced by a stiffer extracellular matrix (ECM). Moreover, Ble also reduced the activation of myofibroblasts induced by TGF-ß1, which is the most potent pro-fibrotic cytokine. Mechanistically, Ble reduced mechanical contraction, which inhibited the assembly of stress fibers, decreased the F/G-actin ratio, and led to the exnucleation of YAP1 and MRTF-A. Finally, we verified its broad-spectrum antifibrotic effect in multiple models of organ fibrosis. Our results highlighted the important role of mechanical contraction in myofibroblast activation and maintenance, rather than just a characteristic of activation, suggesting that it may be a potential target to explore broad-spectrum drugs for the treatment of fibrotic diseases.

RESUMEN

Objective: 1,3-Dioleoyl-2-palmitoylglycerol (OPO) is an ideal structured triglyceride for infant formula, with a similar structure to human milk fat. We conducted this randomized, double-blind controlled, single-center trial to evaluate the effects of an OPO formula in infants. Study Design: One hundred seventy-four healthy term infants <14 days old were assigned to the standard formula-fed group (n = 55), high sn-2 palmitic acid (OPO) formula-fed infants (n = 58), and breastfed (BF) group (n = 61). The primary endpoint was the total saponified fatty acid content in feces at week 6 and week 12. Results: Infants from the OPO group had lower concentrations of fecal saponified fatty acids than those from the standard formula group (p < 0.0001) at week 6 and week 12. The frequencies of crying per day and per night of infants in the OPO group were significantly less than those of infants in the standard formula group (p < 0.0001). After 12 weeks of feeding, the length of infants was significantly higher in the OPO group than in the other two groups (p = 0.002). Infants in the OPO group had a significantly lower stool calcium concentration and a higher stool frequency per day than infants in the standard formula group. Conclusion: In summary, a high concentration of OPO in formula is beneficial to the growth and development of infants.

RESUMEN

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key accelerated aging phenotypes. Restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells. We revealed an epigenetic-metabolism axis contributing to aging and potentially to antiaging therapy.

RESUMEN

Meiosis with a single round of DNA replication and two successive rounds of chromosome segregation requires specific cyclins associated with cyclin-dependent kinases (CDKs) to ensure its fidelity. But how cyclins control the distinctive meiosis is still largely unknown. In this study, we explored the role of cyclin B3 in female meiosis by generating Ccnb3 mutant mice via CRISPR/Cas9. Ccnb3 mutant oocytes characteristically arrested at metaphase I (MetI) with normal spindle assembly and lacked enough anaphase-promoting complex/cyclosome (APC/C) activity, which is spindle assembly checkpoint (SAC) independent, to initiate anaphase I (AnaI). Securin siRNA or CDK1 inhibitor supplements rescued the MetI arrest. Furthermore, CCNB3 directly interacts with CDK1 to exert kinase function. Besides, the MetI arrest oocytes had normal development after intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA), along with releasing the sister chromatids, which implies that Ccnb3 exclusively functioned in meiosis I, rather than meiosis II. Our study sheds light on the specific cell cycle control of cyclins in meiosis.

Asunto(s)

Anafase/fisiología , Segregación Cromosómica , Ciclina B/fisiología , Cinetocoros/fisiología , Meiosis/fisiología , Metafase/fisiología , Oocitos/fisiología , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Desarrollo Embrionario , Femenino , Puntos de Control de la Fase M del Ciclo Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Oocitos/citología

RESUMEN

Trophoblast stem (TS) cells are increasingly used as a model system for studying placentation and placental disorders. However, practical limitations of genetic manipulation have posed challenges for genetic analysis using TS cells. Here, we report the generation of mouse parthenogenetic haploid TS cells (haTSCs) and show that supplementation with FGF4 and inhibition of Rho-associated protein kinase (ROCK) enable the maintenance of their haploidy and developmental potential. The resulting haTSCs have 20 chromosomes, exhibit typical expression features of TS cells, possess the multipotency to differentiate into specialized trophoblast cell types, and can chimerize E13.5 and term placentas. We also demonstrate the capability of the haTSCs to undergo genetic manipulation and facilitate genome-wide screening in the trophoblast lineage. We expect that haTSCs will offer a powerful tool for studying functional genomics and placental biology.

Asunto(s)

Células Madre Embrionarias/citología , Haploidia , Trofoblastos/citología , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Femenino , Factor 4 de Crecimiento de Fibroblastos/farmacología , Cariotipo , Ratones , Cultivo Primario de Células/métodos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo

Asunto(s)

Blastómeros/citología , Ratones/embriología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Blastómeros/metabolismo , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Ratones/genética , Ratones/metabolismo , Ratones Noqueados , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo

RESUMEN

Unisexual reproduction is widespread among lower vertebrates, but not in mammals. Deletion of the H19 imprinted region in immature oocytes produced bimaternal mice with defective growth; however, bipaternal reproduction has not been previously achieved in mammals. We found that cultured parthenogenetic and androgenetic haploid embryonic stem cells (haESCs) display DNA hypomethylation resembling that of primordial germ cells. Through MII oocyte injection or sperm coinjection with hypomethylated haploid ESCs carrying specific imprinted region deletions, we obtained live bimaternal and bipaternal mice. Deletion of 3 imprinted regions in parthenogenetic haploid ESCs restored normal growth of fertile bimaternal mice, whereas deletion of 7 imprinted regions in androgenetic haploid ESCs enabled production of live bipaternal mice that died shortly after birth. Phenotypic analyses of organ and body size of these mice support the genetic conflict theory of genomic imprinting. Taken together, our results highlight the factors necessary for crossing same-sex reproduction barriers in mammals.

Asunto(s)

Metilación de ADN/genética , Haploidia , Células Madre Embrionarias de Ratones/metabolismo , Animales , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Fenotipo

RESUMEN

The recent success of derivation of mammalian haploid embryonic stem cells (haESCs) has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons. Using piggyBac transposon-based insertional mutagenesis, we generated a haploid neural cell library harboring genome-wide mutations for genetic screening. As a proof of concept, we screened for Mn2+-mediated toxicity and identified the Park2 gene. Our findings expand the applications of mouse haploid cell technology to somatic cell types and may also shed light on the mechanisms of ploidy maintenance.

Asunto(s)

Pruebas Genéticas , Genoma , Haploidia , Amidas/farmacología , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Diferenciación Celular/efectos de los fármacos , Diploidia , Ratones , Mitosis/efectos de los fármacos , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Páncreas/citología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo

Asunto(s)

Reprogramación Celular , Regulación de la Expresión Génica , Proteínas Represoras/genética , Animales , Proteínas Cromosómicas no Histona , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

BACKGROUND: Several studies have reported the favorable effect of leucine supplementation on insulin resistance or insulin sensitivity. However, whether or not leucine supplementation improves leptin sensitivity remains unclear. DESIGN: Forty-eight male Sprague-Dawley rats were fed with either a high-fat diet (HFD) or HFD supplemented with 1.5, 3.0, and 4.5% leucine for 16 weeks. At the end of the experiment, serum leptin level was measured by ELISA, and leptin receptor (ObR) in the hypothalamus was examined by immunohistochemistry. The protein expressions of ObR and leptin-signaling pathway in adipose tissues were detected by western blot. RESULTS: No significant differences in body weight and food/energy intake existed among the four groups. Serum leptin levels were significantly lower, and ObR expression in the hypothalamus and adipose tissues was significantly higher in the three leucine groups than in the control group. These phenomena suggested that leptin sensitivity was improved in the leucine groups. Furthermore, the expressions of JAK2 and STAT3 (activated by ObR) were significantly higher, and that of SOCS3 (inhibits leptin signaling) was significantly lower in the three leucine groups than in the control group. CONCLUSIONS: Leucine supplementation improves leptin sensitivity in rats on HFD likely by promoting leptin signaling.

RESUMEN

OBJECTIVE: To investigate the changes in the expression of phospholipase C-gamma1tyr783 (PLC-γ1tyr783) in the condylar cartilage of a young rat during functional mandibular protraction. This work also explores the function of PLC-γ1tyr783 in the rat mandibular condylar cartilage bone remodeling, which could provide experimental evidence for clinical bone ortho- pedic work. METHODS: A total of 60 four-week-old male Sprague-Dawley (SD) rats were used in this study. The rats were divided equally and randomly into experimental group and control group. The functional appliances that were fitted to the upper incisors of the animals in the experimental group were worn 24 h a day after the rats were fed for 7 d with homemade pellet feed. The animals in the experimental group, along with their matched controls, were sacrificed after 1, 3, 7, 14, 21, and 28 d. The bilateral condylar was fixed, decalcified, dehyded, and then conventional paraffin embedded. Immunohisto- chemistry of PLC-γ1tyr783 was applied to observe its express distribution and variation. RESULTS: The expression of PLC-γ1tyr783 decreased gradually in the control group, which showed age-related changes (P > 0.05). On the 14th day, PLC-γ1tyr783 expres- sion in the experimental group was significantly higher than that in the control group. PLC-γ1tyr783 expression began to appear statistically and significantly different between the two groups (P < 0.01). CONCLUSION: PLC-γ1tyr783 is involved in the bone remodeling process of the rat condylar cartilage after functional mandibular-protraction.

Asunto(s)

Remodelación Ósea , Cóndilo Mandibular , Fosfolipasa C gamma , Animales , Cartílago , Masculino , Hidrolasas Diéster Fosfóricas , Ratas , Ratas Sprague-Dawley

RESUMEN

<p><b>OBJECTIVE</b>To investigate the changes in the expression of phospholipase C-gamma1tyr783 (PLC-γ1tyr783) in the condylar cartilage of a young rat during functional mandibular protraction. This work also explores the function of PLC-γ1tyr783 in the rat mandibular condylar cartilage bone remodeling, which could provide experimental evidence for clinical bone ortho- pedic work.</p><p><b>METHODS</b>A total of 60 four-week-old male Sprague-Dawley (SD) rats were used in this study. The rats were divided equally and randomly into experimental group and control group. The functional appliances that were fitted to the upper incisors of the animals in the experimental group were worn 24 h a day after the rats were fed for 7 d with homemade pellet feed. The animals in the experimental group, along with their matched controls, were sacrificed after 1, 3, 7, 14, 21, and 28 d. The bilateral condylar was fixed, decalcified, dehyded, and then conventional paraffin embedded. Immunohisto- chemistry of PLC-γ1tyr783 was applied to observe its express distribution and variation.</p><p><b>RESULTS</b>The expression of PLC-γ1tyr783 decreased gradually in the control group, which showed age-related changes (P > 0.05). On the 14th day, PLC-γ1tyr783 expres- sion in the experimental group was significantly higher than that in the control group. PLC-γ1tyr783 expression began to appear statistically and significantly different between the two groups (P < 0.01).</p><p><b>CONCLUSION</b>PLC-γ1tyr783 is involved in the bone remodeling process of the rat condylar cartilage after functional mandibular-protraction.</p>

Asunto(s)

Animales , Masculino , Ratas , Remodelación Ósea , Cartílago , Cóndilo Mandibular , Fosfolipasa C gamma , Hidrolasas Diéster Fosfóricas , Ratas Sprague-Dawley