RESUMEN
PURPOSE: Long-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation. METHODS: miR-92a and EndMT-related proteins detected by qRT-PCR and Western blot in vitro and in vivo. Adeno-associated virus 6 (AAV6) delivery gene therapy was used to inhibit neointimal formation in vivo. The intimal hyperplasia of vein grafts was measured by HE staining, the expression of EndMT-related protein in vein grafts was measured by immunofluorescence. Immunohistochemistry and luciferase assay were used to detect potential targets of miR-92a. RESULTS: The expression of miR-92a was found to be upregulated in neointimal hyperplasic lesions after vein grafting. Using cultured human umbilical vein endothelial cells (HUVECs), we show that TGF-ß1 treatment of HUVECs significantly increased miR-92a expression and induced EndMT, characterized by suppression of endothelial-specific markers (CD31 and VE-cadherin) and an increase in mesenchymal-specific markers (a-SMA and vimentin), while inhibition of miR-92a expression blunted EndMT in cultured HUVECs. Furthermore, AAV6 mediated miR-92a suppression gene therapy effectively resulted in decreased EndMT and less neointimal formation in vein grafts in vivo. We further identified that integrin alpha 5 (ITGA5) is a potential target gene involved in the development of neointima formation in these vein grafts. CONCLUSION: This data suggests that neointimal formation does not solely rely on vascular smooth muscle cell phenotypic switching but is also related to EndMT, and miR-92a-mediated EndMT is an important mechanism underlying neointimal formation in vein grafts.
Asunto(s)
Endotelio/metabolismo , MicroARNs/metabolismo , Neointima/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , MicroARNs/genética , Neointima/patología , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes. RESULTS: Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1-/- cells and attenuated cytokine-induced cell death in FAT10-/- clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones. CONCLUSION: Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Sistemas CRISPR-Cas , Proteínas Portadoras/genética , Reparación del ADN , Técnicas de Sustitución del Gen/métodos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Ubiquitinas/genética , Línea Celular , Endodesoxirribonucleasas , Mutagénesis InsercionalRESUMEN
CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.
Asunto(s)
Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Reparación del ADN por Unión de Extremidades/genética , Genes Reporteros/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Reparación del ADN por Recombinación/genética , Línea Celular Tumoral , ADN/genética , Roturas del ADN de Doble Cadena , Edición Génica/métodos , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/genética , Células HCT116 , Células HEK293 , Células Madre Embrionarias Humanas/citología , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.
Asunto(s)
Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Células Cultivadas , Silenciador del Gen , Humanos , Ratones , Proteínas Recombinantes de Fusión/química , Factores de Transcripción/genética , Factores de Transcripción p300-CBP/metabolismo , ARN Pequeño no TraducidoRESUMEN
OBJECTIVE: To investigate the role of myosin light chain kinase (MLCK) in intestinal barrier function in a mouse model with nonalcoholic steatohepatitis (NASH). METHODS: The C57BL/6 mice were randomly divided into five groups including control group, nonalcoholic fatty liver (NAFL) group, NAFL administrated with MLCK inhibitor ML-7 group, nonalcoholic steatohepatitis (NASH) group, NASH administrated with ML-7 group. Plasma ALT and AST were tested. The degree of liver steatosis was assessed by hematoxylin-eosin staining on liver tissue sections.Intestinal mucosal tight junction was observed by electron microscope. The expression of MLCK on intestinal mucosa was detected by immunohistochemistry staining. The level of lipopolysaccharide (LPS) in portal vein was determined by enzyme linked immune sorbent assay (ELISA). The protein and mRNA expression of inflammatory cytokines in liver tissue were tested using ELISA and real-time PCR. RESULTS: MLCK expression in intestinal mucosa was increased in NASH group compared with control group (P<0.01). The tight junctions of intestinal barrier were disrupted in NASH group and intercellular space was larger than control group [(26.60 ± 1.20) nm vs (14.90 ± 0.33) nm, P<0.05], which were improved after ML-7 administration [(14.9 0 ± 0.67) nm]. The LPS in portal vein was higher in NASH group than control group [(7.260 ± 3.184) U/L vs (2.962 ± 0.845) U/L, P<0.05], suggesting that the permeability of intestinal barrier was impaired, however the level of LPS was reduced by ML-7 [(3.772 ± 1.033) U/L, P<0.05]. ALT and AST in plasma, TNFα and IL-6 in liver tissue, the mRNA levels of TNFα and NF-κB in liver tissue were all elevated in NASH group compared with control group (all P<0.05), which were reduced by MLCK inhibitor ML-7. CONCLUSION: Epithelia MLCK probably plays a role in intestinal barrier impairment, which is critical to the pathogenesis of NASH.
Asunto(s)
Azepinas/metabolismo , Modelos Animales de Enfermedad , Mucosa Intestinal/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Naftalenos/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Animales , Hígado Graso , Interleucina-6 , Intestinos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Inhibidores de la Síntesis de la Proteína , ARN Mensajero , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfaRESUMEN
Electronic cigarettes (e-cigarettes) have rapidly gained popularity as alternatives to traditional combustible cigarettes. However, their long-term health impact remains uncertain. This study aimed to investigate the effects of chronic exposure to e-cigarette aerosol (ECA) in mice compared to conventional cigarette smoke (CS) exposure. The mice were exposed to air (control), low, medium, or high doses of ECA, or a reference CS dose orally and nasally for eight months. Various cardiovascular and pulmonary assessments have been conducted to determine the biological and prosthetic effects. Histopathological analysis was used to determine structural changes in the heart and lungs. Biological markers associated with fibrosis, inflammation, and oxidative stress were investigated. Cardiac proteomic analysis was applied to reveal the shared and unique protein expression changes in ECA and CS groups, which related to processes such as immune activation, lipid metabolism, and intracellular transport. Overall, chronic exposure to ECA led to adverse cardiovascular and pulmonary effects in mice, although they were less pronounced than those of CS exposure. This study provides evidence that e-cigarettes may be less harmful than combustible cigarettes for the long-term health of the cardiovascular and respiratory systems in mice. However, further human studies are needed to clarify the long-term health risks associated with e-cigarette use.
Asunto(s)
Fumar Cigarrillos , Sistemas Electrónicos de Liberación de Nicotina , Animales , Humanos , Ratones , Aerosoles/toxicidad , Pulmón , ProteómicaRESUMEN
African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies. IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Sistemas CRISPR-Cas , Edición Génica , Replicación Viral , Animales , Virus de la Fiebre Porcina Africana/genética , Porcinos , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/prevención & control , Edición Génica/métodos , Replicación Viral/genética , Animales Modificados Genéticamente/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Genoma Viral/genéticaRESUMEN
BACKGROUND: ß-catenin, acting as the core effector of canonical Wnt signaling pathway, plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models, the ß-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood, partly due to the rapid and complex cell fate transitions during early differentiation. RESULTS: In this study, we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of ß-catenin. Our analysis showed that a truncated ß-catenin lacking both N- and C-terminal domains (ΔN148C) could robustly rescue the DE formation, whereas hyperactive ß-catenin mutants with S33Y mutation or N-terminal deletion (ΔN90) had limited ability to induce DE lineage. Notably, the ΔN148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak ß-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes, whereas the hyperactive ß-catenin mutants activated mesoderm genes. CONCLUSION: Our study uncovered an unconventional regulatory function of ß-catenin through weak transactivation, indicating that the levels of ß-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.
RESUMEN
Branch retinal vein occlusion (BRVO) is the second most frequent retinal vascular disorder. Currently the first-line therapies for BRVO include anti-VEGF and dexamethasone implant treatment, however, with direct or indirect damage on retinal neurons, it has limited effect in improving patients visual acuity. Therefore, novel treatments with neuroprotective effect for BRVO retina were expected. Minocycline is a semisynthetic, broad spectrum tetracycline antibiotic with high penetration through the blood brain barrier. The neuroprotective effects of minocycline have been shown in various central nervous system (CNS) disease. Since both CNS and retina were composed of neurons and glials, it is reasonable to expect a neuroprotective effect by minocycline for BRVO retina. Therefore, the aim of the present study was to study whether minocycline has neuroprotective effect in branch retinal vein occlusion (BRVO) and the possible underlying molecular basis. We created BRVO in rats using laser photocoagulation. The animals were then randomly divided into 4 groups to evaluate the effect of minocycline: group A: minocycline 45 mg/kg intraperitoneal injection (i.p.), group B: minocycline 90 mg/kg i.p., group C: normal saline i.p., group D: sham injection. Fundus photography and fluorescein angiography (FA) were conducted. The changes in thickness of retinal layers were measured with optical coherence tomography (OCT) in vivo. We found that retinal edema occurred predominantly in the inner retinal layers. Intraperitoneal administration of minocycline significantly ameliorated retinal edema in the early stage of BRVO. We performed Full field Electroretinography (ffERG) to evaluate retinal function and found that the reduction of b wave amplitude decreased in the combined maximal response. The expressional levels of apoptosis related genes (Bax, Bcl-2) and inflammation related genes (IL-1 ß, TNF α, MCP-1 and CCR2) were measured by real-time PCR, the results showed that minocycline treatment upregulated Bcl-2 expression and inhibits TNF α expression since early stage of BRVO. We also performed Hematoxylin-Eosin (HE) and immunostaining for Iba 1 (a microgilal marker), active caspase-3, Bax, Bcl-2, IL-1 ß, TNF α and found that minocycline inhibits retinal microglial activation, prevents retinal ganglion cell loss, and inhibits retinal caspase-3 activation. Thus, our study indicates that systemic administration of minocycline ameliorates retinal edema and preserves retinal function in the early stage of BRVO possibly via inhibiting microglia activation and protecting RGC from apoptosis.
Asunto(s)
Antibacterianos/uso terapéutico , Modelos Animales de Enfermedad , Microglía/efectos de los fármacos , Minociclina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Papiledema/prevención & control , Oclusión de la Vena Retiniana/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Citocinas/genética , Electrorretinografía/efectos de los fármacos , Femenino , Angiografía con Fluoresceína , Humanos , Inyecciones Intraperitoneales , Papiledema/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Ratas Endogámicas BN , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/fisiopatología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Oclusión de la Vena Retiniana/genética , Oclusión de la Vena Retiniana/fisiopatología , Tomografía de Coherencia Óptica , Agudeza Visual , Proteína X Asociada a bcl-2/genéticaRESUMEN
Background: Endometrial carcinoma is one of the most common female malignancies worldwide. Based on our preliminary investigation, DUSP1 was identified as a potential biomarker for endometrial carcinoma prognosis, but its function and mechanism remained unclear. Methods: In this study, genes highly correlated with DUSP1 in endometrial cancer were found through correlation analysis, and the promoter sequence of DUSP1 was analyzed by PROMO program. Next-generation phosphorylation mass spectrometry was used to explore new downstream target proteins and pathways of DUSP1 in endometrial carcinoma. The mRNA and protein expression levels were detected by real-time quantitative PCR, immunohistochemistry and Western blotting. The cell survival and proliferation were analyzed by CCK8 assay, cell apoptosis was analyzed by Annexin-V-APC and PI dual staining assay, and the cell invasion was analyzed by Transwell method. Results: (1) There was a high correlation between the expression of DUSP1 and the genes involved in AP-1 complex and its co-expression network. (2) Promoter sequence analysis predicted that the members of AP-1 complex might be the upstream transcriptional regulators of DUSP1. (3) Transfection experiments proved DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating ERK pathway. (4) The results of phosphorylation mass spectrometry showed that overexpression of DUSP1 mainly dephosphorylated EPHA2 in endometrial carcinoma, and co-immunoprecipitation verified the protein interaction between DUSP1 and EPHA2. (5) Overexpression or knockdown of EPHA2 significantly changed the phosphorylation level of EPHA2. (6) The expression of EPHA2 protein was high in patients with more aggressive endometrial cancer. (7) Using EPHA2 inhibitor could significantly slow down the growth rate of tumor cells. Conclusion: (1) There exists a mutual regulation relationship between DUSP1 and AP-1 co-expression network in endometrial carcinoma. (2) It is reported for the first time that DUSP1 phosphatase acts on the ser899 site of EphA2 in endometrial carcinoma. (3) DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating MAPK pathway through directly dephosphorylating ERK, or by dephosphorylating EPHA2.
RESUMEN
BACKGROUND: Steroid receptor-associated and regulated protein (SRARP) suppresses tumor progression and modulates steroid receptor signaling by interacting with estrogen receptors and androgen receptors in breast cancer. In endometrial cancer (EC), progesterone receptor (PR) signaling is crucial for responsiveness to progestin therapy. The aim of this study was to investigate the role of SRARP in tumor progression and PR signaling in EC. METHODS: Ribonucleic acid sequencing data from the Cancer Genome Atlas, Clinical Proteomic Tumor Analysis Consortium, and Gene Expression Omnibus were used to analyze the clinical significance of SRARP and its correlation with PR expression in EC. The correlation between SRARP and PR expression was validated in EC samples obtained from Peking University People's Hospital. SRARP function was investigated by lentivirus-mediated overexpression in Ishikawa and HEC-50B cells. Cell Counting Kit-8 assays, cell cycle analyses, wound healing assays, and Transwell assays were used to evaluate cell proliferation, migration, and invasion. Western blotting and quantitative real-time polymerase chain reaction were used to evaluate gene expression. The effects of SRARP on the regulation of PR signaling were determined by co-immunoprecipitation, PR response element (PRE) luciferase reporter assay, and PR downstream gene detection. RESULTS: Higher SRARP expression was significantly associated with better overall survival and disease-free survival and less aggressive EC types. SRARP overexpression suppressed growth, migration, and invasion in EC cells, increased E-cadherin expression, and decreased N-cadherin and Wnt family member 7A ( WNT7A ) expression. SRARP expression was positively correlated with PR expression in EC tissues. In SRARP -overexpressing cells, PR isoform B (PRB) was upregulated and SRARP bound to PRB. Significant increases in PRE-based luciferase activity and expression levels of PR target genes were observed in response to medroxyprogesterone acetate. CONCLUSIONS: This study illustrates that SRARP exerts a tumor-suppressive effect by inhibiting the epithelial-mesenchymal transition via Wnt signaling in EC. In addition, SRARP positively modulates PR expression and interacts with PR to regulate PR downstream target genes.
Asunto(s)
Neoplasias Endometriales , Receptores de Progesterona , Femenino , Humanos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Proteómica , Línea Celular Tumoral , Neoplasias Endometriales/metabolismo , Proliferación Celular/genética , Luciferasas/genética , Luciferasas/metabolismo , Luciferasas/farmacología , Regulación Neoplásica de la Expresión Génica/genéticaRESUMEN
Introduction: Cryptotanshinone(CTS), a compound derived from the root of Salvia miltiorrhiza, has been linked to various of diseases, particularly pulmonary fibrosis. In the current study, we investigated the benefit of CTS on Sprague-Dawley (SD) rats induced by bleomycin (BLM) and established high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods to compare pharmacokinetics and tissue distribution in subsequent normal and modulated SD rats. Methods: The therapeutic effect of CTS on BLM-induced SD rats was evaluated using histopathology, lung function and hydroxyproline content measurement, revealing that CTS significantly improved SD rats induced by BLM. Additionally, a simple, rapid, sensitive and specific HPLC-MS/MS method was developed to determine the pharmacokinetics of various components in rat plasma. Results: Pharmacokinetic studies indicated that CTS was slowly absorbed by oral administration and had low bioavailability and a slow clearance rate. The elimination of pulmonary fibrosis in 28-day rats was slowed down, and the area under the curve was increased compared to the control group. Long-term oral administration of CTS did not accumulate in vivo, but the clearance was slowed down, and the steady-state blood concentration was increased. The tissue distribution study revealed that CTS exposure in the lungs and liver. Discussion: The lung CTS exposure was significantly higher in the model group than in the control group, suggesting that the pathological changes of pulmonary fibrosis were conducive to the lung exposure of CTS and served as the target organ of CTS.
RESUMEN
OBJECTIVE: To examine the expression of high mobility group A2 (HMGA2), P53 and let-7 family microRNA, to investigate the correlation of HMGA2 and let-7, and to compare the HMGA2 and P53 expressions in human serous ovarian cancer. METHODS: Immunohistochemistry assay was used to examine the expressions of HMGA2 and P53 in 50 paraffin-embedded tissue specimens of human serous ovarian cancer and 4 normal fallopian tube tissues. HMGA2 mRNA and let-7 family microRNA were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction in the corresponding frozen tissues. RESULTS: HMGA2 and P53 were immuno-positive in 70% (35/50) and 78% (39/50) of the ovarian cancer tissues, respectively. HMGA2 was weakly expressed in the ciliated cells, but negative in the secretary cells of the fallopian tube. There was a tendency that the expression of HMGA2 increased with higher pathological grade of the ovarian cancer, but no correlation was observed between the HMGA2 overexpression and clinical stages. HMGA2 mRNA was detected in all the ovarian cancer samples, and its expression level was higher than that of the normal fallopian tube tissues in 72% (36/50) of the ovarian cancer samples. The expression of HMGA2 mRNA was much higher in more malignant SKOV3.ipl cells than in its corresponding SKOV3 cells. All let-7 family members were detectable in all ovarian cancer samples, and their expression were inversely correlated with HMGA2 mRNA expression (r=-0.305,P<0.05). CONCLUSION: HMGA2 can be a biomarker complement to P53, and its high expression has an inclination of more malignancy. The downregulation of let-7 is, but not the only mechanism of HMGA2 overexpression in serous ovarian cancer.
Asunto(s)
Cistadenocarcinoma Seroso/genética , Proteína HMGA2/metabolismo , MicroARNs/metabolismo , Neoplasias Ováricas/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Femenino , Proteína HMGA2/genética , Humanos , MicroARNs/genética , Neoplasias Ováricas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Background: Ulcerative colitis (UC) is one type of inflammatory bowel disease, characterized by inflammation with infiltration and activation of macrophages in colonic tissue. LH011 is a trypsin inhibitor with potential anti-inflammatory effect. Purpose: Here, we aim to assay the effects of LH011 on UC and further investigate the potential mechanisms in vitro and in vivo. Methods: Dextran sulfate sodium (DSS, 3.5%, w/v) was used to induce UC, and lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 cells. LH011 was administrated to mice in vivo or to RAW 264.7 cells in vitro at different concentrations. The cytokines (IL-1ß, IL-6, and TNF-α) and the changes of NF-κB and Nrf2 pathways were detected. Results: The results showed that LH011 improved DSS-induced mice colitis, including loss of weight, disease activity index (DAI), and colonic pathological damage. In addition, LH011 inhibited the expressions of IL-1ß, IL-6, and TNF-α and strengthened the anti-oxidative capacity. Mechanically, LH011 downregulated the nuclear localization of NF-κB p65 and upregulated the protein expression of Nrf2. Conclusion: These results demonstrated that LH011 alleviated inflammation and oxidative stress during UC by inhibiting TLR4/NF-κB and activating Nrf2/Keap1/HO-1 signaling pathways.
RESUMEN
AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose (2 × 109 vg/neonate and 1 × 1010 vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9R338L variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.
Asunto(s)
Hemofilia B , Ratones , Animales , Humanos , Hemofilia B/genética , Hemofilia B/terapia , Edición Génica , Sistemas CRISPR-Cas/genética , Formación de Anticuerpos , Vectores Genéticos/genética , Hemostasis , HígadoRESUMEN
OBJECTIVE: The aim of this study was to investigate whether miR-449a, miR-449b and miR-192 family microRNAs play the same roles in p53 pathway as miR-34 family in ovarian cancer. METHODS: Wild-type p53 ovarian carcinoma cell line A2780 cells were treated with genotoxic agent adriamycin. The reactivation of p53 was detected by Western blot. The expression of miR-449a/b, miR-34a, miR-34b, miR-34c, miR-192 and miR-194 were detected by real-time quantitative PCR. Mutant p53 ovarian cancer cell line SKOV3.ipl cells were transfected with pre-microRNAs and the cell-cycle changes were detected. The expression level of miR-449a/b, miR-34a, miR-34b, miR-34c, miR-192 and miR-194 in serous ovarian carcinomas of varying grade and stage were compared with real-time PCR. RESULTS: The expressions of miR-449a/b, miR-34b and miR-34c were 19-fold to 21-fold elevated after p53 activation by genotoxic agent. Ectopic expression of miR-449b, as well as miR-34c, resulted in cell-cycle arrest in SKOV3.ipl cells. The expression of miR-449a/b was parallel with that of miR-34b, miR-34c, and were significantly lower in late stage and high-grade serous carcinomas than in the normal fallopian tube, early stage and low-grade serous carcinomas. The expression of miR-192, miR-194 and miR-34a did not show evident features in serous ovarian carcinomas and were much lower than miR-449a/b, miR-34b and miR-34c in normal fallopian tube. CONCLUSIONS: As tumor-suppressor microRNAs, miR-449a/b, miR-34b and miR-34c cooperate and play important roles in p53 pathway. Their inactivation may contribute to the carcinogenesis and progression of serous ovarian carcinomas.
Asunto(s)
Cistadenocarcinoma Seroso/metabolismo , MicroARNs/metabolismo , Neoplasias Ováricas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Antibióticos Antineoplásicos/farmacología , Ciclo Celular , Línea Celular Tumoral , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Doxorrubicina/farmacología , Femenino , Humanos , MicroARNs/genética , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , TransfecciónRESUMEN
Gene therapy has entered a new era after decades-long efforts, where the recombinant adeno-associated virus (AAV) has stood out as the most potent vector for in vivo gene transfer and demonstrated excellent efficacy and safety profiles in numerous preclinical and clinical studies. Since the first AAV-derived therapeutics Glybera was approved by the European Medicines Agency (EMA) in 2012, there is an increasing number of AAV-based gene augmentation therapies that have been developed and tested for treating incurable genetic diseases. In the subsequent years, the United States Food and Drug Administration (FDA) approved two additional AAV gene therapy products, Luxturna and Zolgensma, to be launched into the market. Recent breakthroughs in genome editing tools and the combined use with AAV vectors have introduced new therapeutic modalities using somatic gene editing strategies. The promising outcomes from preclinical studies have prompted the continuous evolution of AAV-delivered therapeutics and broadened the scope of treatment options for untreatable diseases. Here, we describe the clinical updates of AAV gene therapies and the latest development using AAV to deliver the CRISPR components as gene editing therapeutics. We also discuss the major challenges and safety concerns associated with AAV delivery and CRISPR therapeutics, and highlight the recent achievement and toxicity issues reported from clinical applications.
Asunto(s)
Dependovirus/genética , Edición Génica/métodos , Terapia Genética/métodos , Vectores Genéticos , Animales , Productos Biológicos/uso terapéutico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Humanos , Proteínas Recombinantes de Fusión/uso terapéutico , Resultado del TratamientoRESUMEN
OBJECTIVE: To identify the differentially expressed microRNAs in the hippocampus of Down Syndrome model mouse Ts65Dn. METHODS: Low molecular weight RNA from hippocampus were tailed and reverse transcribed by extended RT-primer. MicroRNAs primers were arrayed on plates according to the Tm of each primer. PCR were carried out at different annealing temperatures using a gradient real-time PCR instrument. The relative expression level of each microRNAs was calculated using the most stable reference gene as normalizer which was selected by geNorm and Normfinder software. RESULTS: miR-27a was identified as the most stable reference gene by geNorm and Normfinder software. Among the 52 microRNAs detected by real-time PCR array, miR-33 and miR-19a were significantly down-regulated, whereas miR-130 were significantly up-regulated in the hippocampus of Ts65Dn mice as compared with the euploid control mice. The expression of miR-802, which was the trisomy chromosome 16 derived microRNAs, was very low in hippocampus with no difference between the two groups. CONCLUSION: MicroRNAs are dysregulated in the hippocampus of Ts65Dn mice, which may contribute to the reduced neurogenesis of Down syndrome.
Asunto(s)
Síndrome de Down/genética , Hipocampo/metabolismo , MicroARNs/metabolismo , Animales , Ratones , Ratones Mutantes Neurológicos , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Ammonia oxidation is mainly performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Allylthiourea (ATU) has been found to specifically inhibit ammonia oxidation. However, the effect of ATU on AOA and AOB transcription has been infrequently studied. In the present study, we examined the responses of AOA and AOB activity and DNA/cDNA community structure to ATU exposure. The ammonia oxidation activity in the 100-mg/L ATU group was 4.3% of that in the control group after 7 days. When exposed to ATU, the gene abundance of AOA was favored compared with that of AOB, and there were no statistically significant differences in the abundance of AOB amoA in DNA and cDNA between the two groups. Compared with the control group, the gene abundance of AOA significantly increased by 5.23 times, while the transcription of AOA significantly decreased by 0.70 times. Moreover, the transcriptional ratio of AOA in the ATU group was only 0.05 times as high as that in the control group. ATU selectively affected AOB and completely inhibited Nitrosomonas europaea and Bacterium amoA.22.HaldeII.kultur at the genetic level. Under ATU exposure, all AOA clusters were transcribed, but three AOB clusters were not transcribed. Our results indicated that the ammonia oxidation potential of the soil of water level fluctuation areas, based on ATU inhibition, was associated mainly with AOA amoA gene abundance and AOB community shifts in DNA and cDNA.
Asunto(s)
Suelo , Agua , Amoníaco , Archaea/genética , Bacterias/genética , ADN Complementario , Oxidación-Reducción , Filogenia , Microbiología del Suelo , Tiourea/análogos & derivadosRESUMEN
Aerobic ammonia oxidation, an important part of the global nitrogen cycle, is thought to be jointly driven by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in coastal wetlands. However, the activities and contributions of AOA and AOB in coastal wetlands have remained largely unknown. Here, we investigated the oxidation capability of AOA and AOB in four types of typical coastal wetlands (paddy, estuary, shallow and reed wetland) in the Bohai region in China using DNA-based stable-isotope probing (DNA-SIP), quantitative PCR and high-throughput sequencing techniques. We found that the community structure of AOB varied substantially, and the AOA structure was more stable across different coastal wetlands. The rate of AOA was 0.12, 0.84, 0.45 and 0.93 µg N g-1 soil d-1 in paddy, estuary, shallow and reed wetlands, and the rate of AOB was 5.61, 10.72, 0.74 and 1.16 µg N g-1 soil d-1, respectively. We found that the contribution of AOA gradually increased from paddy to estuary to shallow wetland and finally to reed wetland, with values of 2.03%, 7.25%, 37.53% and 44.51%, respectively. Our results provide new insight into the mechanisms of the differences in activities and the contributions of AOA and AOB in different coastal wetlands, and our findings may contribute to further understanding of the global nitrogen cycle.