RESUMO
BACKGROUND: Non-syndromic orofacial cleft (NSOC) is one of the most common craniofacial malformations with complex etiology. This study aimed to explore the role of specific SNPs in ZFP36L2 and its functional relevance in zebrafish models. METHODS: We analyzed genetic data of the Chinese Han population from two previous GWAS, comprising of 2512 cases and 2255 controls. Based on the Hardy-Weinberg Equilibrium (HWE) and minor allele frequency (MAF), SNPs in the ZFP36L2 were selected for association analysis. In addition, zebrafish models were used to clarify the in-situ expression pattern of zfp36l2 and the impact of its Morpholino-induced knockdown. RESULTS: Via association analysis, rs7933 in ZFP36L2 was significantly associated with various non-syndromic cleft lip-only subtypes, potentially conferring a protective effect. Zebrafish embryos showed elevated expression of zfp36l2 in the craniofacial region during critical stages of oral cavity formation. Furthermore, Morpholino-induced knockdown of zfp36l2 led to craniofacial abnormalities, including cleft lip, which was partially rescued by the addition of zfp36l2 mRNA. CONCLUSION: Our findings highlight the significance of ZFP36L2 in the etiology of NSOC, supported by both human genetic association data and functional studies in zebrafish. These results pave the way for further exploration of targeted interventions for craniofacial malformations.
Assuntos
Fenda Labial , Fissura Palatina , Anormalidades Craniofaciais , Animais , Humanos , Fenda Labial/genética , Fissura Palatina/genética , Peixe-Zebra , Predisposição Genética para Doença , Morfolinos , Polimorfismo de Nucleotídeo Único , Genótipo , Fatores de Transcrição/genéticaRESUMO
Objective: Female fertility gradually decreases with the increase in women's age. The underlying reasons include the decline in the quantity and quality of oocytes. Oocyte aging is an important manifestation of the decline in oocyte quality, including in vivo oocyte aging before ovulation and in vitro oocyte aging after ovulation. Currently, few studies have been done to examine oocyte aging, and the relevant molecular mechanisms are not fully understood. Therefore, we used zebrafish as a model to investigate oocyte aging. Three different age ranges of female zebrafish were selected to mate with male zebrafish of the best breeding age. In this way, we studied the effects of maternal age-related oocyte aging on fertility and investigated the potential molecular mechanisms behind maternal age-related fertility decline. Methods: Eight female zebrafish aged between 158 and 195 d were randomly selected for the 6-month age group (180±12) d, 8 female zebrafish aged between 330 and 395 d were randomly selected for the 12-month age group (360±22) d, and 8 female zebrafish aged between 502 and 583 d were randomly selected for the 18-month age group (540±26) d. Male zebrafish of (180±29) d were randomly selected from zebrafish aged between 158 and 195 d and mated with female zebrafish in each group. Each mating experiment included 1 female zebrafish and 1 male zebrafish. Zebrafish embryos produced by the mating experiments were collected and counted. The embryos at 4 hours post-fertilization were observed under the microscope, the total number of embryos and the number of unfertilized embryos were counted, and the fertilization rate was calculated accordingly. The numbers of malformed embryos and dead embryos were counted 24 hours after fertilization, and the rates of embryo malformation and mortality were calculated accordingly. The primary outcome measure was the embryo fertilization rate, and the secondary outcome measures were the number of embryos per spawn (the total number of embryos laid within 1.5 hours after the beginning of mating and reproduction of the zebrafish), embryo mortality, and embryo malformation rate. The outcome measures of each group were compared. The blastocyst embryos of female zebrafish from each group born after mating with male zebrafish in their best breeding period were collected for transcriptomics analysis. Fresh oocytes of female zebrafish in each group were collected for transcriptomics analysis to explore the potential molecular mechanisms of maternal age-related fertility decline. Results: Compared with that of the 6-month group (94.9%±3.6%), the embryo fertilization rate of the 12-month group (92.3%±4.2%) showed no significant difference, but that of the 18-month group (86.8%±5.5%) decreased significantly (P<0.01). In addition, the fertilization rate in the 18-month group was significantly lower than that in the 12-month group (P<0.05). Compared with that of the 6-month group, the embryo mortality of the female zebrafish in the 12-month group and that in the 18-month group were significantly higher than that in the 6-month group (P<0.000 1, P<0.001). There was no significant difference in the number of embryos per spawn or in the embryo malformation rate among the three groups. The results of the transcriptomics analysis of blastocyst embryos showed that some genes, including dusp5, bdnf, ppip5k2, dgkg, aldh3a2a, acsl1a, hal, mao, etc, were differentially expressed in the 12-month group or the 18-month group compared with their expression levels in the 6-month group. According to the KEGG enrichment analysis, these differentially expressed genes (DEGs) were significantly enriched in the MAPK signaling pathway, the phosphatidylinositol signaling system, and the fatty acid degradation and histidine metabolism pathway (P<0.05). The analysis of the expression trends of the genes expressed differentially among the three groups (the 6-month group, the 12-month group, and the 18-month group in turn) showed that the gene expression trends of fancc, fancg, fancb, and telo2, which were involved in Fanconi anemia pathway, were statistically significant (P<0.05). In the results of oocyte transcriptomics analysis, the genes that were differentially expressed in the 12-month group or the 18-month group compared with the 6-month group were mainly enriched in cell adhesion molecules and the protein digestion and absorption pathway (P<0.05). The results of the trends of gene expression in the zebrafish oocytes of the three groups (the 6-month group, the 12-month group, and the 18-month group in turn) showed that three kinds of gene expression trends of declining fertility with growing maternal age had significant differences (P<0.05). Further analysis of the three significantly differential expression trends showed 51 DEGs related to mitochondria and 5 DEGs related to telomere maintenance and DNA repair, including tomm40, mpc2, nbn, tti1, etc. Conclusion: With the increase in the maternal age of the zebrafish, the embryo fertilization rate decreased significantly and the embryo mortality increased significantly. In addition, with the increase in the maternal age of the zebrafish, the expression of mitochondria and telomere-related genes, such as tomm40, mpc2, nbn, and tti1, in female zebrafish oocytes decreased gradually. Maternal age may be a factor contributing to the decrease in oocyte fertilization ability and the increase in early embryo mortality. Maternal age-related oocyte aging affects the fertility and embryo development of the offspring.
Assuntos
Fertilidade , Oócitos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Oócitos/fisiologia , Feminino , Fertilidade/genética , Masculino , Transcriptoma , Idade Materna , Envelhecimento/fisiologia , Envelhecimento/genética , Modelos AnimaisRESUMO
POLR3B gene encodes the 2nd largest catalytic subunit and affects the function of RNA polymerase III enzymes in transcription. Bi-allelic variants in POLR3B pathogenically cause hypomyelinating leukodystrophy-8 (HLD8). Herein, we recruited a family with two patients, who presented clinically with cerebellar atrophy, intellectual disability, hypogonadotropic hypogonadism, and visual problems. We identified the two affected siblings carrying the compound heterozygous variations (c.165_167del; c.1615G>T) in POLR3B by trio-whole-exome sequencing (trio-WES). The qPCR and western blot showed that both transcriptional and translational levels of the mutation (c.165_167del, p.I55_K56delinsM) were sharply attenuated. Following that, a thorough functional examination of a zebrafish line disrupted for human POLR3B validated the pathogenic effects of the two mutations. Our research broadens the spectrum of HLD8-related pathogenic POLR3B mutations and provides new molecular and animal evidence.
Assuntos
Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , RNA Polimerase III , Animais , Humanos , RNA Polimerase III/genética , Irmãos , Peixe-Zebra/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , MutaçãoRESUMO
BACKGROUND: Aminoacyl-tRNA synthetases (ARSs) are enzymes responsible for attaching amino acids to tRNA, which enables protein synthesis. Mutations in isoleucyl-tRNA synthetase (IARS1) have recently been reported to be a genetic cause for growth retardation, intellectual disability, muscular hypotonia, and infantile hepatopathy (GRIDHH). CASE PRESENTATION: In this study, we reported an additional case of compound heterozygous missense variations c.701 T > C (p.L234P) and c.1555C > T (p.R519C) in IARS1, which were identified using medical exome sequencing; c.701 T > C (p.L234P) was a novel variant, and c.1555C > T (p.R519C) was found in GnomAD. Unlike other reported patients, this individual presented prominently with recurrent liver failure, which led to her death at an early age of 19 months. She also had significant growth retardation, muscular hypotonia, chubby and flabby face, recurrent loose stools, and abnormal brain computed tomography (CT), while zinc deficiency and hearing loss were not present. Studies in zebrafish embryo modeling recapitulated some of the key phenotypic traits in embryo development, neurodevelopment, liver development, and myogenesis, demonstrating that these variations caused a loss of gene function in IARS1. CONCLUSIONS: We have found a novel mutation point c.701 T > C (p.L234P) in IARS1. Compound heterozygous mutations of c.701 T > C (p.L234P) and c.1555C > T (p.R519C) in IARS1 are pathogenic, which can cause GRIDHH in child.
Assuntos
Falência Hepática , Hipotonia Muscular , Animais , China , Feminino , Transtornos do Crescimento , Humanos , Falência Hepática/genética , Mutação , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM) is a rare multiple malformation syndrome characterized by malar and mandibular hypoplasia and congenital- or postnatal-onset microcephaly induced by haploinsufficiency of (elongation factor Tu GTP-binding domain-containing 2) EFTUD2. METHODS: We report the case of a 16-month-old boy with MFDM symptoms, including malar and mandibular hypoplasia, microcephaly, micrognathia, midline cleft palate, microtia, auditory canal atresia, severe sensorineural hearing loss, and developmental delay. Whole-exome sequencing (WES) analysis of the patient's family was performed to identify the genetic etiology responsible for this phenotype. RESULTS: We identified a novel de novo missense mutation (c.671G>T, p.Gly224Val) in the EFTUD2. According to the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines, the c.671G>T mutation was classified as likely pathogenic (PS2, PM1, PM2, and PP3). Based on our findings, prenatal diagnosis was performed on the second baby of the proband's parents to exclude the mutation and it was confirmed that the baby did not have the MFDM phenotype after 14 months of follow-up. Furthermore, the zebrafish model confirmed that the EFTUD2 c.671G>T mutation caused a loss of gene function in EFTUD2, and the pathogenicity of the EFTUD2 c.671G>T mutation was classified as pathogenic (PS2, PS3, PM1, and PM2). CONCLUSION: Our results indicate that WES is a useful tool for identifying potentially pathogenic mutations, particularly in rare disorders, and is advantageous for genetic counseling and subsequent prenatal diagnosis. Moreover, the importance of functional assays cannot be underestimated, which could further confirm the pathogenicity of the genetic variants.
Assuntos
Anormalidades Múltiplas , Disostose Mandibulofacial , Microcefalia , Anormalidades Múltiplas/genética , Animais , Humanos , Disostose Mandibulofacial/diagnóstico por imagem , Disostose Mandibulofacial/genética , Microcefalia/diagnóstico por imagem , Microcefalia/genética , Mutação , Mutação de Sentido Incorreto/genética , Fatores de Alongamento de Peptídeos/genética , Fenótipo , Ribonucleoproteína Nuclear Pequena U5/genética , Sequenciamento do Exoma , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Melatonin has been shown with anticancer property and therapeutic potential for tumors. However, there lacks a systematic study on the molecular pathways of melatonin and its antitumor effects in gastrointestinal carcinomas. METHODS: Using the gene expression profiles of four cancer cell lines from three types of gastrointestinal carcinomas before and after melatonin treatment, including gastric carcinoma (GC), colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC), differentially expressed genes (DEGs) and biological pathways influenced by melatonin were identified. The qRT-PCR analyses were performed to validate the effects of melatonin on 5-FU resistance-related genes in CRC. RESULTS: There were 17 pathways commonly altered by melatonin in the three cancer types, including FoxO signaling pathways enriched by the upregulated DEGs and cell cycle signaling pathways enriched by the downregulated DEGs, confirmed the dual role of melatonin to tumor growth, pro-apoptosis and anti-proliferation. DEGs upregulated in the three types of cancer tissues but reversely downregulated by melatonin were commonly enriched in RNA transport, spliceosome and cell cycle signaling pathways, which indicate that melatonin might exert antitumor effects through these pathways. Our results further showed that melatonin can downregulate the expression levels of 5-FU resistance-related genes, such as thymidylate synthase in GC and ATR, CHEK1, BAX and MYC in CRC. The qRT-PCR results demonstrated that melatonin enhanced the sensitivity of CRC 5-FU resistant cells by decreasing the expression of ATR. CONCLUSIONS: Melatonin exerts the effects of pro-apoptosis and anti-proliferation on gastrointestinal carcinomas, and might increase the sensitivity of 5-FU in GC and CRC patients.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Melatonina , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Melatonina/farmacologia , TranscriptomaRESUMO
Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Técnicas de Cultura de Células , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Humanos , Cinesinas , Lamina Tipo A/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Membrana Nuclear/metabolismo , Peixe-Zebra/genéticaRESUMO
Circadian clock and Smad2/3/4-mediated Nodal signaling regulate multiple physiological and pathological processes. However, it remains unknown whether Clock directly cross-talks with Nodal signaling and how this would regulate embryonic development. Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. We found that Clock1a is expressed both maternally and zygotically throughout early zebrafish development. We also noted that Clock1a alterations produce embryonic defects with shortened body length, lack of the ventral tail fin, or partial defect of the eyes. Clock1a regulates the expression of the mesodermal markers ntl, gsc, and eve1 and of the hematopoietic markers scl, lmo2, and fli1a Biochemical analyses revealed that Clock1a stimulates Nodal signaling by increasing expression of Smad2/3/4. Mechanistically, Clock1a activates the smad3a promoter via its E-box1 element (CAGATG). Taken together, these findings provide mechanistic insight into the role of Clock1a in the regulation of mesoderm development and primitive hematopoiesis via modulation of Nodal-Smad3 signaling and indicate that Smad3a is directly controlled by the circadian clock in zebrafish.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário , Mesoderma/metabolismo , Proteína Nodal/agonistas , Transdução de Sinais , Proteína Smad3/agonistas , Proteínas de Peixe-Zebra/agonistas , Peixe-Zebra , Animais , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Embrião não Mamífero/anormalidades , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Hematopoese/efeitos dos fármacos , Humanos , Hibridização In Situ , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesoderma/anormalidades , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Microinjeções , Microscopia de Fluorescência , Morfolinos/farmacologia , Mutação , Proteína Nodal/genética , Proteína Nodal/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affect fertility in both sexes. However, the involvement of CFTR in regulating germ cell development remains largely unknown. Here, we used zebrafish model to investigate the role of CFTR in primordial germ cells (PGCs) development. We generated a cftr frameshift mutant zebrafish line using CRISPR/Cas9 technique and investigated the migration of PGCs during early embryo development. Our results showed that loss of Cftr impairs the migration of PGCs from dome stages onward. The migration of PGCs was also perturbed by treatment of CFTRinh-172, a gating-speciï¬c CFTR channel inhibitor. Moreover, defected PGCs migration in cftr mutant embryos can be partially rescued by injection of WT but not other channel-defective mutant cftr mRNAs. Finally, we observed the elevation of cxcr4b, cxcl12a, rgs14a and ca15b, key factors involved in zebrafish PGCs migration, in cftr-mutant zebrafish embryos. Taken together, the present study revealed an important role of CFTR acting as an ion channel in regulating PGCs migration during early embryogenesis. Defect of which may impair germ cell development through elevation of key factors involved in cell motility and response to chemotactic gradient in PGCs.
Assuntos
Movimento Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Embrião não Mamífero/fisiologia , Células Germinativas/fisiologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenvolvimento Embrionário , Mutação da Fase de Leitura , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
STUDY QUESTION: Do microRNAs (miRNAs) contribute to aberrant progesterone receptor (PGR) expression in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER: miR-196a upregulates MEK/ERK signalling, mediating a downregulation of PGR expression in the eutopic endometrium of women with minimal or mild endometriosis. WHAT IS KNOWN ALREADY: Implantation failure is strongly suggested as an underlying cause for the observed infertility in minimal or mild endometriosis. Progesterone resistance, which is mainly caused by aberrantly expressed progesterone receptor in the eutopic endometrium, is considered as a key factor of decreased endometrial receptivity; thus far, epigenetics, but not miRNA, has been shown to affect PGR expression in the endometrium. STUDY DESIGN SIZE, DURATION: Microarray analysis was used to analyse the eutopic endometrium. The differential expression of miR-196a was validated. Bioinformatics analysis predicted that miR-196a targets the 3'-untranslated region (UTR) of the PGR. The relationship between the miR-196a level and PGR expression was studied and the role of the MEK/ERK signal pathway was investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was extracted from eutopic endometrium samples in three infertile women with mild/minimal endometriosis and three disease-free control subjects. The miRNA and mRNA expression levels were analysed by microarray analysis. The miR-196a expression was validated by qRT-PCR [endometriosis (n = 22) and control (n = 20)], while functional analysis utilised in vitro transfection of endometrial stromal cells (ESCs), induction of decidualization of ESCs, and luciferase reporter assays in 293 T cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 66 dysregulated miRNAs and 357 dysregulated mRNAs were screened by microarray analysis. miR-196a and P-MEK/P-ERK were both found to be significantly upregulated in the eutopic endometrium in patients with mild/minimal endometriosis. PGR and PGR-B mRNA were inhibited by miR-196a overexpression and upregulated by miR-196a inhibition. Luciferase reporter failed to confirm the target regulation of miR-196a on PGR. Transfection of ESCs with a miR-196a mimic led to an increase in the P-MEK/P-ERK protein levels, decrease in the PGR protein levels, and atypical decidualization. Following miR-196a inhibition, the P-MEK/P-ERK protein was downregulated and the PGR protein was upregulated. Inhibition of P-MEK/P-ERK also increased PGR expression. LARGE SCALE DATA: Data are presented in Supplementary Tables SI and SII. LIMITATIONS REASONS FOR CAUTION: This study focused on the role of miR-196a, and therefore does not involve other miRNAs; hence, it is possible that other miRNAs may also be responsible for progestin resistance in endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Our data revealed altered miRNA expression and activated MEK/ERK signalling in the eutopic endometrium in minimal or mild endometriosis. We showed that the miR-196a level is associated with reduced expression of PGR isoforms through MEK/ERK, suggesting that miR-196a and MEK/ERK are both potential biomarkers of endometriosis. These results provide a novel approach to target the mechanisms behind progesterone resistance in endometriosis. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Natural Science Foundation of China (No. 81370693). The authors have no conflicts of interest.
Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Infertilidade Feminina/metabolismo , Sistema de Sinalização das MAP Quinases/genética , MicroRNAs/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Endométrio/anormalidades , Feminino , Regulação da Expressão Gênica , Humanos , Infertilidade Feminina/genética , MicroRNAs/genética , Doenças Uterinas/genética , Doenças Uterinas/metabolismoRESUMO
In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo.
Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/efeitos dos fármacos , Neoplasias da Vesícula Biliar/metabolismo , Ginsenosídeos/química , Panax/química , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose , Caspase 12/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas/citologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fator de Transcrição CHOP/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: Carbonaceous dots (CDs), which have been used for diagnosis, drug delivery and gene delivery, are accumulated in heart at high concentrations. To improve their biocompatibility, polyethylene glycol-modified CDs (PEG-CDs) were prepared. In this study we compared the cardiac toxicity of CDs and PEG-CDs in mouse and zebrafish models. METHODS: Mice were intravenously treated with CDs (size: 4.9 nm, 5 mg·kg(-1)·d(-1)) or PEG-CDs (size: 8.3 nm, 5 mg·kg(-1)·d(-1)) for 21 d. Their blood biochemistry indices, ECG, and histological examination were examined for evaluation of cardiac toxicity. CDs or PEG-CDs was added in incubator of cmlc2 transgenic Zebrafish embryos at 6 hpf, and the shape and size of embryos' hearts were observed at 48 hpf using a fluorescent microscope. Furthermore, whole-mount in situ hybridization was used to examine the expression of early cardiac marker gene (clml2) at 48 hpf. RESULTS: Administration of CDs or PEG-CDs in mice caused mild, but statistically insignificant reduction in serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels detected at 7 d, which were returned to the respective control levels at 21 d. Neither CDs nor PEG-CDs caused significant changes in the morphology of heart cells. Administration of CDs, but not PEG-CDs, in mice caused marked increase of heart rate. Both CDs and PEG-CDs did not affect other ECG parameters. In the zebrafish embryos, addition of CDs (20 µg/mL) caused heart development delay, whereas addition of CDs (80 µg/mL) led to heart malformation. In contrast, PEG-CDs caused considerably small changes in heart development, which was consistent with the results from the in situ hybridization experiments. CONCLUSION: CDs causes greater cardiac toxicity, especially regarding heart development. Polyethylene glycol modification can attenuate the cardiac toxicity of CDs.
Assuntos
Carbono/química , Carbono/toxicidade , Cardiotoxicidade/prevenção & controle , Coração/efeitos dos fármacos , Nanoestruturas/química , Nanoestruturas/toxicidade , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Animais , Animais Geneticamente Modificados , Carbono/administração & dosagem , Modelos Animais de Doenças , Coração/embriologia , Coração/fisiologia , Cardiopatias Congênitas/induzido quimicamente , Frequência Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Nanoestruturas/administração & dosagem , Nanoestruturas/ultraestrutura , Polietilenoglicóis/administração & dosagem , Peixe-ZebraRESUMO
MicroRNAs (miRNAs) are noncoding RNAs with a short length of about 22 nucleotides. As major modulators participating in RNA interference, they affect cellular behaviors by regulating the expression of diverse genes at post-transcriptional levels. miR-15b is a member of the miR-15/16 family, which is broadly expressed in major tissues and specially enriched in the endovascular system of human beings. miR-15/16 affects cellular proliferation, apoptosis, invasion and angiogenesis. In this review, we summarize the role and the underlying mechanism of miR-15b as well as other miR-15/16 family members in different cells, especially in endothelial cells. We focus on the diverse roles of miR-15b in the occurrence, progression and prognosis of vascular diseases, with particular emphasis on preeclampsia, a hypertensive disorder related to endovascular dysfunction in the placenta.
Assuntos
Células Endoteliais/fisiologia , MicroRNAs/fisiologia , Doenças Vasculares/etiologia , Animais , Ciclina D1/genética , Ciclina E/genética , Humanos , Neuropilina-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: To determine the long-term efficacy and safety of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) for treating cervical intraepithelial neoplasia grade 2 (CIN2) as well as the suitability of ALA-PDT in treating of cervical lesions divided into cervical transformation zone type 3. METHODS: We included 81 patients diagnosed with CIN2 at the Department of Gynecology of the Affiliated Hospital of Qingdao University with data collected between January 2019 and January 2021 following ALA-PDT. Furthermore, we analyzed the superiority of ALA-PDT in fertility preservation among women of childbearing age based on follow-up data from 11 patients with fertility requirements. RESULTS: Our findings confirmed the long-term efficacy of ALA-PDT for CIN2 treatment, with an overall efficacy of 95.83 % (23/24) at follow-up of 25-36 months. Moreover, the cervical transformation zone type 3 improvement and human papillomavirus (HPV)-negative efficacy were 69.2 % (18/26) and 82.4 % (14/17), respectively. ALA-PDT is recommended for consenting patients with cervical transformation zone type 3. Additionally, women without primary infertility could experience natural pregnancy and full-term birth of more than one baby following ALA-PDT for CIN2 treatment, with a satisfaction rate of ≈100 %. CONCLUSIONS: ALA-PDT is recommendable for treating high-grade squamous intraepithelial lesions, especially in patients with fertility requirements.
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Ácido Aminolevulínico , Fotoquimioterapia , Fármacos Fotossensibilizantes , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Fotoquimioterapia/métodos , Ácido Aminolevulínico/uso terapêutico , Displasia do Colo do Útero/tratamento farmacológico , Fármacos Fotossensibilizantes/uso terapêutico , Adulto , Seguimentos , Neoplasias do Colo do Útero/tratamento farmacológico , Pessoa de Meia-Idade , Adulto JovemRESUMO
The use of aspartame as an artificial sweetener is prevalent in a wide range of everyday food products, potentially leading to health complications such as obesity, diabetes mellitus, autism spectrum disorders, and neurodegeneration. Aspartame has also been detected in natural water bodies at a concentration of 0.49 µg/L, yet research on its ecotoxicological effects on aquatic life remains scarce. This study aimed to investigate the potential negative effects of environmentally relevant concentrations of aspartame on the development of various tissues and organs in zebrafish embryos. We used a zebrafish model to treat embryos with aspartame at environmental concentration and those higher than in the environment-up to 1000 times. We observed that after exposure to aspartame body length increased, pigmentation was delayed, and neutrophil production inhibited in zebrafish. Furthermore, transcriptome analysis revealed that early exposure of zebrafish embryos to aspartame affected the transcriptomics of various systems, primarily by downregulating genes related to immune cell production, eye and optic nerve development, nervous system development, and growth hormone-related transcription. Most of the genes associated with ferroptosis were upregulated. This study provides new insights into the ecotoxicological effects of aspartame on aquatic environments.
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Noncompaction of the ventricular myocardium (NVM), the third most diagnosed cardiomyopathy, is characterized by prominent trabeculae and intratrabecular recesses. However, the genetic etiology of 40%-60% of NVM cases remains unknown. Here, we identify two infants with NVM, in a nonconsanguineous family, with a typical clinical presentation of persistent bradycardia since the prenatal period. A homozygous missense variant (R223L) of RCAN family member 3 (RCAN3) is detected in both infants using whole-exome sequencing. In the zebrafish model, marked cardiac dysfunction is detected in rcan3 deficiency (MO-rcan3ATG-injected) and rcan-/- embryos. Developmental dysplasia of both endocardial and myocardial layers is also detected in rcan3-deficient embryos. RCAN3 R223L variant mRNAs can not rescue heart defects caused by rcan3 knockdown or knockout; however, hRCAN3 mRNAs rescue these phenotypes. RNA-seq experiments show that several genes involved in cardiomyopathies are significantly regulated through multiple signaling pathways in the rcan3-knockdown zebrafish model. In human cardiomyocytes, RCAN3 deficiency results in reduced proliferation and increased apoptosis, together with an abnormal mitochondrial ultrastructure. Thus, we suggest that RCAN3 is a susceptibility gene for cardiomyopathies, especially NVM and that the R223L mutation is a potential loss-of-function variant.
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Peixe-Zebra , Animais , Feminino , Humanos , Lactente , Masculino , Cardiomiopatias/genética , Cardiomiopatias/patologia , Sequenciamento do Exoma , Ventrículos do Coração/patologia , Miocárdio Ventricular não Compactado Isolado/genética , Miocárdio Ventricular não Compactado Isolado/patologia , Mutação de Sentido Incorreto/genética , Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Linhagem , Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
BACKGROUND: Understanding the pathogenesis of unexplained recurrent pregnancy loss is paramount for advancing effective treatments. Various biological processes, including spermatogenesis and embryo development, are tightly regulated by N6-methyladenosine modifications. However, few studies have focused on the impact of sperm N6-methyladenosine modifications on embryonic development. Therefore, we aimed to study altered N6-methyladenosine-mediated messenger RNA methylation modifications in the spermatozoa of male partners from couples experiencing unexplained recurrent pregnancy loss, to identify potential diagnostic markers and explore their potential molecular mechanisms in pregnancy loss and embryogenesis. METHODS: Methylated RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing were conducted on the spermatozoa of men from couples in the 'unexplained recurrent pregnancy loss' group (n = 6), and the fertility control group (n = 6). To identify the role of the detected key genes, zebrafish model embryos were studied, and multi-omics (transcriptomics, proteomics, and metabolomics) analyses helped to explore the molecular mechanism of abnormal embryogenesis. FINDINGS: Comparing unexplained recurrent pregnancy loss with the fertility control group, 217 N6-methyladenosine peaks were significantly upregulated, and 40 were downregulated in the spermatozoa. The combined analyses of spermatozoa-methylated RNA immunoprecipitation sequencing and RNA sequencing indicated that N6-methyladenosine methylation and the expression of SEMA5A, MT-ATP6, ZNF662, and KDM4C were significantly different. In zebrafish embryos, the altered expression of the four genes increased embryonic mortality and malformations by disturbing several key signaling pathways and zygotic genome activation. INTERPRETATION: This study highlights the paternal epigenome, which could be one of the reasons for faulty embryogenesis leading to pregnancy loss. The N6-methyladenosine modification, the most prevalent RNA modification, contributes to the exploration and understanding of the paternal epigenome in the maintenance of pregnancy and fetal growth and development. The four genes identified in this study may serve as potential diagnostic markers and elucidate novel molecular mechanisms of embryogenesis.
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To investigate the effect of keratin 23 (KRT23) on the anticancer activity of melatonin (MLT) against gastric cancer (GC) cells, microarray analysis was applied to screen differentially expressed genes in AGS GC cells following MLT treatment. Western blotting was used to detect the expression of KRT23 in GC cells and normal gastric epithelial cell line GES1. KRT23 knockout was achieved by CRISPR/Cas9. Assays of cell viability, colony formation, cell cycle, electric cellsubstrate impedance sensing and western blotting were conducted to reveal the biological functions of KRT23knockout cells without or with MLT treatment. Genes downregulated by MLT were enriched in purine metabolism, pyrimidine metabolism, genetic information processing and cell cycle pathway. Expression levels of KRT23 were downregulated by MLT treatment. Expression levels of KRT23 in AGS and SNU216 GC cell lines were significantly higher compared with normal gastric epithelial cell line GES1. KRT23 knockout led to reduced phosphorylation of ERK1/2 and p38, arrest of the cell cycle and inhibition of GC cell proliferation. Moreover, KRT23 knockout further enhanced the inhibitory activity of MLT on the tumor cell proliferation by inhibiting the phosphorylation of p38/ERK. KRT23 knockout contributes to the antitumor effects of MLT in GC via suppressing p38/ERK phosphorylation. In the future, KRT23 might be a potential prognostic biomarker and a novel molecular target for GC.
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Melatonina , Neoplasias Gástricas , Humanos , Melatonina/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Queratinas/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Infertility gives rise to a lot of social and psychological problems. At present, assisted reproductive technology (ART) is an important way to solve infertility. However, the live birth rate of in vitro fertilization and embryo transfer (IVF-ET) is less than 50 %. Medium is essential for the culture of embryos in vitro. Therefore, we want to explore whether the composition of the culture medium affects the survival rate of embryos. Gentamicin (GM) is an aminoglycoside antibiotic that is used to treat various bacterial infections. It is widely used in IVF medium, but it is not known whether it has a toxicity effect on embryonic development. Here, we used zebrafish embryos to investigate the embryotoxicity of GM which is an ingredient in culture medium. Our results found that there was no significant effect on the zebrafish embryo development, including survival rate, malformation rate and developmental time course, while zebrafish embryos were treated with GM at the culture medium concentration (10 mg/L, 17.8 µM) compared with the control group. To research the potential embryotoxicity of GM, we treated zebrafish embryos with GM with high concentration (range from 17.8 µM to 3000 µM). The results showed that the lethal concentration of 50 % (LC50) at 48-h post-fertilization (hpf) value of zebrafish embryos for GM was 1150 µM; the survival rate and malformation rate of zebrafish embryos were significantly changed in a dose-dependent manner. Furthermore, transcriptomics, metabolomics and epigenomics (m6A-MeRIP-seq) were used to investigate the molecular mechanism of embryotoxicity, and results showed cell cycle, dorso-ventral axis formation and collecting duct acid secretion pathway were altered significantly in treated embryos. In conclusion, there are no adverse effects on embryonic development with the working concentration of GM in human culture medium, suggesting that GM is safe for embryo culture at working concentration.