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1.
EMBO J ; 40(4): e105202, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33410511

RESUMO

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused ß-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.

2.
Am J Hum Genet ; 108(2): 337-345, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33434492

RESUMO

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.


Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Anormalidades Congênitas/genética , Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Ductos Mesonéfricos/crescimento & desenvolvimento , Adulto , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Códon sem Sentido , Feminino , Estudos de Associação Genética , Pleiotropia Genética , Proteínas Homeobox A10/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX8/genética , Herança Paterna , Penetrância , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Ductos Mesonéfricos/anormalidades
3.
Front Oncol ; 10: 900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32850303

RESUMO

Background: N6-methyladenosine (m6A) RNA methylation, associated with cancer initiation and progression, is dynamically regulated by the m6A RNA regulators. However, its role in liver carcinogenesis is poorly understood. Methods: Three hundred seventy-one hepatocellular carcinoma (HCC) patients from The Cancer Genome Atlas database with sequencing and copy number variations/mutations data were included. Survival analysis was performed using Cox regression model. We performed gene set enrichment analysis to explore the functions associated with different HCC groups. Finally, we used a machine-learning model on selected regulators for developing a risk signature (m6Ascore) The prognostic value of m6Ascore was finally validated in another two GEO datasets. Results: We demonstrated that 11 m6A RNA regulators are significantly differentially expressed among 371 HCC patients stratified by clinicopathological features (P<0.001). We then identified two distinct HCC clusters by applying consensus clustering to m6A RNA regulators. Compared with the cluster2 subgroup, the cluster1 subgroup correlates with poorer prognosis (P < 0.001). Moreover, the cell cycle, splicesome and notch signaling pathway are significantly enriched in the cluster1 subgroup. We further derived m6Ascore, using four m6A regulators, predicting HCC prognosis well at three (AUC = 0.7) or 5 years (AUC=0.7) in validation. The prognostic value of m6Ascore also was validated successfully in two GEO datasets (P < 0.05). Finally, we discovered that mutations and copy number variations of m6A regulators, conferring worse survival, are strongly associated with TP53 mutations in HCC. Conclusions: We find a significant relationship between the alterations and different expressions causing increased m6A level and worse survival, especially in TP53-mutated HCC patients. Genetic alterations of m6A genes might cooperate with TP53 and its regulator targets in the HCC pathogenesis. Our m6Ascore may be applied in the clinical trials for patient stratification in HCC.

4.
Onco Targets Ther ; 13: 6819-6826, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764969

RESUMO

Solitary fibrous tumors (SFTs) can occur in several locations outside the pleura, but rarely in the sinonasal tract, and particularly not in the nasopharynx. Herein, we describe an unusual case of giant cell-rich SFT (GCRSFT) occurring in the nasopharynx. A 64-year-old man experienced dizziness and headache for more than 10 years with no obvious cause. Computed tomography (CT) scan showed a 3.9 cm × 2 cm tumor on the posterior lateral wall of the left nasopharynx, and angiography revealed a hypervascular tumor fed by branches of the left carotid artery. Hence, preoperative embolization was performed, and then the tumor was endoscopically resected. The symptoms were relieved after the resection, and postoperative head CT and video laryngoscopy showed that the tumor was completely resected. We next characterized the specific pathological characteristics of the resected tumor. Histologically, the tumor was characterized by varying cellular proliferation of cytologically bland spindle cells within a collagenous stroma, with prominent interspersed branching vessels. Mitotic activity was low (2/50HPF), and there was no evidence of pleomorphism or tumor necrosis. Moreover, multinucleated giant cells with deep nuclear staining and distributed in pseudovascular spaces were found within the tumor. We ruled out the possibility that our case was giant cell fibroblastoma (GCF) by immunohistochemical analysis, showing that the tumor cells were positive for CD34, CD99, STAT6, and BCL-2, and that the Ki-67 labeling index was 3%, indicating that our case was SFT and not GCF. The patient's condition is generally good after a 14-month follow-up. This report serves to broaden the morphologic spectrum of GCRSFT and will help clinicians and pathologists better understand this entity to prevent misdiagnosis.

5.
Kidney Int ; 98(4): 1020-1030, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32450157

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUTs) are the most common cause of chronic kidney disease in children. Human 16p11.2 deletions have been associated with CAKUT, but the responsible molecular mechanism remains to be illuminated. To explore this, we investigated 102 carriers of 16p11.2 deletion from multi-center cohorts, among which we retrospectively ascertained kidney morphologic and functional data from 37 individuals (12 Chinese and 25 Caucasian/Hispanic). Significantly higher CAKUT rates were observed in 16p11.2 deletion carriers (about 25% in Chinese and 16% in Caucasian/Hispanic) than those found in the non-clinically ascertained general populations (about 1/1000 found at autopsy). Furthermore, we identified seven additional individuals with heterozygous loss-of-function variants in TBX6, a gene that maps to the 16p11.2 region. Four of these seven cases showed obvious CAKUT. To further investigate the role of TBX6 in kidney development, we engineered mice with mutated Tbx6 alleles. The Tbx6 heterozygous null (i.e., loss-of-function) mutant (Tbx6+/‒) resulted in 13% solitary kidneys. Remarkably, this incidence increased to 29% in a compound heterozygous model (Tbx6mh/‒) that reduced Tbx6 gene dosage to below haploinsufficiency, by combining the null allele with a novel mild hypomorphic allele (mh). Renal hypoplasia was also frequently observed in these Tbx6-mutated mouse models. Thus, our findings in patients and mice establish TBX6 as a novel gene involved in CAKUT and its gene dosage insufficiency as a potential driver for kidney defects observed in the 16p11.2 microdeletion syndrome.

6.
J Med Genet ; 57(6): 371-379, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31888956

RESUMO

BACKGROUND: Congenital vertebral malformations (CVMs) manifest with abnormal vertebral morphology. Genetic factors have been implicated in CVM pathogenesis, but the underlying pathogenic mechanisms remain unclear in most subjects. We previously reported that the human 16p11.2 BP4-BP5 deletion and its associated TBX6 dosage reduction caused CVMs. We aim to investigate the reciprocal 16p11.2 BP4-BP5 duplication and its potential genetic contributions to CVMs. METHODS AND RESULTS: Patients who were found to carry the 16p11.2 BP4-BP5 duplication by chromosomal microarray analysis were retrospectively analysed for their vertebral phenotypes. The spinal assessments in seven duplication carriers showed that four (57%) presented characteristics of CVMs, supporting the contention that increased TBX6 dosage could induce CVMs. For further in vivo functional investigation in a model organism, we conducted genome editing of the upstream regulatory region of mouse Tbx6 using CRISPR-Cas9 and obtained three mouse mutant alleles (Tbx6up1 to Tbx6up3 ) with elevated expression levels of Tbx6. Luciferase reporter assays showed that the Tbx6up3 allele presented with the 160% expression level of that observed in the reference (+) allele. Therefore, the homozygous Tbx6up3/up3 mice could functionally mimic the TBX6 dosage of heterozygous carriers of 16p11.2 BP4-BP5 duplication (approximately 150%, ie, 3/2 gene dosage of the normal level). Remarkably, 60% of the Tbx6up3/up3 mice manifested with CVMs. Consistent with our observations in humans, the CVMs induced by increased Tbx6 dosage in mice mainly affected the cervical vertebrae. CONCLUSION: Our findings in humans and mice consistently support that an increased TBX6 dosage contributes to the risk of developing cervical CVMs.

8.
Mol Cell ; 77(2): 368-383.e7, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31677973

RESUMO

Interphase chromatin is hierarchically organized into higher-order architectures that are essential for gene functions, yet the biomolecules that regulate these 3D architectures remain poorly understood. Here, we show that scaffold attachment factor B (SAFB), a nuclear matrix (NM)-associated protein with RNA-binding functions, modulates chromatin condensation and stabilizes heterochromatin foci in mouse cells. SAFB interacts via its R/G-rich region with heterochromatin-associated repeat transcripts such as major satellite RNAs, which promote the phase separation driven by SAFB. Depletion of SAFB leads to changes in 3D genome organization, including an increase in interchromosomal interactions adjacent to pericentromeric heterochromatin and a decrease in genomic compartmentalization, which could result from the decondensation of pericentromeric heterochromatin. Collectively, we reveal the integrated roles of NM-associated proteins and repeat RNAs in the 3D organization of heterochromatin, which may shed light on the molecular mechanisms of nuclear architecture organization.


Assuntos
Heterocromatina/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas Associadas à Matriz Nuclear/genética , RNA Satélite/genética , Receptores Estrogênicos/genética , Animais , Linhagem Celular , Cromatina/genética , Genoma/genética , Humanos , Camundongos
9.
Biochem Biophys Res Commun ; 522(1): 198-204, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31757422

RESUMO

In nematode Caenorhabditis elegans, an intestinal signaling cascade in canonical Wnt/ß-catenin signaling pathway, including ß-catenin transcriptional factor BAR-1, has been identified to be involved in the control of response to simulated microgravity. However, the downstream target(s) of BAR-1 in regulating the response to simulated microgravity are still unclear. In this study, we found that BAR-1 and its direct target of Wnt effector POP-1 functioned upstream of a Meis TALE-class transcription factor UNC-62 to regulate the response to simulated microgravity. Moreover, UNC-62 regulated the response to simulated microgravity by suppressing the function of FOXO transcriptional factor DAF-16 and its target (mitochondrial Mn-SOD/SOD-3) in insulin signaling pathway. Therefore, canonical Wnt/ß-catenin signaling mediates a protective intestinal response to simulated microgravity by inducing a nucleus-mitochondria communication. Our results provide an important molecular basis for intestinal Wnt/ß-catenin signaling in response to simulated microgravity in organisms.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas do Citoesqueleto/metabolismo , Transdução de Sinais , Via de Sinalização Wnt , Animais , Mucosa Intestinal/metabolismo , Simulação de Ausência de Peso , beta Catenina/metabolismo
10.
Ecotoxicol Environ Saf ; 187: 109848, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31670182

RESUMO

Dimethyl terephthalate (DMT) is a primary ingredient widely used in the manufacture of polyesters and industrial plastics; its environmental fate is of concern due to its global use. Microorganisms play key roles in the dissipation of DMT from the environment; however, the enzymes responsible for the initial transformation of DMT and the possible altered toxicity due to this biotransformation have not been extensively studied. To reduce DMT toxicity, we identified the esterase gene dmtH involved in the initial transformation of DMT from the AOPP herbicide-transforming strain Sphingobium sp. C3. DmtH shows 24-41% identity with α/ß-hydrolases and belongs to subfamily V of bacterial esterases. The purified recombinant DmtH was capable of transforming DMT to mono-methyl terephthalate (MMT) and potentially transforming other p-phthalic acid esters, including diallyl terephthalate (DAT) and diethyl terephthalate (DET). Using C. elegans as an assay model, we observed the severe toxicity of DMT in inducing reactive oxygen species (ROS) production, decreasing locomotion behavior, reducing lifespan, altering molecular basis for oxidative stress, and inducing mitochondrial stress. In contrast, exposure to MMT did not cause obvious toxicity, induce oxidative stress, and activate mitochondrial stress in nematodes. Our study highlights the usefulness of Sphingobium sp. C3 and its esterase DmtH in transforming p-phthalic acid esters and reducing the toxicity of DMT to organisms.


Assuntos
Poluentes Ambientais/toxicidade , Esterases/genética , Genes Bacterianos , Ácidos Ftálicos/toxicidade , Sphingomonadaceae/metabolismo , Animais , Biodegradação Ambiental , Biotransformação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Poluentes Ambientais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácidos Ftálicos/metabolismo , Plásticos/química , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética
11.
J Exp Clin Cancer Res ; 38(1): 443, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31666112

RESUMO

In the original publication of this article [1], the author would like to revise Figure 4.

12.
Cell Death Dis ; 10(11): 796, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641099

RESUMO

CCL14 is a member of CC chemokines and its role in hepatocellular carcinoma (HCC) is still unknown. In this study, CCL14 expression were analyzed by tissue microarray (TMA) including 171 paired tumor and peritumor tissues of patients from Zhongshan Hospital of Fudan University. We found for the first time that CCL14 was downregulated in HCC tumor tissues compared with peritumor tissues (P = 0.01). Meanwhile, CCL14 low expression in HCC tumor tissues is associated with a poor prognosis (P = 0.035). CCL14 also displayed its predictive value in high differentiation (P = 0.026), liver cirrhosis (P = 0.003), and no tumor capsule (P = 0.024) subgroups. The underlying mechanisms were further investigated in HCC cell lines by CCL14 overexpression and knock-down in vitro. We found overexpression of CCL14 suppressed proliferation and promoted apoptosis of HCC cells. Finally, the effect was confirmed by animal xenograft tumor models in vivo. The results shown overexpression of CCL14 lead to inhibiting the growth of tumor in nude mice. Interestingly, our data also implied that CCL14 played these effects by inhibiting the activation of Wnt/ß-catenin pathway. These findings suggest CCL14 is a novel prognostic factor of HCC and serve as a tumor suppressor.


Assuntos
Carcinoma Hepatocelular/metabolismo , Quimiocinas CC/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Apoptose/fisiologia , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Regulação para Baixo , Feminino , Células Hep G2 , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Prognóstico
13.
Ecotoxicol Environ Saf ; 186: 109782, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31614302

RESUMO

Considering the short life-cycle property, Caenorhabditis elegans is a suitable animal model to evaluate the long-term effects of microgravity stress on organisms. Canonical Wnt/ß-catenin signaling is evolutionarily conserved in various organisms. We here investigated the response of canonical Wnt/ß-catenin signaling pathway to microgravity stress in nematodes. We observed the noticeable response of canonical Wnt/ß-catenin signaling to microgravity stress. In contrast, we did not detect the obvious response of non-canonical Wnt/ß-catenin signaling to microgravity stress. The canonical ß-catenin BAR-1 acted in the intestine to regulate the response to simulated microgravity. Moreover, in the intestine, we identified a signaling cascade of canonical Wnt/ß-catenin signaling pathway in response to simulated microgravity, and this signaling cascade contained Frizzled receptor MIG-1, Disheveled protein DSH-2, GSK3A/GSK-3, and ß-catenin transcriptional factor BAR-1. Our data suggests an important protective response of canonical Wnt/ß-catenin signaling to simulated microgravity in nematodes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas do Citoesqueleto/metabolismo , Intestinos/patologia , Estresse Fisiológico , Ausência de Peso , Via de Sinalização Wnt/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Modelos Biológicos
14.
Mol Cell ; 76(4): 646-659.e6, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31543422

RESUMO

Eukaryotic chromosomes contain compartments of various functions, which are marked by and enriched with specific histone modifications. However, the molecular mechanisms by which these histone marks function in chromosome compartmentalization are poorly understood. Constitutive heterochromatin is a largely silent chromosome compartment characterized in part by H3K9me2 and 3. Here, we show that heterochromatin protein 1 (HP1), an H3K9me2 and 3 "reader," interacts with SUV39H1, an H3K9me2 and 3 "writer," and with TRIM28, an abundant HP1 scaffolding protein, to form complexes with increased multivalent engagement of H3K9me2 and 3-modified chromatin. H3K9me2 and 3-marked nucleosomal arrays and associated complexes undergo phase separation to form macromolecule-enriched liquid droplets. The droplets are reminiscent of heterochromatin as they are highly dense chromatin-containing structures that are resistant to DNase and exclude the general transcription factor TFIIB. Our data suggest a general mechanism by which histone marks regulate chromosome compartmentalization by promoting phase separation.


Assuntos
Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Gotículas Lipídicas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HEK293 , Heterocromatina/genética , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Complexos Multiproteicos , Nucleossomos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
15.
J Exp Clin Cancer Res ; 38(1): 409, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533816

RESUMO

BACKGROUND: ATPase associated with a variety of cellular activities (AAA ATPase) family members are closely linked to tumor formation and progression. However, their roles in hepatocellular carcinoma (HCC) largely remain unclear. METHODS: Bioinformatic analyses of public databases were used to excavate the potential AAA ATPases that may contribute to HCC, and thyroid hormone receptor interactor 13 (TRIP13) was selected to following researches because of its most prominently differential expression. Western blot, qRT-PCR and immunohistochemistry were used to detect the expression of TRIP13 in HCC tissues, and then the relationship between TRIP13 expression and clinicopathological parameters were evaluated. Finally, its functions and potential mechanisms were investigated through a series gain- and loss-of-function strategies both in vitro and in vivo. RESULTS: TRIP13 was significantly overexpressed in HCC tissues and high level of TRIP13 was closely correlated with a worse clinical outcome. Functionally, elevated TRIP13 facilitated cell proliferation, migration, invasion, and promoted cellular epithelial-mesenchymal transition (EMT) in vitro, while promote tumor growth and lung metastasis in vivo. Mechanistically, TRIP13 interacted with ACTN4 and positively regulated its expression, thus activating the AKT/mTOR pathway to drive tumor progression. Moreover, miR-192-5p served as an upstream regulator of TRIP13 by directly binding to TRIP13 mRNA 3' UTR, which may partially explain the high expression of TRIP13 in HCC. CONCLUSION: Our findings identified TRIP13 as a promising candidate oncogene in HCC, and TRIP13 induced cell migration, invasion and metastasis of HCC through the AKT/mTOR signaling via interacting with ACTN4.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Actinina/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Transdução de Sinais , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
16.
J Cell Physiol ; 234(11): 19875-19885, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30989656

RESUMO

Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is abnormally overexpressed in multiple cancers and closely correlated with tumor-promoting effects, such as high proliferation. However, how UHRF1 functions in intrahepatic cholangiocarcinoma (ICC) has not yet been determined. Herein, we found that UHRF1 is overexpressed in ICC tissues. Downregulated UHRF1 attenuated the transition of the G1/S cell cycle and then suppressed cell proliferation in vitro and tumor growth in vivo. Moreover, upstream regulators of the UHRF1 expression were predicted, and we found that direct binding of miR-124-3p inhibited the UHRF1 expression. Elevated miR-124-3p suppressed proliferation and led to the arrest of the cell cycle. Furthermore, the expression of UHRF1 was positively correlated with PCNA. Clinically, we showed that elevated UHRF1 was associated with poor prognosis, and served as an independent prognostic factor in ICC patients. Together, these findings demonstrate that UHRF1, regulated by miR-124-3p, acts as a tumor promoter by promoting cell proliferation in ICC.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
17.
Mol Genet Genomics ; 294(2): 493-500, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30604070

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUT) are a wide range of congenital structural renal defects. CAKUT is the leading cause of chronic renal failure and end-stage renal disease in children. Studies in humans and animal models have confirmed the large genetic contribution to CAKUT. The previous evidence suggested that human TBX6 coding mutations might cause CAKUT via gene-dosage insufficiency. However, the potential involvement of TBX6 noncoding mutations in CAKUT remains to be elucidated. Here, we described DNA sequencing and copy-number analysis of TBX6 in 269 Chinese subjects with CAKUT. Interestingly, we identified two heterozygous noncoding variants of TBX6 in sporadic subjects with CAKUT: one is c.769-7delT, from a subject with duplex renal and collecting system, and the other is a 3' untranslated region (3'-UTR) variant (c.1392C>T) from a subject with unilateral renal hypoplasia. These two TBX6 noncoding variants are novel and extremely rare, respectively, in human populations archived in the ExAC database. The mini-gene splicing assay showed that the TBX6 c.769-7delT variant significantly reduced the splicing efficiency of TBX6 intron 5 when compared to the wild-type control. In this work, we identified a novel splicing variant of TBX6 in human CAKUT. Our experimental observations suggested that the TBX6 noncoding variant can affect gene expression and may potentially be involved in human CAKUT.


Assuntos
Sítios de Splice de RNA/genética , Análise de Sequência de DNA , Proteínas com Domínio T/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Adolescente , Criança , Feminino , Humanos , Rim/fisiopatologia , Masculino , Mutação , Fenótipo , Anormalidades Urogenitais/fisiopatologia , Refluxo Vesicoureteral/fisiopatologia , Sequenciamento Completo do Exoma
18.
Genet Med ; 21(7): 1548-1558, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30636772

RESUMO

PURPOSE: To characterize clinically measurable endophenotypes, implicating the TBX6 compound inheritance model. METHODS: Patients with congenital scoliosis (CS) from China(N = 345, cohort 1), Japan (N = 142, cohort 2), and the United States (N = 10, cohort 3) were studied. Clinically measurable endophenotypes were compared according to the TBX6 genotypes. A mouse model for Tbx6 compound inheritance (N = 52) was investigated by micro computed tomography (micro-CT). A clinical diagnostic algorithm (TACScore) was developed to assist in clinical recognition of TBX6-associated CS (TACS). RESULTS: In cohort 1, TACS patients (N = 33) were significantly younger at onset than the remaining CS patients (P = 0.02), presented with one or more hemivertebrae/butterfly vertebrae (P = 4.9 × 10‒8), and exhibited vertebral malformations involving the lower part of the spine (T8-S5, P = 4.4 × 10‒3); observations were confirmed in two replication cohorts. Simple rib anomalies were prevalent in TACS patients (P = 3.1 × 10‒7), while intraspinal anomalies were uncommon (P = 7.0 × 10‒7). A clinically usable TACScore was developed with an area under the curve (AUC) of 0.9 (P = 1.6 × 10‒15). A Tbx6-/mh (mild-hypomorphic) mouse model supported that a gene dosage effect underlies the TACS phenotype. CONCLUSION: TACS is a clinically distinguishable entity with consistent clinically measurable endophenotypes. The type and distribution of vertebral column abnormalities in TBX6/Tbx6 compound inheritance implicate subtle perturbations in gene dosage as a cause of spine developmental birth defects responsible for about 10% of CS.


Assuntos
Dosagem de Genes , Padrões de Herança , Escoliose/congênito , Escoliose/genética , Proteínas com Domínio T/genética , Animais , Estudos de Coortes , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Genéticos , Escoliose/classificação , Escoliose/patologia , Coluna Vertebral/patologia
19.
Hum Mol Genet ; 28(4): 539-547, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30307510

RESUMO

Congenital vertebral malformations (CVMs) are associated with human TBX6 compound inheritance that combines a rare null allele and a common hypomorphic allele at the TBX6 locus. Our previous in vitro evidence suggested that this compound inheritance resulted in a TBX6 gene dosage of less than haploinsufficiency (i.e. <50%) as a potential mechanism of TBX6-associated CVMs. To further investigate this pathogenetic model, we ascertained and collected 108 Chinese CVM cases and found that 10 (9.3%) of them carried TBX6 null mutations in combination with common hypomorphic variants at the second TBX6 allele. For in vivo functional verification and genetic analysis of TBX6 compound inheritance, we generated both null and hypomorphic mutations in mouse Tbx6 using the CRISPR-Cas9 method. These Tbx6 mutants are not identical to the patient variants at the DNA sequence level, but instead functionally mimic disease-associated TBX6 variants. Intriguingly, as anticipated by the compound inheritance model, a high penetrance of CVM phenotype was only observed in the mice with combined null and hypomorphic alleles of Tbx6. These findings are consistent with our experimental observations in humans and supported the dosage effect of TBX6 in CVM etiology. In conclusion, our findings in the newly collected human CVM subjects and Tbx6 mouse models consistently support the contention that TBX6 compound inheritance causes CVMs, potentially via a gene dosage-dependent mechanism. Furthermore, mouse Tbx6 mutants mimicking human CVM-associated variants will be useful models for further mechanistic investigations of CVM pathogenesis in the cases associated with TBX6.


Assuntos
Anormalidades Congênitas/genética , Escoliose/genética , Coluna Vertebral/anormalidades , Proteínas com Domínio T/genética , Adolescente , Alelos , Animais , Sistemas CRISPR-Cas/genética , Criança , Pré-Escolar , Anormalidades Congênitas/diagnóstico por imagem , Anormalidades Congênitas/fisiopatologia , Modelos Animais de Doenças , Feminino , Haploinsuficiência , Humanos , Lactente , Masculino , Camundongos , Mutação , Fenótipo , Escoliose/diagnóstico por imagem , Escoliose/fisiopatologia , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/fisiopatologia
20.
Genomics ; 111(6): 1745-1751, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30529537

RESUMO

The copy number variation (CNV) is an important genetic marker in cancer and other diseases. To detect CNVs of specific genetic loci, the multiplex ligation-dependent probe amplification (MLPA) is an appropriate approach, but the experimental optimization and probe synthesis are still great challenges. The multiplex competitive PCR is an alternative method for CNV detection. However, the construction of internal competitive template and establishment of a stable multiplex PCR system are the main limiting factors for this method. Here, we introduce a novel multiplex fluorescent competitive PCR (NMFC-PCR) for detecting CNVs. In this method, the blunt hairpin primers are used to rapidly establish a stable multiplex PCR system due to the reduction of non-specific amplification, and limited cycles' amplification is used to obtain the internal competitive template instead of artificial synthesis. With this method, we tested 21 clinical samples with potential LIM homeobox 1 (LHX1) or T-box 6 (TBX6) deletion. Every three segments located on the LHX1 and TBX6 were selected as the target regions, while two segments located on X-chromosome and five segments located on autosome were selected as the reference regions for detecting CNVs. The results showed that the gender information of 21 samples can be accurately inferred by the copy number ratio (CNR) of X-chromosomal reference region to autosomal reference region (X/A), and 2 samples had one copy of LHX1 and 9 samples had one copy of TBX6. To evaluate the accuracy of NMFC-PCR, 5 random samples with CNV were also detected by array-based comparative genomic hybridization (aCGH), and the results of aCGH were consistent with the NMFC-PCR results. To further assess the performance of NMFC-PCR, 60 normal samples were simultaneously tested. The results showed that the gender results were exactly the same as known information, and CNVs of LHX1 or TBX6 were not found. In conclusion, the method is a cheap, efficient, accurate, and convenient competitive PCR method for CNV detection.


Assuntos
Cromossomos Humanos X/genética , Variações do Número de Cópias de DNA , Loci Gênicos , Reação em Cadeia da Polimerase Multiplex , Hibridização Genômica Comparativa , Feminino , Humanos , Proteínas com Homeodomínio LIM/genética , Masculino , Proteínas com Domínio T/genética , Fatores de Transcrição/genética
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