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1.
Oral Dis ; 26(1): 53-61, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605415

RESUMO

OBJECTIVE: Proteasome activator 28γ (PA28γ) upregulation plays a critical role in the carcinogenesis of many malignancies, including oral cancer. We aim to screen the related genes of PA28γ and investigate their function in oral mucosa carcinogenesis. MATERIALS AND METHODS: Bioinformatics analysis was performed to screen the related genes of PA28γ. Immunohistochemical analysis was carried out to validate their correlation in oral squamous cell carcinoma (OSCC) and detect their expression levels in the whole process of oral mucosa carcinogenesis. The Kaplan-Meier method was used for estimating the overall survival, and the Cox models were constructed to predict the prognosis. RESULTS: U2 small nuclear RNA auxiliary factor 1 (U2AF1) was screened out, and the correlation between U2AF1 and PA28γ was further validated in OSCC. The expression levels of PA28γ and U2AF1 were gradually increased from normal to oral potentially malignant disorders (OPMD) to OSCC tissues. Overall survival was significantly shorter in patients with high U2AF1 expression and the combined application of U2AF1 and PA28γ notably improved the accuracy of prognosis prediction. CONCLUSION: U2AF1 and PA28γ might play pivotal roles in the progression of OPMD, which may provide insights into the development of new therapeutic strategies to prevent OPMD from becoming malignant.


Assuntos
Autoantígenos/genética , Carcinogênese , Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Complexo de Endopeptidases do Proteassoma/genética , Fator de Processamento U2AF/genética , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos Endogâmicos C57BL , Mucosa Bucal
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(9): 776-782, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31750817

RESUMO

Objective To prepare inducible lupus model mice and investigate the effect of nuclear autoantigenic sperm protein (NASP) gene mutation on the autoimmune response of mice with induced systemic lupus erythematosus (SLE). Methods The 3-month wild-type B6 (B6-WT) mice were used as a control group and the NASP mutant B6 (B6-NASPM) mice as an experimental group. Mouse spleen lymphocytes were activated with concanavalin A (ConA), and the DNA was extracted as autoantigen. B6-WT mice and B6-NASPM mice were immunized three times. Serum anti-double stranded DNA (dsDNA) IgG levels were detected by ELISA. Renal lesions were detected by HE staining. Immunohistochemical staining was performed to detect the deposition of IgG and complement C3 in the renal tissues. Flow cytometry was applied to compare the spleen lymphocyte subsets in B6-WT and B6-NASPM mice and to explore the mechanism of NASP gene mutation affecting the immune response in mice. Results Compared with B6-WT mice, B6-NASPM mice showed no significant changes in body weight, kidney index and spleen index; serum anti-dsDNA IgG levels significantly increased; glomerular cell proliferation was obvious and the deposition of IgG and C3 in the renal tissues increased. The proportion of spleen CD3+ T cells and natural killer (NK) cells decreased, while the proportion of CD19+ B cells and regulatory B cells (Breg) increased. Conclusion Mutation in the NASP gene can increase the levels of anti-dsDNA IgG antibodies, promote cell proliferation in the glomerulus of the kidney, deposition of IgG antibodies and complement C3, alter the proportion of immune cells in the spleen and aggravate the autoimmune response in lupus model mice.


Assuntos
Autoantígenos/genética , Autoimunidade , Lúpus Eritematoso Sistêmico/genética , Proteínas Nucleares/genética , Animais , Anticorpos Antinucleares/sangue , Proteínas de Ciclo Celular , Complemento C3/imunologia , Modelos Animais de Doenças , Glomérulos Renais/imunologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Mutação , Baço/imunologia
3.
Nat Commun ; 10(1): 4307, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541088

RESUMO

To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-ß and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).


Assuntos
Autoantígenos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Metáfase/fisiologia , Proteínas de Transporte Vesicular/metabolismo , alfa Carioferinas/metabolismo , Autoantígenos/genética , Cristalografia por Raios X , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Humanos , Fusão de Membrana , Proteínas de Membrana/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
4.
Nucleic Acids Res ; 47(17): 9368-9385, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31400113

RESUMO

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.


Assuntos
Autoantígenos/genética , RNA Helicases DEAD-box/genética , Herpesvirus Humano 8/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Argonauta/genética , Regulação Viral da Expressão Gênica/genética , Células HeLa , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , RNA Viral/genética , Replicação Viral/genética
5.
EMBO J ; 38(14): e101109, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304627

RESUMO

Centriolar satellites are small electron-dense granules that cluster in the vicinity of centrosomes. Satellites have been implicated in multiple critical cellular functions including centriole duplication, centrosome maturation, and ciliogenesis, but their precise composition and assembly properties have remained poorly explored. Here, we perform in vivo proximity-dependent biotin identification (BioID) on 22 human satellite proteins, to identify 2,113 high-confidence interactions among 660 unique polypeptides. Mining this network, we validate six additional satellite components. Analysis of the satellite interactome, combined with subdiffraction imaging, reveals the existence of multiple unique microscopically resolvable satellite populations that display distinct protein interaction profiles. We further show that loss of satellites in PCM1-depleted cells results in a dramatic change in the satellite interaction landscape. Finally, we demonstrate that satellite composition is largely unaffected by centriole depletion or disruption of microtubules, indicating that satellite assembly is centrosome-independent. Together, our work offers the first systematic spatial and proteomic profiling of human centriolar satellites and paves the way for future studies aimed at better understanding the biogenesis and function(s) of these enigmatic structures.


Assuntos
Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Proteômica/métodos , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Deleção de Genes , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mapas de Interação de Proteínas , Espectrometria de Massas em Tandem
6.
Chin Med J (Engl) ; 132(15): 1823-1832, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31306228

RESUMO

BACKGROUND: Collagen type IV (COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations. Previous studies revealed several novel mutations, including three COL4A3 missense mutations (G619R, G801R, and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA); however, the mutation mechanisms that lead to podocyte injury remain unclear. This study aimed to further explore the mutation mechanisms that lead to podocyte injury. METHODS: Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid, then stably transfected into human podocytes. Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)- and apoptosis-related mRNA and protein levels. Then, human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor). The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. RESULTS: Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes. Moreover, the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group. Compared to the other test groups, the COL4A3 c.4317delA group showed excessive ERS and apoptosis. Podocytes treated with MG132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation. MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group. Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired; this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group. CONCLUSIONS: We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation. Furthermore, proteasome pathway intervention might become a potential treatment for collagen type IV-related nephropathy caused by a severely truncated COL4A3 mutation.


Assuntos
Autoantígenos/genética , Colágeno Tipo IV/genética , Estresse do Retículo Endoplasmático/fisiologia , Mutação/genética , Podócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Brefeldina A/farmacologia , Estresse do Retículo Endoplasmático/genética , Mutação da Fase de Leitura/genética , Humanos , Lentivirus/genética , Leupeptinas/farmacologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto/genética , Podócitos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética
7.
Mol Med Rep ; 20(2): 951-958, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173237

RESUMO

Prostate cancer (PCa) is the most common malignancy among males worldwide, and is one of the leading causes of cancer­related mortality. MicroRNAs (miRs) are a type of endogenous, noncoding RNA that serve a key role in pathological processes, and have been demonstrated to be involved in the formation and progression of PCa. Previous studies have reported that miR­106b acts as an oncogene; however, the specific effects of miR­106b on PCa have not been fully elucidated. The present study aimed to investigate the role and underlying molecular mechanisms of miR­106b in the initiation and progression of PCa. In this study, miR­106b was reported to be overexpressed and la­related protein 4B (LARP4B) was downregulated in PCa tissues compared with paracancerous tissues. In addition, LARP4B was identified as a target gene of miR­106b by bioinformatics prediction analysis and a dual luciferase reporter gene assay. Furthermore, MTT, wound healing and Transwell assays were performed to evaluate PCa cell viability, and migration and invasive abilities. The data revealed that inhibition of miR­106b significantly suppressed the viability, migration and invasion of PCa cells. In addition, inhibition of miR­106b significantly suppressed the mRNA and protein expression of cancer­related genes, including matrix metalloproteinase­2, cluster of differentiation 44 and Ki­67, and increased that of the tumor suppressor, mothers against decapentaplegic homolog 2. Collectively, the findings of the present study indicated that miR­106b may target LAR4B to inhibit cancer cell viability, migration and invasion, and may be considered as a novel therapeutic target in PCa.


Assuntos
Autoantígenos/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , Ribonucleoproteínas/genética , Idoso , Antagomirs/genética , Antagomirs/metabolismo , Autoantígenos/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo
8.
World Neurosurg ; 131: e23-e31, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31238169

RESUMO

BACKGROUND: Intracranial aneurysm (IA) represents a cerebrovascular disorder that featured by dilation or bulging of the weakened blood vessel wall. When it ruptures, an IA leads to subarachnoid hemorrhage with high disability and mortality rates. Despite the numerous studies focusing on IA ruptures, little research on IA pathogenesis has been reported. In this study, we aimed to reveal key genes related to IA formation. METHODS: Four datasets from Gene Expression Omnibus data were downloaded, normalized, and separated into the IA group and the normal vessel control group for analyses. We screened for differentially expressed genes (DEGs) between groups and conducted functional enrichment, pathway enrichment, and gene set enrichment analysis analyses among significant DEGs. RESULTS: according to our analyses, significant DEGs majorly associate with smooth muscle system and the complement system. Among all DEGs, 5 down-regulated genes (MYH11, CNN1, MYOCD, ACTA1, and LMOD1) and 3 up-regulated genes (C1QB, C3AR1, and VSIG4) are most relevant in IA formation. CONCLUSIONS: Key DEGs identified in this study are related to IA pathogenesis. Among identified DEGs, LMOD1 is the most significant and merits more attention.


Assuntos
Aneurisma Intracraniano/genética , Actinas/genética , Autoantígenos/genética , Proteínas de Ligação ao Cálcio/genética , Estudos de Casos e Controles , Complemento C1q/genética , Proteínas do Sistema Complemento/genética , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Proteínas Nucleares/genética , Receptores de Complemento/genética , Transativadores/genética , Regulação para Cima , Remodelação Vascular/genética
9.
Dokl Biochem Biophys ; 485(1): 115-118, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201628

RESUMO

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.


Assuntos
Autoantígenos/metabolismo , Cadeias HLA-DRB1/metabolismo , Esclerose Múltipla/metabolismo , Proteína Básica da Mielina/metabolismo , Peptídeos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Feminino , Cadeias HLA-DRB1/química , Cadeias HLA-DRB1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Proteína Básica da Mielina/química , Proteína Básica da Mielina/genética , Peptídeos/química , Peptídeos/genética
10.
PLoS Genet ; 15(6): e1008187, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31226128

RESUMO

Active adult stem cells maintain a bipotential state with progeny able to either self-renew or initiate differentiation depending on extrinsic signals from the surrounding microenvironment. However, the intrinsic gene regulatory networks and chromatin states that allow adult stem cells to make these cell fate choices are not entirely understood. Here we show that the transcription factor DNA Replication-related Element Factor (DREF) regulates adult stem cell maintenance in the Drosophila male germline. A temperature-sensitive allele of DREF described in this study genetically separated a role for DREF in germline stem cell self-renewal from the general roles of DREF in cell proliferation. The DREF temperature-sensitive allele caused defects in germline stem cell self-renewal but allowed viability and division of germline stem cells as well as cell viability, growth and division of somatic cyst stem cells in the testes and cells in the Drosophila eye. Germline stem cells mutant for the temperature sensitive DREF allele exhibited lower activation of a TGF-beta reporter, and their progeny turned on expression of the differentiation factor Bam prematurely. Results of genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, genetically antagonize the role of DREF in germline stem cell maintenance. Taken together, these data suggest that DREF contributes to intrinsic components of the germline stem cell regulatory network that maintains competence to self-renew.


Assuntos
Adenosina Trifosfatases/genética , Células-Tronco Adultas/metabolismo , Autoantígenos/genética , Proteínas de Drosophila/genética , Proteína 4 de Ligação ao Retinoblastoma/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Masculino , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Nicho de Células-Tronco/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fator de Crescimento Transformador beta/genética
11.
BMC Bioinformatics ; 20(Suppl 4): 120, 2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999843

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. METHODS: We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3'UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples. RESULTS: For each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3'UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region. CONCLUSIONS: Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3'UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.


Assuntos
Proteínas Argonauta/metabolismo , Autoantígenos/metabolismo , Imunoprecipitação da Cromatina/métodos , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Argonauta/genética , Autoantígenos/genética , Sítios de Ligação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células MCF-7 , MicroRNAs/genética , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Máquina de Vetores de Suporte
12.
PLoS One ; 14(4): e0215215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30986258

RESUMO

The close physical proximity between the Golgi and the centrosome is a unique feature of mammalian cells that has baffled scientists for years. Several knockdown and overexpression studies have linked the spatial relationship between these two organelles to the control of directional protein transport, directional migration, ciliogenesis and mitotic entry. However, most of these conditions have not only separated these two organelles, but also caused extensive fragmentation of the Golgi, making it difficult to dissect the specific contribution of Golgi-centrosome proximity. In this study, we present our results with stable retinal pigment epithelial (RPE-1) cell lines in which GM130 was knocked out using a CRISPR/Cas9 approach. While Golgi and centrosome organization appeared mostly intact in cells lacking GM130, there was a clear separation of these organelles from each other. We show that GM130 may control Golgi-centrosome proximity by anchoring AKAP450 to the Golgi. We also provide evidence that the physical proximity between these two organelles is dispensable for protein transport, cell migration, and ciliogenesis. These results suggest that Golgi-centrosome proximity per se is not necessary for the normal function of RPE-1 cells.


Assuntos
Centrossomo/metabolismo , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/citologia , Deleção de Genes , Complexo de Golgi/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Epitélio Pigmentado da Retina/citologia
13.
Mol Med Rep ; 19(6): 4561-4568, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942447

RESUMO

Osteoarthritis (OA) is a common inflammatory joint disease. MicroRNAs (miRNAs/miRs) have been reported to be involved in the pathogenesis of OA; however, the role of miRNAs in OA remains largely unexplained. The purpose of the present study was to investigate the expression and role of miR­195­5p in OA, and to further explore the mechanism. The expression level of miR­195­5p was measured using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). TargetScan and a luciferase reporter assay were used to reveal the associations between miR­195­5p and REGγ (also known as PSME3). To investigate the role of miR­195­5p in OA, a cell model of OA was established by treating ATDC5 cells with lipopolysaccharide (LPS). Then an MTT assay was conducted to detect cell proliferation ability, and an Annexin V­fluorescein isothiocyanate/propidium iodide apoptosis detection kit was used to measure cell apoptosis. In addition, the levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α were determined using ELISA. Furthermore, gene and protein expression was measured via RT­qPCR and western blot assay, respectively. The results revealed that miR­195­5p was significantly upregulated in the articular cartilage tissues of patients with OA and in LPS stimulated ATDC5 cells. REGγ was a direct target of miR­195­5p. The repressed cell proliferation ability and enhanced cell apoptosis of ATDC5 cells induced by LPS were reversed by miR­195­5p downregulation. Furthermore, LPS stimulation significantly upregulated the levels of IL­1ß, IL­6 and TNF­α, while miR­195­5p downregulation markedly reduced the expression of inflammatory factors induced by LPS. The results also revealed that a miR­195­5p inhibitor inhibited the LPS induced repression of the Wnt/ß­catenin signaling pathway and activation of nuclear factor (NF)­κB signaling pathway in ATDC5 cells. Notably, the results of the present study also indicated that all of the effects of the miR­195­5p inhibitor on ATDC5 cells were reversed by REGγ silencing. In conclusion, the results indicated that the miR­195­5p inhibitor served a protective role in OA by inhibiting chondrocyte apoptosis and inflammatory responses by regulating the Wnt/ß­catenin and NF­κB signaling pathways.


Assuntos
Autoantígenos/genética , MicroRNAs/genética , Osteoartrite/terapia , Complexo de Endopeptidases do Proteassoma/genética , Adulto , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoartrite/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Via de Sinalização Wnt
14.
Mol Genet Genomic Med ; 7(5): e647, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30883042

RESUMO

BACKGROUND: Alport syndrome is an inherited renal disease caused by mutations in COL4A3, COL4A4, or COL4A5 genes. Coexisting mutations in either two of the three genes in Alport patients have been reported recently. However, the effect of heterozygous mutations in COL4A3 or COL4A4 genes in X-linked Alport syndrome (XLAS) patients is unclear. METHODS: Using targeted next-generation sequencing, six unrelated Chinese children were identified to have a combination of a pathogenic variant in COL4A5 and a heterozygous mutation in COL4A3 or COL4A4. They were three males and three females. Another three XLAS males each with only one pathogenic variant in COL4A5 were included. The clinical data were analyzed and compared between the males in two groups (group 1, males with a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4; group 2, males with only one pathogenic variant in COL4A5). RESULTS: Patients with XLAS who also had heterozygous pathogenic COL4A3 or COL4A4 variants accounted for 1% of Alport syndrome. In this study, three children showed coexisting pathogenic variants in COL4A5 and COL4A3. Two children showed pathogenic variants in COL4A5 and COL4A4. One child had pathogenic variants in the three COL4A3-5 genes, in which the pathogenic variant in COL4A5 was de novo and the pathogenic variants in COL4A4 and COL4A3 were inherited independently (in trans). The site and type of mutations in COL4A5 were similar between the two groups. It was revealed that males in group 1 presented more severe proteinuria than males in group 2 (p < 0.05). CONCLUSION: The present study provides further evidence for complicated genotype in Alport syndrome. For the first time, we reported a case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Moreover, we found that heterozygous pathogenic COL4A3 or COL4A4 variants are likely to make XLAS disease more serious.


Assuntos
Autoantígenos/genética , Colágeno Tipo IV/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heterozigoto , Nefrite Hereditária/genética , Criança , Pré-Escolar , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Masculino , Nefrite Hereditária/patologia , Fenótipo
15.
J Biochem ; 166(2): 163-173, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30918974

RESUMO

Collagen type XVII (COL17) is expressed in various tissues and its aberrant expression is associated with tumour progression. In this study, we investigated the regulation of COL17 expression in oral squamous cell carcinoma (OSCC) using the cell lines NA, SAS, Ca9-22, and Sa3. COL17 was induced upon p53 activation by cisplatin in SAS; however, this effect was more limited in NA and hardly in Ca9-22 and Sa3, with mutated p53. Moreover, COL17 was found to be regulated by miR203a-3p in all cell lines. Our data suggest that COL17 expression in OSCC cell lines is regulated by p53 and miR203a-3p.


Assuntos
Autoantígenos/metabolismo , Carcinoma de Células Escamosas/metabolismo , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Colágenos não Fibrilares/metabolismo , Autoantígenos/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/patologia , Colágenos não Fibrilares/genética , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/metabolismo
16.
PLoS Genet ; 15(3): e1008075, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30917130

RESUMO

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease.


Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 15/genética , Duplicações Segmentares Genômicas , Animais , Autoantígenos/genética , Instabilidade Cromossômica , Evolução Molecular , Dosagem de Genes , Rearranjo Gênico , Variação Genética , Proteínas da Matriz do Complexo de Golgi/genética , Hominidae/genética , Humanos , Família Multigênica , Filogenia , Primatas/genética , Recombinação Genética , Especificidade da Espécie
17.
Biomed Res Int ; 2019: 9218903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915365

RESUMO

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.


Assuntos
Autoantígenos/genética , Hipotireoidismo Congênito/genética , Iodeto Peroxidase/genética , Proteínas de Ligação ao Ferro/genética , Mutação/genética , Relação Estrutura-Atividade , Adolescente , Substituição de Aminoácidos/genética , Autoantígenos/química , Bangladesh/epidemiologia , Criança , Pré-Escolar , Simulação por Computador , Hipotireoidismo Congênito/epidemiologia , Hipotireoidismo Congênito/patologia , Feminino , Genótipo , Humanos , Iodeto Peroxidase/química , Proteínas de Ligação ao Ferro/química , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Fenótipo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia
18.
J Exp Clin Cancer Res ; 38(1): 138, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30922366

RESUMO

BACKGROUND: SOX11 is a transcription factor that plays an important role in mantle cell lymphoma development. However, its functional role in head and neck squamous cell carcinoma (HNSCC) remains unknown. METHODS: Protein expression was measured with Western blotting, immunohistochemistry or quantitative proteomics, and gene expression was measured with quantitative RT-PCR. Functional role of SOX11 in HNSCC was evaluated with MTS/apoptosis, migration, invasion assays and a xenograft model. A SOX11-targeting gene, SDCCAG8, was confirmed with chromatin immunoprecipitation (ChIP), luciferase reporter and rescue assays. RESULTS: SOX11 was up-regulated in recurrent versus primary HNSCC and in highly invasive versus low invasive HNSCC cell lines. Silencing SOX11 in HNSCC cell lines significantly inhibited the cell proliferation, migration, invasion and resistance to Cisplatin, and vice versa. Quantitative proteomic analysis of SOX11-silencing HNSCC cells revealed a number of differentially expressed proteins, including a down-regulated tumor antigen SDCCAG8. Silencing of SDCCAG8 in HNSCC cells also significantly inhibited the cell proliferation, migration and invasion, and vice versa. ChIP assays demonstrated that endogenous SOX11 strongly bound to Sdccag8 gene promoter in highly invasive HNSCC cells. When over-expressed in low invasive HNSCC cells, wild type SOX11 but not mutant SOX11 induced the promoter activity of Sdccag8 and significantly induced the expression of SDCCAG8. However, exogenous mutant SOX11 abolished the expression of SDCCAG8 in highly invasive HNSCC cells. In addition, the inhibitory effects of SOX11 knockdown were partially rescued by over-expression of SDCCAG8 in HNSCC cells. CONCLUSION: Collectively, our findings indicate SOX11 promotes HNSCC progression via the regulation of SDCCAG8.


Assuntos
Autoantígenos/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição SOXC/metabolismo , Regulação para Cima , Animais , Autoantígenos/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Fatores de Transcrição SOXC/genética
19.
Dis Markers ; 2019: 8705989, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881523

RESUMO

Mutations in the COL4A3 gene are frequently reported to be associated with various types of hereditary nephropathy. COL4A3 encodes the α3 chain of type IV collagen, which is the main structural protein in the basement membrane. Mutations in this gene are always related to kidney performance, and deafness and ocular lesion have also been reported. In this study, using next-generation sequencing, we investigated the DNA of a family visiting a clinic for hearing loss. A new missense mutation was found in COL4A3 of 5 patients, c.3227C>T (p.P1076L). Based on these results, we predict that the mutation is pathogenic and leads to abnormal collagen IV. Here, we report for the first time on this autosomal dominant syndrome, characterized by hearing loss and eye abnormalities, but without renal damage, in all carriers. Since the oldest patient in the trial was less than 50 years old, however, we recommend that renal examination be reviewed regularly. Our results reveal expansion in the mutation spectrum of the COL4A3 gene and phenotypic spectrum of collagen IV disease. Our study suggests that next-generation sequencing is an economical and effective method and may help in the accurate diagnosis and treatment of these patients.


Assuntos
Autoantígenos/genética , Colágeno Tipo IV/genética , Perda Auditiva/genética , Hematúria/genética , Nefrite Hereditária/genética , Fenótipo , Adolescente , Adulto , Feminino , Perda Auditiva/patologia , Hematúria/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Nefrite Hereditária/patologia , Linhagem
20.
Nucleic Acids Res ; 47(8): 4272-4291, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30820564

RESUMO

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.


Assuntos
Autoantígenos/química , Poli A/química , Proteínas de Ligação a Poli(A)/química , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Motivos de Aminoácidos , Autoantígenos/genética , Autoantígenos/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Poli A/genética , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Especificidade por Substrato , Termodinâmica
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