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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 518-525, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292056

RESUMO

Objective To investigate the expression of microRNA-383 (miR-383) in viability classification of growing follicles and its biological function on proliferation and cycle of granulosa cells. Methods The expression of miR-383 in ovaries tissues, follicles and granulosa cells extracted from Kunming mice after birth of 12, 21 days, 21 days [after injection of pregnant mare serum gonadotropin (PMSG) for 48 hours] and 21 days [after injection of PMSG for 48 hours and (human chorionic gonadotrophin) hCG for 6 hours] were detected by real-time quantitative PCR. The miR-383 mimics or miR-383 inhibitor were transfected into granulosa cells extracted from Kunming mice after birth of 21 days, respectively as miR-383 mimics group and miR-383 inhibitor group. Meanwhile, we established negative control (NC) group. MTT assay was used to detect the proliferation and flow cytometry was used to detect cell cycle of granulosa cells. And the levels of cyclin D1, cyclin B, CDK1, CDK2, and CDK4 protein were tested by Western blot analysis. Results Compared with small preantral follicles, miR-383 level in big preantral follicles and big antral follicles were both lower, but miR-383 in small antral follicles was higher. Similarly, miR-383 of granulosa cells in big preantral follicles and small antral follicles were lower than that of granulosa cells in small preantral follicles, but up-regulated in granulosa cells of big antral follicles. Compared with granulosa cells in the NC group, the proliferation activity of granulosa cells in miR-383 mimics group was lower, and there was more granulosa cells in G0/G1 phase, less in G2/M phase in miR-383 mimics group, besides cyclin D1, cyclinB, CDK1, CDK2, CDK4 proteins in miR-383 mimics group were all lower. Meanwhile, the proliferation activity of granulosa cells in miR-383 inhibitor group was higher, and there was less granulosa cells in G0/G1 phase, more in G2/M phase in miR-383 inhibitor group, besides cyclinD1, cyclinB, CDK1, CDK2, CDK4 proteins in miR-383 inhibitor group were all higher than those proteins in the NC group. Conclusion miR-383 inhibits the proliferation of granulosa cells by down-regulating of cycle-related proteins and blocking granulosa cells in G0/G1 phase.


Assuntos
Proteínas de Ciclo Celular/genética , Células da Granulosa/citologia , MicroRNAs/genética , Animais , Regulação para Baixo , Feminino , Camundongos , Folículo Ovariano , Gravidez
2.
Exp Parasitol ; 204: 107727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31344389

RESUMO

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Citocinese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Trypanosoma rangeli/citologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Citocinese/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Citometria de Fluxo , Imunofluorescência , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Fatores de Tempo , Trypanosoma rangeli/efeitos dos fármacos , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/genética , Tripanossomíase/parasitologia
3.
Nat Commun ; 10(1): 2908, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266948

RESUMO

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity.


Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Pluripotentes/química , Ligação Proteica , Domínios Proteicos , Especificidade da Espécie
4.
Nat Commun ; 10(1): 2910, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266951

RESUMO

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinases Ciclina-Dependentes/genética , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Histochem Cell Biol ; 152(3): 207-215, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250100

RESUMO

Faithful chromosome segregation during the cell cycle is ensured by the spindle assembly checkpoint (SAC). Although SAC activity is highly conserved and most organisms share common SAC components, additional proteins that regulate SAC activity to ensure high fidelity chromosome segregation are present in higher eukaryotes. Zw10 is one of these additional SAC components. Although Zw10 has been demonstrated to be involved in SAC activity during mitosis, little is known about its role during oocyte meiosis. Here, we report that Zw10 is localized at the kinetochore and is required for SAC activation during meiotic maturation. Knockdown of Zw10 led to precocious polar body extrusion by impairing Mad2 recruitment at kinetochores. Moreover, Zw10 knockdown impaired chromosome alignment and kinetochore-microtubule attachment, increasing the incidence of aneuploidy. Furthermore, Zw10 expression decreased with maternal age, suggesting that Zw10 is associated with the age-related increase in the incidence of aneuploidy. Together, our results demonstrate that Zw10 is localized at kinetochores and functions as an essential SAC component in mouse oocytes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Oócitos/citologia , Oócitos/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Feminino , Pontos de Checagem da Fase M do Ciclo Celular/genética , Meiose/genética , Camundongos
6.
Genes Dev ; 33(13-14): 782-798, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171699

RESUMO

Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of ∼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.


Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Desmetilação do DNA , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 833-838, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204940

RESUMO

OBJECTIVE: To explore the expression level of PLK1 in mantle cell lymphoma(MCL), and the effect of silencing PLK1 gene by RNA interference on the cell proliferation, apoptosis, and cell cycle. METHODS: S-P immunohistochemistry technique was used to detect the expression of PLK1 in tissues of 42 patients with MCL and 30 patients with reactive proliferative lymphodenitis(RPL), their expression levels were compared and analyzed. The Jeko-1 cells were transfected with lentivirus contaiming PLK-1 shRNA, then the mRNA and protein expression of PLK-1 was detected by real-time guantitative PCR and Western blot nespectively, and the silencing efficacy of PLK-1 shRNA was identificd. The cell proliferation was detected by CCK method, the cell apoptosis was detected by Annexin V/PI double staining, the cell cycle was detected by PI single staining, the changes of apoptosis-related proteins BAX, BCL-2 and Caspase 3 were detected by Western blot. RESULTS: The positive expression rate of PLK-1 in tissue of MCL patients was 66.67%(28/42), which was significanfly higher than 20%(6/30) in tissue of RPL patients (P<0.05). The PLK-1 positive expression correlated with B symptom, IPI score, Ann-Arbor stage(P<0.05). After infection of Jeko-1 cells with lentivirus containing PLK-1 shRNA for 72 hours, the mRNA and protein expressions of PLK-1 were significantly down-regulated(P<0.05), the proliferation rate of cells in group of PLK-1 shRNA was significanly lower than that in control and Neg shRNA groups(P<0.05); the apoptosis rate of cells in PLK-1 shRNA group was (27.42±3.44)%, which was significantly higher than that in control group (1.23±0.42)% and Neg shRNA group (2.07±0.58) % (P<0.05). The cell cycle analysis showed that the cell ratio in G2/M phase of PLK-1 shRNA group was (27.21±3.59) %, which was higher than that in control group (13.28±2.63)% and Neg shRNA group (14.34±2.37) %. The detection of apoptosis-related proteins showed that the expression of BAX was up-regulated, the expression of BCL-2 was down-regnlated and the expression of caspase 3 was up-regulated. CONCLUSION: The PLK-l overexpression appears in tissue of MCL patients. The silencing PLK-1 gene can inhibit the proliferation of Jeko-1 cells, induce the apopotosis of Jeko-1 cells and arrestes cell cycle in G2/M phase.


Assuntos
Proteínas de Ciclo Celular/genética , Linfoma de Célula do Manto , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Linfoma de Célula do Manto/genética , RNA Interferente Pequeno
8.
Nat Commun ; 10(1): 2612, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197141

RESUMO

Primary microcephaly is caused by mutations in genes encoding centrosomal proteins including WDR62 and KIF2A. However, mechanisms underlying human microcephaly remain elusive. By creating mutant mice and human cerebral organoids, here we found that WDR62 deletion resulted in a reduction in the size of mouse brains and organoids due to the disruption of neural progenitor cells (NPCs), including outer radial glia (oRG). WDR62 ablation led to retarded cilium disassembly, long cilium, and delayed cell cycle progression leading to decreased proliferation and premature differentiation of NPCs. Mechanistically, WDR62 interacts with and promotes CEP170's localization to the basal body of primary cilium, where CEP170 recruits microtubule-depolymerizing factor KIF2A to disassemble cilium. WDR62 depletion reduced KIF2A's basal body localization, and enhanced KIF2A expression partially rescued deficits in cilium length and NPC proliferation. Thus, modeling microcephaly with cerebral organoids and mice reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly, disruption of which contributes to microcephaly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Neuroglia/citologia , Neuroglia/patologia , Organoides/patologia , Fosfoproteínas/genética , RNA Interferente Pequeno/metabolismo
9.
Gastroenterology ; 157(3): 744-759.e4, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31154022

RESUMO

BACKGROUND & AIMS: Many genetic and environmental factors, including family history, dietary fat, and inflammation, increase risk for colon cancer development. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates systemic lipid homeostasis. We explored the role of intestinal PPARα in colon carcinogenesis. METHODS: Colon cancer was induced in mice with intestine-specific disruption of Ppara (PparaΔIE), Pparafl/fl (control), and mice with disruption of Ppara that express human PPARA (human PPARA transgenic mice), by administration of azoxymethane with or without dextran sulfate sodium (DSS). Colons were collected from mice and analyzed by immunoblots, quantitative polymerase chain reaction, and histopathology. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses were performed on urine and colons. We used molecular biology and biochemical approaches to study mechanisms in mouse colons, primary intestinal epithelial cells, and colon cancer cell lines. Gene expression data and clinical features of patients with colorectal tumors were obtained from Oncomine, and human colorectal-tumor specimens and adjacent normal tissues were collected and analyzed by immunohistochemistry. RESULTS: Levels of Ppara messenger RNA were reduced in colon tumors from mice. PparaΔIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation-related metabolites in urine and colons from PparaΔIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from PparaΔIE mice, compared with colon tumors from control mice. Depletion of PPARα reduced the expression of retinoblastoma protein, resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and retinoblastoma protein and higher levels of DNMT1 and PRMT6 than normal colon tissues. CONCLUSIONS: Loss of PPARα from the intestine promotes colon carcinogenesis by increasing DNMT1-mediated methylation of P21 and PRMT6-mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA messenger RNA and protein than nontumor tissues. Agents that activate PPARα might be developed for chemoprevention or treatment of colon cancer.


Assuntos
Adenocarcinoma/prevenção & controle , Colo/enzimologia , Neoplasias do Colo/prevenção & controle , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Proteínas Nucleares/metabolismo , PPAR alfa/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Anticarcinógenos/farmacologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/efeitos dos fármacos , Bases de Dados Genéticas , Modelos Animais de Doenças , Fenofibrato/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , PPAR alfa/agonistas , PPAR alfa/deficiência , PPAR alfa/genética , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais
10.
Life Sci ; 232: 116597, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31238052

RESUMO

LncRNA SNHG3 (SNHG3) is involved in tumor development and progression, but little is known about how SNHG3 functions in laryngeal carcinoma (LC). Real time-PCR (RT-PCR) was used to estimate the expression of SNHG3 in LC tissues and cell lines TU212, TU686, and Hep-2. Cell viability, migration, and invasion were evaluated. Our results showed increased SNHG3 in LC tissues and cell lines. Loss of function of SNHG3 reduced cell viability, migration, and invasion of TU212 and TU686 cells. Western blot analyses demonstrated that the protein levels of MMP2 and MMP9 decreased after SNHG3 silencing. Additionally, bioinformatics software predicted that SNHG3 could sponge miR-384 at the 3'-UTR with complementary binding sites, which was validated by a dual-luciferase reporter assay. RT-PCR analysis revealed that knockdown of SNHG3 upregulated miR-384 expression and that overexpression of miR-384 decreased SNHG3. Furthermore, a dual-luciferase reporter assay showed that miR-384 could bind to the 3'-UTR of WEE1, and inhibition of miR-384 markedly increased WEE1 expression. The mRNA and protein levels of WEE1 were downregulated upon deletion of SNGH3. Suppression of WEE1 partly abolished the tumorigenic migration and invasion potential of the miR-384 inhibitor in migration and invasion. Inhibition of miR-384 partially reversed the biological activities of SNHG3 in TU212 and TU686 cells. Collectively, our results indicate that SNHG3 regulated LC cell migration and invasion via the miR-384/WEE1 axis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Carcinogênese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Neoplasias Laríngeas/patologia , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética
11.
Anticancer Res ; 39(5): 2325-2335, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092424

RESUMO

BACKGROUND/AIM: The minichromosome maintenance proteins (MCMs) may be potential biomarkers of cancer cell proliferation. They are essential to initiate DNA replication. The aim of the study was to investigate the level of MCM5 expression in benign lesions (BLs) and laryngeal squamous cell cancer (LSCC). MATERIALS AND METHODS: Immunohistochemical (IHC) analysis was carried out on 83 LSCCs and 10 BLs. Western-blot, immunofluorescence analysis (IF) and real-time PCR (RT-PCR) were performed using HEp-2 cancer cells and HaCaT keratinocytes. RESULTS: The expression of MCM5 was higher in LSCC than in the BLs (p<0.0001) and was higher in subsequent malignancies of LSCC. Positive correlations were demonstrated between the expression levels of MCM5 and the Ki-67 antigen. In vitro studies have confirmed that the expression of MCM5 is elevated in cancer cells. CONCLUSION: MCM5 protein may be used as a potential marker of cancer cell proliferation in LSCC.


Assuntos
Proteínas de Ciclo Celular/genética , Antígeno Ki-67/genética , Neoplasias Laríngeas/genética , Neoplasias/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Idoso , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
12.
Nat Genet ; 51(5): 815-823, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31043753

RESUMO

We sought to identify susceptibility genes for high-grade serous ovarian cancer (HGSOC) by performing a transcriptome-wide association study of gene expression and splice junction usage in HGSOC-relevant tissue types (N = 2,169) and the largest genome-wide association study available for HGSOC (N = 13,037 cases and 40,941 controls). We identified 25 transcriptome-wide association study significant genes, 7 at the junction level only, including LRRC46 at 19q21.32, (P = 1 × 10-9), CHMP4C at 8q21 (P = 2 × 10-11) and a PRC1 junction at 15q26 (P = 7 × 10-9). In vitro assays for CHMP4C showed that the associated variant induces allele-specific exon inclusion (P = 0.0024). Functional screens in HGSOC cell lines found evidence of essentiality for three of the new genes we identified: HAUS6, KANSL1 and PRC1, with the latter comparable to MYC. Our study implicates at least one target gene for 6 out of 13 distinct genome-wide association study regions, identifying 23 new candidate susceptibility genes for HGSOC.


Assuntos
Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/genética , Processamento Alternativo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Bases de Dados Genéticas , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Modelos Genéticos , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Transcriptoma
13.
Carbohydr Polym ; 216: 97-106, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047086

RESUMO

Here, we identified inulin-type oligosaccharides with 3-13 degrees of polymerization from Morinda officinalis. Radical-scavenging assays showed that Inulins 4-7 had modest anti-oxidative effects. Inulins 4 and 5 dose-dependently increased human umbilical vein endothelial cell survival during hypoxia/re-oxygenation (H/R)-induced injury, and Inulin 5 promoted angiogenesis. Triplicate assays with the Affymetrix Human Transcriptome Array 2.0 showed that Inulin 5 exposure up-regulated genes associated with cell cycle progression, apoptosis, DNA replication and repair, ubiquitin-mediated proteolysis, the mitogen-activated protein kinase pathway, and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-signaling pathway. Flow cytometry, reverse transcription-quantitative polymerase chain reaction, and western blot experiments verified the microarray results and demonstrated that Inulin 5 influenced cell cycle progression and the PI3K-protein kinase B (PKB)-endothelial nitric oxide synthase (eNOS) pathway. Thus, inulin-type oligosaccharides from M. officinalis roots may protect against H/R-induced injury, primarily through an anti-oxidative effect, and promote angiogenesis by activating the PI3K-PKB-eNOS-signaling pathway.


Assuntos
Depuradores de Radicais Livres/farmacologia , Hipóxia/tratamento farmacológico , Inulina/farmacologia , Morinda/química , Oligossacarídeos/farmacologia , Indutores da Angiogênese/isolamento & purificação , Indutores da Angiogênese/farmacologia , Sequência de Carboidratos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Depuradores de Radicais Livres/isolamento & purificação , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Inulina/isolamento & purificação , Peso Molecular , Óxido Nítrico Sintase Tipo III/metabolismo , Oligossacarídeos/isolamento & purificação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
14.
Biosci Biotechnol Biochem ; 83(9): 1697-1702, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31062645

RESUMO

An enhancer located upstream of the transcriptional start site of Ccnb1ip1 containing two GGA-rich regions and a 14-GGA repeat (GGA)14 region has been previously identified. Three copies of four GGA repeats in the c-myb promoter that form a tetrad:heptad:heptad:tetrad (T:H:H:T) dimerized G-quadruplex (G4) structure reportedly functions as both a transcriptional repressor and activator. Here, the secondary structures of the two GGA-rich and (GGA)14 regions were analyzed using circular dichroism spectral analysis, which indicated that the two GGA-rich DNAs formed parallel-type G4 structures, whereas (GGA)14 DNA formed the T:H:H:T dimerized G4 structure. Reporter assays demonstrated that individual regions did not show enhancer activity; however, the deletion of the (GGA)14 region resulted in 1.5-fold higher enhancer activity than that of the whole enhancer. These results indicate that the (GGA)14 region that forms the T:H:H:T dimerized G4 structure functions as a negative regulator of the Ccnb1ip1 enhancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Elementos Facilitadores Genéticos , Quadruplex G , Sequências Repetitivas de Ácido Nucleico , Animais , Dicroísmo Circular , Humanos , Camundongos , Células NIH 3T3
16.
J Dairy Sci ; 102(7): 6263-6275, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31103297

RESUMO

Domestic yaks (Bos grunniens) and domestic Taurus cattle (Bos taurus) are closely related. An interesting phenomenon in interspecific crossings is male sterility in the F1 hybrid (yattle) and F2 backcross, with no late meiotic cells or spermatids in the seminiferous tubules. The mammalian Y chromosome is crucial for spermatogenesis and male fertility. This study investigated the copy number variations and mRNA of Y-transitional region genes TSPY2 (testis specific protein, Y-linked 2 and testis-specific Y-encoded protein 3-like) and PRAMEY (preferentially expressed antigen in melanoma, Y-linked), and Y-ampliconic region genes TSPY (testis-specific Y-encoded protein 1-like), ZNF280BY (zinc finger protein 280B, Y-linked) and HSFY (heat-shock transcription factor, Y-linked) in mature testes from Taurus cattle, yaks, and yattle. Phylogenetic trees divided 33 copies of TSPY into major 2 types (TSPY-T1 and TSPY-T2), 19 copies of TSPY2 into 2 types (TSPY2-T1 and T2), and 8 copies of PRAMEY into 4 types (PRAMEY-T1 to T4). Searching by the Basic Local Alignment Search Tool of the TSPY2 coding sequences in GenBank revealed that TSPY2 was conserved in Bovidae. The TSPY2-T2 sequences were absent, whereas PRAMEY-T2 and PRAMEY-T4 were amplified on the yak Y chromosome. The average copy numbers of TSPY-T2 and ZNF280BY were significantly different between cattle and yaks. The TSPY-T2, TSPY2, PRAMEY, ZNF280BY, and HSFY genes were uniquely or predominantly expressed in testes. Reverse-transcription quantitative PCR showed that the TSPY-T2, PRAMEY-T2, HSFY, ZNF280BY, protamine 1 (PRM1), and protamine 2 (PRM2) genes were almost not expressed in yattle. The PRM1 and PRM2 genes are used as positive markers for spermatozoa. Thus, our results showed that the genomic structure of the Y-transitional and Y-ampliconic region differed between Taurus cattle and yaks. Dysregulated expression of Y-ampliconic region genes TSPY-T2, HSPY, ZNF280BY, and Y-transitional region gene PRAMEY-T2 may be associated with hybrid male sterility in yattle.


Assuntos
Antígenos de Neoplasias/genética , Bovinos/genética , Proteínas de Ciclo Celular/genética , Ligação Genética/genética , Hibridização Genética/genética , Cromossomo Y/genética , Animais , Cruzamentos Genéticos , Variações do Número de Cópias de DNA , Expressão Gênica , Regulação da Expressão Gênica , Variação Genética/genética , Infertilidade Masculina/genética , Masculino , Filogenia , RNA Mensageiro/análise , Espermatogênese/genética , Testículo/metabolismo
17.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035388

RESUMO

Transactivation of p21 (cyclin-dependent kinase inhibitor 1A, CDKN1A) is closely related to the recruitment of transcription cofactors at the p53 responsive elements (p53REs) in its promoter region. Human chromatin remodeling enzyme INO80 can be recruited to the p53REs of p21 promoter and negatively regulates p21. As one of the key subunits of the INO80 complex, YY1 has also been confirmed to bind to the p53RE sites of p21 promoter. Importantly, YY1 was recently reported to be bound and stabilized by BCCIP (BRCA2 and CDKN1A-interacting protein). Therefore, we hypothesized that the YY1/BCCIP complex plays an important role in regulating the transactivation of p21. Here we present evidence that the YY1/BCCIP complex coordinatively regulates p53RE-mediated p21 transactivation. We first confirmed the cross-interaction between YY1, BCCIP, and p53, suggesting an intrinsic link between three proteins in the regulation of p21 transcription. In dual luciferase assays, YY1 inhibited p53RE-mediated luciferase activity, whereas BCCIP revealed the opposite effect. More interestingly, the region 146-270 amino acids of YY1, which bound to BCCIP, increased p53-mediated luciferase activity, indicating the complexity of the YY1/BCCIP complex in co-regulating p21 transcription. Further in-depth research confirmed the co-occupancy of YY1/BCCIP with p53 at the p53RE-proximal region of p21. Lentiviral-mediated knockdown of BCCIP inhibited the recruitment of p53 and YY1 at the p53RE proximal region of p21; however, this phenomenon was reversed by expressing exogenous YY1, suggesting the collaborative regulation of YY1/BCCIP complex in p53RE-mediated p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Células HCT116 , Humanos , Proteínas Nucleares/genética , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Fator de Transcrição YY1/genética
18.
Korean J Parasitol ; 57(2): 185-189, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31104412

RESUMO

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.


Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Fase G2/genética , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/genética , Regulação para Cima , Antiprotozoários/metabolismo , Afidicolina/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Giardia lamblia/efeitos dos fármacos , Nocodazol/metabolismo , Análise de Sequência de RNA
19.
Mol Med Rep ; 19(6): 4935-4945, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059086

RESUMO

Previous reports have indicated a potential link between microRNA (miR)­214 and hypoxia. In the present study, the biological functions and potential mechanisms of miR­214 were determined, as well as its correlation with HIF­1α signaling in non­small cell lung cancer (NSCLC) cells. Quantitative polymerase chain reaction revealed that miR­214 expression was upregulated in lung cancer tissues compared with adjacent normal tissues. miR­214 mimics were transfected into A549 cells, and MTT, colony formation, invasion and wound healing assays were performed. It was demonstrated that miR­214 mimic transfection promoted the invasion, proliferation and migration of A549 cells. Furthermore, miR­214 inhibitor transfection decreased H1299 cell invasion, proliferation and migration. Next, the association between miR­214 expression and the HIF­1α signaling cascade was examined. It was demonstrated that miR­214 mimics upregulated the expression of hypoxia­inducible factor (HIF)­1α, vascular endothelial growth factor (VEGF), adenylate kinase 3 and matrix metalloproteinase (MMP)2, whereas miR­214 inhibitor downregulated the expression of these factors. Using prediction software, it was demonstrated that tumor suppressor ING4 was a target of miR­214. A luciferase reporter assay confirmed that ING4 was a direct target of miR­214. There was a negative correlation between ING4 and miR­214 expression in lung cancer tissues. In addition, ING4 siRNA and plasmid was transfected into cells in order to validate its effect on HIF­1α, MMP2 and VEGF expression. ING4 overexpression downregulated HIF­1α and its targets MMP2 and VEGF, while ING4 siRNA upregulated HIF­1α, MMP2 and VEGF. In conclusion, it was demonstrated that miR­214 targeted ING4 in lung cancer cells, and upregulated the HIF­1α cascade, leading to MMP2 and VEGF upregulation. This approach may help to clarify the role of miRNA in non­small lung cancer and may be a new therapeutic target for non­small lung cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células A549 , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Adulto , Idoso , Antagomirs/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Fator A de Crescimento do Endotélio Vascular/genética
20.
Int J Mol Sci ; 20(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060254

RESUMO

Acrylamide (AA) and glycidamide (GA) can be produced in carbohydrate-rich food when heated at a high temperature, which can induce a malignant transformation. It has been demonstrated that GA is more mutagenic than AA. It has been shown that the proliferation rate of some cancer cells are increased by treatment with GA; however, the exact genes that are induced by GA in most cancer cells are not clear. In the present study, we demonstrated that GA promotes the growth of prostate cancer cells through induced protein expression of the cell cycle regulator. In addition, we also found that GA promoted the migratory ability of prostate cancer cells through induced epithelial-to-mesenchymal transition (EMT)-associated protein expression. In order to understand the potential prognostic relevance of GA-mediated regulators of the cell cycle and EMT, we present a three-gene signature to evaluate the prognosis of prostate cancer patients. Further investigations suggested that the three-gene signature (CDK4, TWIST1 and SNAI2) predicted the chances of survival better than any of the three genes alone for the first time. In conclusion, we suggested that the three-gene signature model can act as marker of GA exposure. Hence, this multi-gene panel may serve as a promising outcome predictor and potential therapeutic target in prostate cancer patients.


Assuntos
Proteínas de Ciclo Celular/genética , Transição Epitelial-Mesenquimal/genética , Compostos de Epóxi/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Biomarcadores Tumorais , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/genética , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Prognóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Transdução de Sinais , Transcriptoma
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