RESUMO
The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Replicação do DNA , Proteínas Intrinsicamente Desordenadas/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Fase G1 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Complexo de Reconhecimento de Origem/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Estabilidade Proteica , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismoRESUMO
A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2-7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1.
Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Animais , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/ultraestrutura , Ligação Proteica , Subunidades Proteicas , Relação Estrutura-AtividadeRESUMO
BACKGROUND: CDC6 is an oncogenic protein whose expression level fluctuates during the cell cycle. Although several E3 ubiquitin ligases responsible for the ubiquitin-mediated proteolysis of CDC6 have been identified, the deubiquitination pathway for CDC6 has not been investigated. METHODS: The proteome-wide deubiquitinase (DUB) screening was used to identify the potential regulator of CDC6. Immunofluorescence, protein half-life and deubiquitination assays were performed to determine the protein stability of CDC6. Gain- and loss-of-function experiments were implemented to analyse the impacts of OUTD6A-CDC6 axis on tumour growth and chemosensitivity in vitro. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced conditional Otud6a knockout (CKO) mouse model and tumour xenograft model were performed to analyse the role of OTUD6A-CDC6 axis in vivo. Tissue specimens were used to determine the association between OTUD6A and CDC6. RESULTS: OTUD6A interacts with, depolyubiquitinates and stabilizes CDC6 by removing K6-, K33-, and K48-linked polyubiquitination. Moreover, OTUD6A promotes cell proliferation and decreases sensitivity to chemotherapy by upregulating CDC6. CKO mice are less prone to BCa tumorigenesis induced by BBN, and knockdown of OTUD6A inhibits tumour progression in vivo. Furthermore, OTUD6A protein level has a positive correlation with CDC6 protein level, and high protein levels of OTUD6A and CDC6 are associated with poor prognosis in patients with bladder cancer. CONCLUSIONS: We reveal an important yet missing piece of novel DUB governing CDC6 stability. In addition, our findings propose a model for the OTUD6A-CDC6 axis that provides novel insights into cell cycle and chemosensitivity regulation, which may become a potential biomarker and promising drug target for cancer treatment.
Assuntos
Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares , Ubiquitinação , Animais , Humanos , Camundongos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Camundongos Knockout , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Neoplásica da Expressão Gênica , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Modelos Animais de DoençasRESUMO
Strict regulation of DNA replication is of fundamental significance for the maintenance of genome stability. Licensing of origins of DNA replication is a critical event for timely genome duplication. Errors in replication licensing control lead to genomic instability across evolution. Here, we present accumulating evidence that aberrant replication licensing is linked to oncogene-induced replication stress and poses a major threat to genome stability, promoting tumorigenesis. Oncogene activation can lead to defects in where along the genome and when during the cell cycle licensing takes place, resulting in replication stress. We also discuss the potential of replication licensing as a specific target for novel anticancer therapies.
Assuntos
Replicação do DNA , DNA/genética , Instabilidade Genômica/genética , Estresse Fisiológico/genética , HumanosRESUMO
Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, leads to the ubiquitination of >40 of its target substrates. Since a variety of point mutations in the substrate-binding domain of SPOP have been identified in cancers, including prostate and endometrial cancers, the pathological roles of those cancer-associated SPOP mutants have been extensively elucidated. In this study, we evaluated the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells expressing wild-type SPOP gene. SPOP knockdown using siRNA in HaCaT cells dramatically reduced cell growth and arrested their cell cycles at G1/S phase. The expression of DNA replication licensing factors CDT1 and CDC6 in HaCaT cells drastically decreased on SPOP knockdown as their translation was inhibited. CDT1 and CDC6 downregulation induced p21 expression without p53 activation. Our results suggest that SPOP is essential for DNA replication licensing in non-cancerous keratinocyte HaCaT cells.
Assuntos
Neoplasias do Endométrio , Células HaCaT , Masculino , Feminino , Humanos , Células HaCaT/metabolismo , Células HaCaT/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Ubiquitinação , Neoplasias do Endométrio/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
BACKGROUND: Cell division cycle 6 (CDC6) is a key licensing factor in the assembly of pre-replicative complexes at origins of replication. The role of CDC6 in the pathogenesis of in diffuse larger B-cell lymphoma (DLBCL) remains unknown. We aim to investigate the effects of CDC6 on the proliferation, apoptosis and cell cycle regulation in DLBCL cells, delineate its underlying mechanism, and to correlate CDC6 expression with clinical characteristics and prognosis of patients with DLBCL. METHODS: Initial bioinformatic analysis was performed to screen the potential role of CDC6 in DLBCL. Lentiviral constructs harboring CDC6 or shCDC6 was transfected to overexpress or knockdown CDC6 in SUDHL4 and OCI-LY7 cells. The cell proliferation was evaluated by CCK-8 assay, cell apoptosis was detected by Annexin-V APC/7-AAD double staining, and cell cycle was measured by flow cytometry. Real time quantitative PCR and western blot was used to characterize CDC6 expression and its downstream signaling pathways. The clinical data of DLBCL patients were retrospectively reviewed, the CDC6 expression in DLBCL or lymph node reactive hyperplasia tissues was evaluated by immunohistochemistry. RESULTS: In silico data suggest that CDC6 overexpression is associated with inferior prognosis of DLBCL. We found that CDC6 overexpression increased SUDHL4 or OCI-LY7 cell proliferation, while knockdown of CDC6 inhibited cell proliferation in a time-dependent manner. Upon overexpression, CDC6 reduced cells in G1 phase and did not affect cell apoptosis; CDC6 knockdown led to significant cell cycle arrest in G1 phase and increase in cell apoptosis. Western blot showed that CDC6 inhibited the expression of INK4, E-Cadherin and ATR, accompanied by increased Bcl-2 and deceased Bax expression. The CDC6 protein was overexpressed DLBCL compared with lymph node reactive hyperplasia, and CDC6 overexpression was associated with non-GCB subtype, and conferred poor PFS and OS in patients with DLBCL. CONCLUSION: CDC6 promotes cell proliferation and survival of DLBCL cells through regulation of G1/S cell cycle checkpoint and apoptosis. CDC6 is overexpressed and serves as a novel prognostic marker in DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Hiperplasia , Estudos Retrospectivos , Linhagem Celular Tumoral , Linfoma Difuso de Grandes Células B/patologia , Prognóstico , Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
BACKGROUND: Endocrine-resistant breast cancers have elevated expression of XBP1, where it drives endocrine resistance by controlling the expression of its target genes. Despite the in-depth understanding of the biological functions of XBP1 in ER-positive breast cancer, effectors of endocrine resistance downstream of XBP1 are poorly understood. The aim of this study was to identify the XBP1-regulated genes contributing to endocrine resistance in breast cancer. METHODS: XBP1 deficient sub-clones in MCF7 cells were generated using the CRISPR-Cas9 gene knockout strategy and were validated using western blot and RT-PCR. Cell viability and cell proliferation were evaluated using the MTS assay and colony formation assay, respectively. Cell death and cell cycle analysis were determined using flow cytometry. Transcriptomic data was analysed to identify XBP1-regulated targets and differential expression of target genes was evaluated using western blot and qRT-PCR. Lentivirus and retrovirus transfection were used to generate RRM2 and CDC6 overexpressing clones, respectively. The prognostic value of the XBP1-gene signature was analysed using Kaplan-Meier survival analysis. RESULTS: Deletion of XBP1 compromised the upregulation of UPR-target genes during conditions of endoplasmic reticulum (EnR) stress and sensitized cells to EnR stress-induced cell death. Loss of XBP1 in MCF7 cells decreased cell growth, attenuated the induction of estrogen-responsive genes and sensitized them to anti-estrogen agents. The expression of cell cycle associated genes RRM2, CDC6, and TOP2A was significantly reduced upon XBP1 deletion/inhibition in several ER-positive breast cancer cells. Expression of RRM2, CDC6, and TOP2A was increased upon estrogen stimulation and in cells harbouring point-mutants (Y537S, D538G) of ESR1 in steroid free conditions. Ectopic expression of RRM2 and CDC6 increased cell growth and reversed the hypersensitivity of XBP1 KO cells towards tamoxifen conferring endocrine resistance. Importantly, increased expression of XBP1-gene signature was associated with poor outcome and reduced efficacy of tamoxifen treatment in ER-positive breast cancer. CONCLUSIONS: Our results suggest that RRM2 and CDC6 downstream of XBP1 contribute to endocrine resistance in ER-positive breast cancer. XBP1-gene signature is associated with poor outcome and response to tamoxifen in ER-positive breast cancer.
Assuntos
Neoplasias da Mama , Tamoxifeno , Humanos , Feminino , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estrogênios/farmacologia , Células MCF-7 , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5' RACE and 3' RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5'RACE, and 464 bp of 3'RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2'- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality.
Assuntos
RNA Longo não Codificante , Animais , Bovinos , RNA Longo não Codificante/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Adipócitos/metabolismo , Divisão Celular , Proliferação de Células/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Cell division cycle 6 (CDC6) has been proven to be associated with the initiation and progression of human multiple tumors. However, it's role in glioma, which is ranked as one of the common primary malignant tumor in the central nervous system and is associated with high morbidity and mortality, is unclear. METHODS: In this study, we explored CDC6 gene expression level in pan-cancer. Furthermore, we focused on the relationships between CDC6 expression, its prognostic value, potential biological functions, and immune infiltrates in glioma patients. We also performed vitro experiments to assess the effect of CDC6 expression on proliferative, apoptotic, migrant and invasive abilities of glioma cells. RESULTS: As a result, CDC6 expression was upregulated in multiple types of cancer, including glioma. Moreover, high expression of CDC6 was significantly associated with age, IDH status, 1p/19q codeletion status, WHO grade and histological type in glioma (all p < 0.05). Meanwhile, high CDC6 expression was associated with poor overall survival (OS) in glioma patients, especially in different clinical subgroups. Furthermore, a univariate Cox analysis showed that high CDC6 expression was correlated with poor OS in glioma patients. Functional enrichment analysis indicated that CDC6 was mainly involved in pathways related to DNA transcription and cytokine activity, and Gene Set Enrichment Analysis (GSEA) revealed that MAPK pathway, P53 pathway and NF-κB pathway in cancer were differentially enriched in glioma patients with high CDC6 expression. Single-sample gene set enrichment analysis (ssGSEA) showed CDC6 expression in glioma was positively correlated with Th2 cells, Macrophages and Eosinophils, and negative correlations with plasmacytoid dendritic cells, CD8 T cells and NK CD56bright cells, suggesting its role in regulating tumor immunity. Finally, CCK8 assay, flow cytometry and transwell assays showed that silencing CDC6 could significantly inhibit proliferation, migration, invasion, and promoted apoptosis of U87 cells and U251 cells (p < 0.05). CONCLUSION: In conclusion, high CDC6 expression may serve as a promising biomarker for prognosis and correlated with immune infiltrates, presenting to be a potential immune therapy target in glioma.
Assuntos
Neoplasias Encefálicas , Glioma , Biomarcadores , Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular/genética , Glioma/patologia , Humanos , NF-kappa B , Proteínas Nucleares/genética , PrognósticoRESUMO
Radiotherapy (RT) is a conventional cancer therapeutic modality. However, cancer cells tend to develop radioresistance after a period of treatment. Diagnostic markers and therapeutic targets for radiosensitivity are severely lacking. Our recently published studies demonstrated that the cell division cycle (CDC6) is a critical molecule contributing to radioresistance, and maybe a potential therapeutic target to overcome radioresistance. In the present study, we for the first time reported that Norcantharidin (NCTD), a demethylated form of cantharidin, re-sensitized radioresistant cancer cells to overcome radioresistance, and synergistically promoted irradiation (IR)-induced cell killing and apoptosis by inducing CDC6 protein degradation. Mechanistically, NCTD induced CDC6 protein degradation through the ubiquitin-proteasome pathways. By using small interfering RNA (siRNA) interference or small compound inhibitors, we further determined that NCTD induced CDC6 protein degradation through a neddylation-dependent pathway, but not through Huwe1, Cyclin F, and APC/C-mediated ubiquitin-proteasome pathways. We screened the six most relevant Cullin subunits (CUL1, 2, 3, 4A, 4B, and 5) using siRNAs. The knockdown of Cullin1 but not the other five cullins remarkably elevated CDC6 protein levels. NCTD promoted the binding of Cullin1 to CDC6, thereby promoting CDC6 protein degradation through a Cullin1 neddylation-mediated ubiquitin-proteasome pathway. NCTD can be used in combination with radiotherapy to achieve better anticancer efficacy, or work as a radiosensitizer to overcome cancer radioresistance.
Assuntos
Proteínas de Ciclo Celular , Neoplasias , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismoRESUMO
MicroRNAs have emerged as essential regulators in the biological process of liver regeneration by modulating the post-transcriptional expression of the target genes. In the present study, we found miR-20a expression is decreased remarkably in three rodent liver regeneration models using miRNA PCR array and Venn diagram analysis. Inhibition of miR-20a expression enhanced hepatocytes proliferation in vivo and in vitro. In contrast, overexpression of miR-20a reduces hepatocytes proliferation and subsequently impaired liver regeneration in the mouse PHx model. Moreover, we have identified TCF4 as a target gene of miR-20a using the PCR Array and luciferase assay. Next, mice with TCF4 deficiency were used to establish the PHx model and subjected to the examination of liver regeneration capacity. We found TCF4-deficient mice exhibited impaired liver regeneration compared with control. Given that TCF4 acts as a transcription factor, we sort to elucidate the downstream genes involved in liver regeneration. Promoter analysis and Chip assay confirmed that TCF4 enhances CDC2 and CDC6 expression through binding to the promoter region and leads to the proliferation and cell cycle progression in hepatocytes. In conclusion, this study provides evidence that the miR20a-TCF4-CDC2/6 axis plays an essential role during liver regeneration.
Assuntos
Regulação da Expressão Gênica , Hepatectomia/métodos , Hepatócitos/patologia , Regeneração Hepática , MicroRNAs/genética , Fator de Transcrição 4/metabolismo , Animais , Proliferação de Células , Hepatócitos/metabolismo , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Fator de Transcrição 4/genéticaRESUMO
Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.
Assuntos
Proteínas de Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Meiose/genética , Proteínas Nucleares/genética , Oócitos/crescimento & desenvolvimento , Anáfase/genética , Animais , Centrossomo , Feminino , Pontos de Checagem da Fase M do Ciclo Celular/genética , Metáfase/genética , Camundongos , Oócitos/metabolismo , Fuso Acromático/genéticaRESUMO
Cdc6 is a key replication licencing factor with a pivotal role in regulating the process of DNA replication, rendering it an important investigatory focus in tumourigenesis. Indeed, Cdc6 overexpression has been found to be a feature in certain tumours and has been associated as an early event in malignancies. With a focus on pancreatic cancer, there are evidence of its convergence in downstream pathways implicated in major genetic alterations found in pancreatic cancer, primarily KRAS. There is also data of its direct influence on protumourigenic processes as a transcriptional regulator, repressing the key tumour suppressor loci CDH1 (E-Cadherin) and influencing epithelial to mesenchymal transition (EMT). Moreover, gene amplification of Cdc6 as well as of E2F (an upstream regulator of Cdc6) have also been found to be a key feature in tumours overexpressing Cdc6, further highlighting this event as a potential driver of tumourigenesis. In this review, we summarise the evidence for the role of Cdc6 overexpression in cancer, specifically that of pancreatic cancer. More importantly, we recapitulate the role of Cdc6 as part of the DNA damage response and on senescence-an important antitumour barrier-in the context of pancreatic cancer. Finally, recent emerging observations suggest that the potential of the subcellular localisation of Cdc6 in inducing senescence. In this regard, we speculate and hypothesise potentially exploitable mechanisms in the context of inducing senescence via a novel pathway involving cytoplasmic retention of Cdc6 and Cyclin E.
Assuntos
Antineoplásicos/farmacologia , Carcinogênese/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Ciclina E/metabolismo , Citoplasma/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Amplificação de Genes , Humanos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Oncogênicas/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Background: Illustrating the pathogenesis of hepatocellular carcinoma (HCC) pathogenesis as well as identifying specific biomarkers are of great significance. Methods: The original CEL files were obtain from Gene Expression Omnibus, then affymetrix package was used to preprocess the CEL files, the function of DEGs were investigated by multiple bioinformatics approach. Finally, typical HCC cell lines and tissue samples were using to validate the role of CDC6 in vitro. Bioinformatics software was used to predict potential microRNA of CDC6. Luciferase assay was used to verify the interactions between CDC6 and microRNA. Results: A total of 445 DEGs were identified in HCC tissues based on two GEO datasets. GSEA results showed that the significant enriched gene sets were only associated with cell cycle signaling pathway. In the co-expression analysis, there were 370 hub genes from the blue modules were screened. We integrated DEGs, hub genes, TCGA cohort and GSEA analyses to further obtain 10 upregulated genes for validation. These genes were overexpressed in HCC tissues and negatively associated with overall and disease-free survival in HCC patients and related to immune cell infiltration in HCC microenvironments. Finally, Cell Division Cycle 6 (CDC6) was highlighted as one of the most probable genes among the 10 candidates participating in cancer process. The expression of CDC6 either in public datasets and HCC tissues sample were commonly high than the non-cancerous counterpart. Furthermore, we recognized that miR-215-5p, could directly bind to the 3'UTR of CDC6. In addition, CDC6 promoted proliferation via regulation of G1 phase checkpoint and was negative regulated by miR-215-5p to involve in the proliferation of HCC. Conclusion: Our study suggested that CDC6 served as a potential therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Proteínas Nucleares/genética , Regiões 3' não Traduzidas/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/terapia , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/terapia , Recidiva Local de Neoplasia/genética , Proteínas Nucleares/antagonistas & inibidores , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/genética , Análise de Sobrevida , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
Preeclampsia (PE) is a serious disease that occurs after 20 weeks during pregnancy. There are some aberrant microRNAs (miRNAs) that are associated with the etiology of PE. As discovered by scholars, there was an increased level of miR-215-5p in plasma of PE patients compared with the control group; nonetheless, there is still no knowledge of the mechanism of miR-215-5p in PE. We carried out the comparison of the expression levels of miR-215-5p, and the supposed target gene cell division cycle 6 (CDC6) in 30 placentas from PE patients as well as 30 placentas from normal pregnant women. The verification of the impacts of miR-215-5p and CDC6 was carried out by functional assays in HTR-8/SVneo cells transfected with the miR-215-5p mimic or siR-CDC6. As indicated by findings, miR-215-5p showed an apparent increase; conversely, CDC6 was inhibited in the experiment group. The upregulation of miR-215-5p inhibited both the migration and invasive potential of trophoblasts, besides decreasing the G1-S transition and downregulating CDC6 in HTR-8/SVneo cells; nonetheless, it did not significantly impact the cell proliferation. Furthermore, siR-CDC6 replicated the functions of the miR-215-5p mimic. Also, the miR-215-5p mimic and siR-CDC6 both decreased the epithelial-mesenchymal transition (EMT) with additional E-cadherin level and decreased the expressions of N-cadherin as well as vimentin in trophoblast cells. To conclude, miR-215-5p decreased not only the migration but also the invasion of trophoblasts through regulating CDC6, which indicated that miR-215-5p might be associated with the etiology of PE. SIGNIFICANCE OF THE STUDY: More and more attention has been paid on the roles of miRNAs in the pathogenesis of PE. However, there is no study of miR-215-5p in the etiology of PE. We first investigated the mechanism of miR-215-5p in placental tissues and HTR-8/SVneo cells. It was suggested that miR-215-5p decreased the abilities of migration and invasion of trophoblasts through regulating CDC6 in PE. miR-215-5p might be used as an target for the early diagnosis and treatment of PE in the future.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Fase G1 , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Pré-Eclâmpsia/metabolismo , Fase S , Trofoblastos/metabolismo , Adulto , Proteínas de Ciclo Celular/genética , Linhagem Celular , Feminino , Humanos , MicroRNAs/genética , Proteínas Nucleares/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologiaRESUMO
BACKGROUND: Cell division cycle 6 (cdc6) is a key factor of DNA replication initiation license system and a proto-oncogene. It has been detected in some tumor tissues to aid cancer diagnosis in many research projects. However, it remains unclear that if cdc6 could be detected in the peripheral blood, just like liquid biopsy, in solid tumor patients. The aim of this study is to investigate the possibility of cdc6 as a biomarker for circulating tumor cells in patients with lung cancer. METHODS: We first detected the expression of cdc6 in peripheral blood mononuclear cells (PBMCs) and tumor cells by in situ hybridization with cdc6 RNA probe. Then, we examined the expression of cdc6 in PBMCs from health individual, mononuclear cells from cord blood, or A549 cell line by RT-qPCR. Furthermore, we used RT-qPCR to test the cdc6 expression in PBMCs from tumor patients (test group) and non-tumor individuals as a control group. Chi-square test with Fisher's exact test was used to analyze the statistical significance of the difference. P < .05 is considered as statistically significant difference. RESULTS: When compared the cdc6 expression in cells from difference sources, we found that A549 tumor cell line had the strongest expression of cdc6, samples from cord blood showed the least expression level, indicating the detection strategy of RT-qPCR is reasonable. Using this method, we studied whether cdc6 in Peripheral blood could be detected as a biomarker by examining cdc6 expression from PBMCs of patients with lung cancer. We found 20% of patients with lung cancer were cdc6 positive in PBMCs, whereas only 4.26% was found positive in the control group (P = .039, P < .05). CONCLUSION: Cell division cycle 6 has a potential to be used as a circulating tumor cell biomarker for lung cancer. Further study in clinical application is still broad needed.
Assuntos
Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Proteínas Nucleares/genética , Células A549 , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Sangue Fetal/citologia , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ/métodos , Pneumopatias/genética , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Projetos Piloto , Proto-Oncogene MasRESUMO
Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.
Assuntos
Replicação do DNA , Células Eucarióticas/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Ativação Enzimática , Humanos , Proteínas de Manutenção de Minicromossomo/análise , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/análise , Complexo de Reconhecimento de Origem/metabolismo , Origem de ReplicaçãoRESUMO
Rapid proliferation and metastasis of breast cancers resulted in poor prognosis in clinic. Recent studies have proved that long noncoding RNAs (lncRNAs) were involved in tumor progression. In this study, we aimed to determine the roles and mechanisms of lncRNA-cell division cycle 6 (CDC6) in regulating proliferation and metastasis of breast cancer. Clinically, lncRNA-CDC6 was highly expressed in tumor tissues and was positively correlated with clinical stages of breast cancers. Functionally, the ectopic expression of lncRNA-CDC6 promoted proliferation via regulation of G1 phase checkpoint, and further promoting the migration capability. Moreover, lncRNA-CDC6 could function as competitive endogenous RNA (ceRNA) via directly sponging of microRNA-215 (miR-215), which further regulating the expression of CDC6. Taken together, our results proved that lncRNA-CDC6 could function as ceRNA and promote the proliferation and metastasis of breast cancer cells, which provided a novel prognostic marker for breast cancers in clinic.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Progressão da Doença , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Transdução de SinaisRESUMO
Execution of the meiotic and mitotic cell division programs requires distinct gene expression patterns. Unlike mitotic cells, meiotic cells reduce ploidy by following one round of DNA replication with two rounds of chromosome segregation (meiosis I and meiosis II). However, the mechanisms by which cells prevent DNA replication between meiosis I and meiosis II are not fully understood. Here, we show that transcriptional repression of two essential DNA replication genes, CDC6 and SLD2, is associated with production of shorter meiosis-specific RNAs containing the 3' end of both genes. Despite the short CDC6 RNA coding for a short protein (Cdc6short), this protein is not essential for meiosis and it does not have either a positive or negative impact on DNA replication. Production of CDC6short mRNA does not require the upstream CDC6 promoter (PCDC6) and is not a processed form of the full-length RNA. Instead, CDC6short depends on transcription initiation from within the ORF upon repression of PCDC6. Finally, using CDC6 genes from related yeast, we show that repression of full-length CDC6 mRNA is evolutionarily conserved and that this repression is consistently associated with production of unique short CDC6 RNAs. Together, these data demonstrate that meiotic cells transcriptionally repress full-length CDC6 and SLD2, and that inactivation of PCDC6 results in heterogeneous transcription initiation from within the CDC6 ORF.
Assuntos
Proteínas de Ciclo Celular/genética , Meiose , Isoformas de RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces/genética , Fatores de Elongação da Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Códon de Iniciação , Evolução Molecular , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Isoformas de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
In nature, cells face a variety of stresses that cause physical damage to the plasma membrane and cell wall. It is well established that evolutionarily conserved cell cycle checkpoints monitor various cellular perturbations, including DNA damage and spindle misalignment. However, the ability of these cell cycle checkpoints to sense a damaged plasma membrane/cell wall is poorly understood. To the best of our knowledge, our recent paper described the first example of such a checkpoint, using budding yeast as a model. In this review, we will discuss this important question as well as provide hypothetical explanations to be tested in the future.