RESUMEN
In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.
Asunto(s)
Cromatina , Replicación del ADN , Fase G1 , Complejo de Reconocimiento del Origen , Origen de Réplica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicación del ADN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Fase G1/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Fase S/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dominios Proteicos/genética , Sitios de Unión , Unión Proteica , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.
Asunto(s)
Ciclo Celular , Marcación de Gen , Proteína 2 Homóloga a MutS , Recombinasa Rad51 , Proteína Recombinante y Reparadora de ADN Rad52 , Humanos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Ciclo Celular/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 2 Homóloga a MutS/genética , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Recombinación Homóloga , Roturas del ADN de Doble Cadena , Reparación del ADN , Reparación del ADN por Unión de Extremidades , Fase G1/genéticaRESUMEN
The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fase G1/genética , Regiones Promotoras Genéticas , Meiosis , Regulación Fúngica de la Expresión Génica , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children. Due to drug resistance to radiotherapy and chemotherapy, mainly due to the existence of cancer stem cells (CSCs), some children still have a poor prognosis. Therefore, researchers have focused their attention on CSCs. Our research group successfully constructed cancer stem cell-like cells named Piwil2-iCSCs by reprogramming human preputial fibroblasts (FBs) with the PIWIL2 gene in the early stage, and Piwil2-iCSCs were confirmed to induce the formation of embryonic tumors. PiRNAs, noncoding small RNAs that interact with PIWI proteins, play important roles in a variety of tumors. Therefore, our study aimed to explore the role of differentially expressed (DE) piRNAs derived from sequencing of Piwil2-iCSCs in NB. METHODS: The DE piRNAs in Piwil2-iCSCs were screened using high-throughput sequencing and further verified in NB tissues and cells. An unknown piRNA, named piRNA-MW557525, showed obvious downregulation in NB. Thus we studied the effect of piRNA-MW557525 on the biological behavior of NB through in vitro and in vivo experiments. On this basis, we successfully constructed a stably transfected NB cell line overexpressing piRNA-MW557525 and performed transcriptome sequencing to further explore the mechanism of piRNA-MW557525 in NB. RESULTS: In vitro, piRNA-MW557525 inhibited NB cell proliferation, migration and invasion and induced apoptosis; in vivo, piRNA-MW557525 significantly reduced the volume and weight of tumors and inhibited their proliferation, migration and invasion. piRNA-MW557525 overexpression induced G0/G1 phase arrest in NB cells via activation of the P53-P21-CDK2-Cyclin E signaling pathway thus inhibiting NB growth. CONCLUSIONS: Our findings show that piRNA-MW557525 functions as a tumor suppressor gene in NB and may serve as an innovative biomarker and possible therapeutic target for NB.
Asunto(s)
Neuroblastoma , ARN de Interacción con Piwi , Niño , Humanos , Proteína p53 Supresora de Tumor/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Fase G1/genética , Proliferación Celular/genética , Transducción de Señal/genética , Línea Celular Tumoral , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMEN
BACKGROUND: Cyclin D1 overexpression may contribute to development of various cancers, including breast cancer, and thus may serve as a key cancer diagnostic marker and therapeutic target. In our previous study, we generated a cyclin D1-specific single-chain variable fragment antibody (ADκ) from a human semi-synthetic single-chain variable fragment library. ADκ specifically interacted with recombinant and endogenous cyclin D1 proteins through an unknown molecular basis to inhibit HepG2 cell growth and proliferation. RESULTS: Here, using phage display and in silico protein structure modeling methods combined with cyclin D1 mutational analysis, key residues that bind to ADκ were identified. Notably, residue K112 within the cyclin box was required for cyclin D1-ADκ binding. In order to elucidate the molecular mechanism underlying ADκ anti-tumor effects, a cyclin D1-specific nuclear localization signal-containing intrabody (NLS-ADκ) was constructed. When expressed within cells, NLS-ADκ interacted specifically with cyclin D1 to significantly inhibit cell proliferation, induce G1-phase arrest, and trigger apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. Moreover, the NLS-ADκ-cyclin D1 interaction blocked binding of cyclin D1 to CDK4 and inhibited RB protein phosphorylation, resulting in altered expression of downstream cell proliferation-related target genes. CONCLUSION: We identified amino acid residues in cyclin D1 that may play key roles in the ADκ-cyclin D1 interaction. A nuclear localization antibody against cyclin D1 (NLS-ADκ) was constructed and successfully expressed in breast cancer cells. NLS-ADκ exerted tumor suppressor effects via blocking the binding of CDK4 to cyclin D1 and inhibiting phosphorylation of RB. The results presented here demonstrate anti-tumor potential of intrabody-based cyclin D1-targeted breast cancer therapy.
Asunto(s)
Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Quinasa 4 Dependiente de la Ciclina/genética , Fase G1/genética , Fosforilación , Ciclina D1/inmunologíaRESUMEN
Abstract EphrinB2 plays a critical role in tumor growth. In this study, we studied the antitumor activity of imperatorin derivative IMP-1 in renal cell carcinoma (RCC) by regulating EphrinB2 pathway.. Results showed that IMP-1 inhibited the proliferation of 786-O cells in a dose- and time-dependent manner. More importantly, knockdown and transfection of EphrinB2 altered the inhibitory effect of IMP-1 on the activity of 786-O cells. IMP-1 arrested 786-O cell cycle at G0/G1 phase by decreasing the expression of cyclin D1 and cyclin E. Moreover, IMP-1 regulated Bcl-2 family proteins' expression, thus inducing apoptosis of 786-O cells. IMP-1 down-regulated the expression of EphrinB2, Syntenin1 and PICK1. Then, IMP-1 decreased the phosphorylation of Erk1/2 and AKT. In all, IMP-1 could regulate the EphrinB2 pathway in order to inhibit 786-O cell growth by arresting the cell cycle at G0/G1 phase and inducing cell apoptosis. Thus, IMP-1 may present as a potential strategy for RCC treatment.
Asunto(s)
Carcinoma de Células Renales/patología , Neoplasias/clasificación , Fase G1/genética , Ciclina D1/efectos adversos , Ciclina E/efectos adversosRESUMEN
DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138's participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/metabolismo , Ubiquitina , Proteínas Portadoras/genética , ADN , Fase G1/genética , Humanos , Ligasas , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Pluripotent embryonic stem cells have a unique cell cycle structure with a suppressed G1/S restriction point and little differential expression across the cell cycle phases. Here, we evaluate the link between G1/S restriction point activation, phasic gene expression, and cellular differentiation. Expression analysis reveals a gain in phasic gene expression across lineages between embryonic days E7.5 and E9.5. Genetic manipulation of the G1/S restriction point regulators miR-302 and P27 respectively accelerates or delays the onset of phasic gene expression in mouse embryos. Loss of miR-302-mediated p21 or p27 suppression expedites embryonic stem cell differentiation, while a constitutive Cyclin E mutant blocks it. Together, these findings uncover a causal relationship between emergence of the G1/S restriction point with a gain in phasic gene expression and cellular differentiation.
Asunto(s)
MicroARNs , Animales , Ciclo Celular , Puntos de Control del Ciclo Celular , Diferenciación Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G1/genética , Expresión Génica , Ratones , MicroARNs/genéticaRESUMEN
DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 murine and human cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, which has important implications for DNA DSB repair in quiescent cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas F-Box , Animales , ADN/genética , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteína Quinasa Activada por ADN/genética , Proteínas F-Box/genética , Fase G1/genética , Humanos , RatonesRESUMEN
BACKGROUND: The oncogenic drivers of triple-negative breast cancer (TNBC), which is characterized by worst prognosis compared with other subtypes, are poorly understood. Although next-generation sequencing technology has facilitated identifying potential targets, few of the findings have been translated into daily clinical practice. The present study is aimed to explore ZNF703 (Zinc finger 703) function and its underlying mechanism in TNBC. METHODS: ZNF703 expressions in tissue microarray were retrospectively examined by immunohistochemistry. The cell proliferation by SRB assay and colony formation assay, as well as cell cycle distribution by flow cytometry were assessed. The protein levels associated with possible underlying molecular mechanisms were evaluated by western blotting. Kaplan-Meier analysis was used to plot survival analysis. RESULTS: Our data suggest that ZNF703 expressed in 34.2% of triple-negative human breast tumors by immunohistochemistry. In vitro, ZNF703 knockdown had potent inhibitory effects on TNBC cell proliferation and cell cycle, with cyclin D1, CDK4, CDK6, and E2F1 downregulated, while Rb1 upregulated. Moreover, Kaplan-Meier analysis showed that high mRNA expression of ZNF703 was correlated to worse overall survival (HR for high expression was 3.04; 95% CI, 1.22 to 7.57, P = 0.017). CONCLUSIONS: Taken together, the results identified that targeting ZNF703 contributed to the anti-proliferative effects in TNBC cells, due to induced G1-phase arrest. This study is the first to identify ZNF703 as a potentially important protein that is involved in TNBC progression.
Asunto(s)
Proteínas Portadoras/metabolismo , Ciclo Celular/genética , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Femenino , Fase G1/genética , Humanos , Estimación de Kaplan-Meier , Pronóstico , Estudios Retrospectivos , Neoplasias de la Mama Triple Negativas/mortalidad , Regulación hacia Arriba/genéticaRESUMEN
Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions.
Asunto(s)
Fase G1/genética , Regulación Fúngica de la Expresión Génica , Nitrógeno/metabolismo , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , División Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Immunoblotting , Unión Proteica , RNA-Seq/métodos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Dysregulation of the cell cycle contributes to tumor progression. Cell division cycleassociated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleavedcaspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NFκB signaling proteins (NFκBp105/p50, IKKα/ß, and phoNFκBp65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.
Asunto(s)
Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , FN-kappa B/metabolismo , Neoplasias de la Próstata/genética , Apoptosis/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Fase G1/genética , Humanos , Masculino , Fase de Descanso del Ciclo Celular/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors in women. Accumulating studies have been reported that long non-coding RNA (lncRNA) SNHG5 is highly expressed in BC. However, the specific molecular mechanism of SNHG5 in BC is unclear. METHODS: Gene and protein expressions in BC cell were detected by qRT-PCR and western blotting. The proliferation and cell cycle were measured using colony formation assay and flow cytometry analysis, separately. The glucose consumption and lactate production were determined by using the glucose assay kit and lactate assay kit. A dual-luciferase reporter assay was performed to measure the interaction between miR-299 and SNHG5 or BACH1. RESULTS: SNHG5 and BACH1 expressions were increased in BC cell while miR-299 level was decreased. SNHG5 increased BACH1 expression by directly targeting miR-299. SNHG5 silencing or miR-299 overexpression suppressed the proliferation of BC cell, arrested the cell cycle in the G1 cell phase, and decreased the glucose consumption and lactate production of BC cell. However, inhibition of miR-299 or overexpression of BACH1 could reverse the inhibitory effects of sh-SNHG5 on cell proliferation and glycolysis in BC. CONCLUSION: SNHG5 promoted the BC cell growth and glycolysis through up-regulating BACH1 expression via targeting miR-299. These findings may improve the diagnostic and therapeutic approaches to BC.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Neoplasias de la Mama/patología , MicroARNs/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proliferación Celular , Femenino , Fase G1/genética , Regulación Neoplásica de la Expresión Génica , Glucólisis/genética , Humanos , Ácido Láctico/metabolismo , ARN Largo no Codificante/metabolismoRESUMEN
Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II)-transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4- or TetR-VP16-activated promoters downstream of a mistranslating tRNASer variant that misincorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in the G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.
Asunto(s)
Biosíntesis de Proteínas/genética , ARN Polimerasa II/genética , ARN de Transferencia/genética , Transcripción Genética/genética , Codón/genética , Células Eucariotas/fisiología , Fase G1/genética , Prolina/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa III/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Gastric cancer (GC) is a malignant tumor with high mortality, but research on its molecular mechanisms remain limited. This study is the first to explore the biological role of nuclear factor NFE2L3 (nuclear factor, erythroid 2 like 3) in GC. We used Western blot and RT-qPCR to detect gene expression at the protein or mRNA level. Short hairpin RNA (shRNA) transfection was used to inhibit NFE2L3 expression. CCK-8 and colony formation assays were used to detect cell proliferation. Cell migration, invasion, cell cycle and apoptosis were detected by Transwell assays and flow cytometry. The results showed that NFE2L3 was highly expressed in gastric cancer tissues and promoted gastric cancer cell proliferation and metastasis. Inhibiting NFE2L3 expression blocks the cell cycle and increases the proportion of apoptotic cells, whereas NFE2L3 expression promotes the epithelial-mesenchymal transformation (EMT) process. In summary, NFE2L3 is highly expressed in gastric cancer and promotes gastric cancer cell proliferation and metastasis and the EMT process.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Apoptosis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/genética , Transformación Celular Neoplásica/patología , Regulación hacia Abajo/genética , Fase G1/genética , Técnicas de Silenciamiento del Gen , Ontología de Genes , Silenciador del Gen , Humanos , Metástasis de la Neoplasia , Mapas de Interacción de Proteínas/genética , Fase de Descanso del Ciclo Celular/genética , Regulación hacia Arriba/genéticaRESUMEN
Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small number of two-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are unstable and they are prone to transiting back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA sequencing on isolated 2CLCs that underwent 2C-like state exit and re-entry, and revealed a step-by-step transitional process between 2C-like and pluripotent states. Mechanistically, we found that cell cycle played an important role in mediating these transitions by regulating assembly of the nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions.
Asunto(s)
Ciclo Celular/genética , Diferenciación Celular/genética , División Celular/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Línea Celular , Nucléolo Celular/metabolismo , Fase G1/genética , Fase G2/genética , Perfilación de la Expresión Génica/métodos , Heterocromatina/genética , Heterocromatina/metabolismo , Ratones , RNA-Seq/métodos , Análisis de la Célula Individual/métodosRESUMEN
Oncogenic c-Myc is a master regulator of G1/S transition. Long non-coding RNAs (lncRNAs) emerge as new regulators of various cell activities. Here, we found that lncRNA SnoRNA Host Gene 17 (SNHG17) was elevated at the early G1-phase of cell cycle. Both gain- and loss-of function studies disclosed that SNHG17 increased c-Myc protein level, accelerated G1/S transition and cell proliferation, and consequently promoted tumor cell growth in vitro and in vivo. Mechanistically, the 1-150-nt of SNHG17 physically interacted with the 1035-1369-aa of leucine rich pentatricopeptide repeat containing (LRPPRC) protein, and disrupting this interaction abrogated the promoting role of SNHG17 in c-Myc expression, G1/S transition, and cell proliferation. The effect of SNHG17 in stimulating cell proliferation was attenuated by silencing c-Myc or LRPPRC. Furthermore, silencing SNHG17 or LRPPRC increased the level of ubiquitylated c-Myc and reduced the stability of c-Myc protein. Analysis of human hepatocellular carcinoma (HCC) tissues revealed that SNHG17, LRPPRC, and c-Myc were significantly upregulated in HCC, and they showed a positive correlation with each other. High level of SNHG17 or LRPPRC was associated with worse survival of HCC patients. These data suggest that SNHG17 may inhibit c-Myc ubiquitination and thus enhance c-Myc level and facilitate proliferation by interacting with LRPPRC. Our findings identify a novel SNHG17-LRPPRC-c-Myc regulatory axis and elucidate its roles in G1/S transition and tumor growth, which may provide potential targets for cancer therapy.
Asunto(s)
Proliferación Celular/genética , Fase G1/genética , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/metabolismo , Fase S/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Células HEK293 , Humanos , Masculino , Ratones , Modelos Biológicos , Proteínas de Neoplasias/genética , Fosforilación , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-myc/genética , ARN Largo no Codificante/genética , Regulación hacia Arriba/genéticaRESUMEN
Cell division is thought to be initiated by cyclin-dependent kinases (Cdks) inactivating key transcriptional inhibitors. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate the Whi5 protein, thereby releasing the transcription factor SBF and committing cells to division. We report that Whi5 is a poor substrate of Cln3-Cdk1, which instead phosphorylates the RNA polymerase II subunit Rpb1's C-terminal domain on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters and Cln3's function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, we propose that Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to promote transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these two processes.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , División Celular/fisiología , Ciclinas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , División Celular/genética , Ciclinas/genética , Fase G1/genética , Fase G1/fisiología , Regulación Fúngica de la Expresión Génica , Fosforilación , Regiones Promotoras Genéticas , Dominios Proteicos , ARN Polimerasa II/química , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismoRESUMEN
Objective To investigate the effect of lysine-specific demethylase 3A (KDM3A) on the invasion and migration of MDA-MB-231 breast cancer cells. Methods The mRNA and the protein expressions of KDM3A in MDA-MB-231 breast cancer cells and MCF-10A normal breast cells were detected by real-time quantitative PCR and Western blotting, respectively; the KDM3A level of MDA-MB-231 cells was knocked down by lentivirus infection of KDM3A short hairpin RNA (shKDM3A). The change of invasion and migration ability of MDA-MB-231 cells was detected by TranswellTM assay, and the change in the cell cycle was detected by flow cytometry. Results The expression of KDM3A in MDA-MB-231 breast cancer cells was significantly increased compared with that in MCF-10A epithelial cells; after KDM3A knockdown, the invasion and migration abilities of MDA-MB-231 cells were significantly decreased, and the cell cycle was arrested in the G0/G1 phase. Conclusion Knockdown of KDM3A inhibits the invasion and migration of MDA-MB-231 breast cancer cells and arrests the cell cycle in G0/G1 phase.
Asunto(s)
Neoplasias de la Mama , Lisina , Neoplasias de la Mama/genética , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Fase G1/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Invasividad Neoplásica/genéticaRESUMEN
Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.