RESUMO
Rosmarinic acid (RA) is an important medicinal metabolite and a potent food antioxidant. We discovered that exposure to high light intensifies the accumulation of RA in the leaves of perilla (Perilla frutescens (L.) Britt). However, the molecular mechanism underlying RA synthesis in response to high light stress remains poorly understood. To address this knowledge gap, we conducted a comprehensive analysis employing transcriptomic sequencing, transcriptional activation, and genetic transformation techniques. High light treatment for 1 and 48â h resulted in the upregulation of 592 and 1,060 genes, respectively. Among these genes, three structural genes and 93 transcription factors exhibited co-expression. Notably, NAC family member PfNAC2, GBF family member PfGBF3, and cinnamate-4-hydroxylase gene PfC4H demonstrated significant co-expression and upregulation under high light stress. Transcriptional activation analysis revealed that PfGBF3 binds to and activates the PfNAC2 promoter. Additionally, both PfNAC2 and PfGBF3 bind to the PfC4H promoter, thereby positively regulating PfC4H expression. Transient overexpression of PfNAC2, PfGBF3, and PfC4H, as well as stable transgenic expression of PfNAC2, led to a substantial increase in RA accumulation in perilla. Consequently, PfGBF3 acts as a photosensitive factor that positively regulates PfNAC2 and PfC4H, while PfNAC2 also regulates PfC4H to promote RA accumulation under high light stress. The elucidation of the regulatory mechanism governing RA accumulation in perilla under high light conditions provides a foundation for developing a high-yield RA system and a model to understand light-induced metabolic accumulation.
Assuntos
Cinamatos , Depsídeos , Regulação da Expressão Gênica de Plantas , Luz , Proteínas de Plantas , Ácido Rosmarínico , Depsídeos/metabolismo , Cinamatos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Perilla frutescens/genética , Perilla frutescens/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Regiões Promotoras Genéticas/genéticaRESUMO
Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.
Assuntos
Brônquios , Carcinoma de Células Escamosas , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p27 , Células Epiteliais , Neoplasias Pulmonares , Nitrosaminas , Humanos , Nitrosaminas/toxicidade , Brônquios/metabolismo , Brônquios/patologia , Brônquios/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação para Baixo/efeitos dos fármacos , Carcinógenos/toxicidadeRESUMO
Nonphotochemical quenching (NPQ) is an important photoprotective mechanism that quickly dissipates excess light energy as heat. NPQ can be induced in a few seconds to several hours; most studies of this process have focused on the rapid induction of NPQ. Recently, a new, slowly induced form of NPQ, called qH, was found during the discovery of the quenching inhibitor suppressor of quenching 1 (SOQ1). However, the specific mechanism of qH remains unclear. Here, we found that hypersensitive to high light 1 (HHL1)-a damage repair factor of photosystem II-interacts with SOQ1. The enhanced NPQ phenotype of the hhl1 mutant is similar to that of the soq1 mutant, which is not related to energy-dependent quenching or other known NPQ components. Furthermore, the hhl1 soq1 double mutant showed higher NPQ than the single mutants, but its pigment content and composition were similar to those of the wildtype. Overexpressing HHL1 decreased NPQ in hhl1 to below wildtype levels, whereas NPQ in hhl1 plants overexpressing SOQ1 was lower than that in hhl1 but higher than that in the wildtype. Moreover, we found that HHL1 promotes the SOQ1-mediated inhibition of plastidial lipoprotein through its von Willebrand factor type A domain. We propose that HHL1 and SOQ1 synergistically regulate NPQ.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Temperatura Alta , Luz , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Mutação , Fotoquímica , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Plastídeos/metabolismo , Domínios Proteicos , Fator de von Willebrand/químicaRESUMO
Dendrobium officinale is edible and has medicinal and ornamental functions. Polysaccharides and flavonoids, including anthocyanins, are important components of D. officinale that largely determine the nutritional quality and consumer appeal. There is a need to study the molecular mechanisms regulating anthocyanin and polysaccharide biosynthesis to enhance D. officinale quality and its market value. Here, we report that high light (HL) induced the accumulation of polysaccharides, particularly mannose, as well as anthocyanin accumulation, resulting in red stems. Metabolome and transcriptome analyses revealed that most of the flavonoids showed large changes in abundance, and flavonoid and polysaccharide biosynthesis was significantly activated under HL treatment. Interestingly, DoHY5 expression was also highly induced. Biochemical analyses demonstrated that DoHY5 directly binds to the promoters of DoF3H1 (involved in anthocyanin biosynthesis), DoGMPP2, and DoPMT28 (involved in polysaccharide biosynthesis) to activate their expression, thereby promoting anthocyanin and polysaccharide accumulation in D. officinale stems. DoHY5 silencing decreased flavonoid- and polysaccharide-related gene expression and reduced anthocyanin and polysaccharide accumulation, whereas DoHY5 overexpression had the opposite effects. Notably, naturally occurring red-stemmed D. officinale plants similarly have high levels of anthocyanin and polysaccharide accumulation and biosynthesis gene expression. Our results reveal a previously undiscovered role of DoHY5 in co-regulating anthocyanin and polysaccharide biosynthesis under HL conditions, improving our understanding of the mechanisms regulating stem color and determining nutritional quality in D. officinale. Collectively, our results propose a robust and simple strategy for significantly increasing anthocyanin and polysaccharide levels and subsequently improving the nutritional quality of D. officinale.
Assuntos
Dendrobium , Flavonoides , Flavonoides/metabolismo , Antocianinas/metabolismo , Dendrobium/genética , Dendrobium/química , Dendrobium/metabolismo , Polissacarídeos/metabolismo , Perfilação da Expressão GênicaRESUMO
BACKGROUND: Long non-coding RNAs (lncRNAs) have been confirmed to play important roles in various cancers including bladder cancer (BC). The precise expression pattern of lncRNA small nucleolar RNA host gene 18 (SNHG18) in BC and its mechanisms of action have not been fully explored. MATERIALS AND METHODS: The expression of SNHG18 was evaluated by RT-qPCR in bladder cancer clinical samples and human bladder cancer cell lines, and stable cell lines overexpressing SNHG18 were constructed. The effect of SNHG18 on the proliferation of bladder cancer cells was detected by soft agar colony formation test, ATP activity test and subcutaneous tumorigenesis model in nude mice. The specific mechanism of SNHG18 inhibition of bladder cancer proliferation was studied by flow cytometry, western blotting, dual luciferase reporter gene assay and protein degradation assay. RESULTS: We found that SNHG18 is significantly downregulated in BC tissues and cell lines. Kaplan-Meier analysis showed that SNHG18 expression is positively correlated with survival in BC patients. Ectopic overexpression of SNHG18 significantly inhibited the proliferation of BC cells in vitro and in vivo. Further mechanistic investigations demonstrated that SNHG18 inhibited c-Myc expression by modulating the ubiquitination-proteasome pathway and that c-Myc is the critical transcription factor that mediates SNHG18 inhibition of BC growth by directly binding to the p21 promoter, which was attributed with significant p21 accumulation. CONCLUSIONS: SNHG18 promotes the transcription and expression of p21 by inhibiting c-Myc expression, leading to G0-G1 arrest and inhibiting the proliferation of bladder cancer cells. These findings highlight a novel cell cycle regulatory mechanism involving the SNHG18/c-Myc/p21 pathway in BC pathogenesis and could potentially lead to new lncRNA-based diagnostics and/or therapeutics for BC.
RESUMO
BACKGROUND: Long non-coding RNAs play an important role in the development of colorectal cancer (CRC), while many CRC-related lncRNAs have not yet been identified. METHODS: The relationship between the expression of LINC00955 (Long Intergenic Non-protein Coding RNA 955) and the prognosis of colorectal cancer patients was analyzed using the sequencing results of the TCGA database. LINC00955 expression levels were measured using qRT-PCR. The anti-proliferative activity of LINC00955 was evaluated using CRC cell lines in vitro and xenograft models in nude mice in vivo. The interaction of TRIM25-Sp1-DNMT3B-PHIP-CDK2 was analyzed by western blotting, protein degradation experiment, luciferase, RNA-IP, RNA pull-down assays and immunohistochemically analysis. The biological roles of LINC00955, tripartite motif containing 25 (TRIM25), Sp1 transcription factor (Sp1), DNA methyltransferase 3 beta (DNMT3B), pleckstrin homology domain interacting protein (PHIP), cyclin dependent kinase 2 (CDK2) in colorectal cancer cells were analyzed using ATP assays, Soft agar experiments and EdU assays. RESULTS: The present study showed that LINC00955 is downregulated in CRC tissues, and such downregulation is associated with poor prognosis of CRC patients. We found that LINC00955 can inhibit CRC cell growth both in vitro and in vivo. Evaluation of its mechanism of action showed that LINC00955 acts as a scaffold molecule that directly promotes the binding of TRIM25 to Sp1, and promotes ubiquitination and degradation of Sp1, thereby attenuating transcription and expression of DNMT3B. DNMT3B inhibition results in hypomethylation of the PHIP promoter, in turn increasing PHIP transcription and promoting ubiquitination and degradation of CDK2, ultimately leading to G0/G1 growth arrest and inhibition of CRC cell growth. CONCLUSIONS: These findings indicate that downregulation of LINC00955 in CRC cells promotes tumor growth through the TRIM25/Sp1/DNMT3B/PHIP/CDK2 regulatory axis, suggesting that LINC00955 may be a potential target for the therapy of CRC.
Assuntos
Neoplasias Colorretais , Fator de Transcrição Sp1 , Animais , Humanos , Camundongos , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Metilação , Camundongos Nus , RNA , Fator de Transcrição Sp1/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Photosynthesis involves a series of redox reactions and is the major source of reactive oxygen species in plant cells. Fluctuating light (FL) levels, which occur commonly in natural environments, affect photosynthesis; however, little is known about the specific effects of FL on the redox regulation of photosynthesis. Here, we performed global quantitative mapping of the Arabidopsis thaliana cysteine thiol redox proteome under constant light and FL conditions. We identified 8857 redox-switched thiols in 4350 proteins, and 1501 proteins that are differentially modified depending on light conditions. Notably, proteins related to photosynthesis, especially photosystem I (PSI), are operational thiol-switching hotspots. Exposure of wild-type A. thaliana to FL resulted in decreased PSI abundance, stability, and activity. Interestingly, in response to PSI photodamage, more of the PSI assembly factor PSA3 dynamically switches to the reduced state. Furthermore, the Cys199 and Cys200 sites in PSA3 are necessary for its full function. Moreover, thioredoxin m (Trx m) proteins play roles in redox switching of PSA3, and are required for PSI activity and photosynthesis. This study thus reveals a mechanism for redox-based regulation of PSI under FL, and provides insight into the dynamic acclimation of photosynthesis in a changing environment.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteômica , Luz , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Oxirredução , Compostos de Sulfidrila/metabolismoRESUMO
Light is a key environmental cue regulating photomorphogenesis and photosynthesis in plants. However, the molecular mechanisms underlying the interaction between light signaling pathways and photosystem function are unknown. Here, we show that various monochromatic wavelengths of light cooperate to regulate PSII function in Arabidopsis (Arabidopsis thaliana). The photoreceptors cryptochromes and phytochromes modulate the expression of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173), which is required for PSII biogenesis by regulating PSII core protein D1 synthesis mediated by the transcription factor ELONGATED HYPOCOTYL5 (HY5). HY5 directly binds to the ACGT-containing element ACE motif and G-box cis-element present in the HCF173 promoter and regulates its activity. PSII activity was decreased significantly in hy5 mutants under various monochromatic wavelengths of light. Interestingly, we demonstrate that HY5 also directly regulates the expression of the genes associated with PSII assembly and repair, including ALBINO3, HCF136, HYPERSENSITIVE TO HIGH LIGHT1, etc., which is required for the functional maintenance of PSII under photodamaging conditions. Moreover, deficiency of HY5 broadly decreases the accumulation of other photosystem proteins besides PSII proteins. Thus, our study reveals an important role of light signaling in both biogenesis and functional regulation of the photosystem and provides insight into the link between light signaling and photosynthesis in land plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Transdução de Sinal Luminoso/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso/genética , Fotossíntese/genética , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Photosystem II (PSII), a multisubunit protein complex of the photosynthetic electron transport chain, functions as a water-plastoquinone oxidoreductase, which is vital to the initiation of photosynthesis and electron transport. Although the structure, composition, and function of PSII are well understood, the mechanism of PSII biogenesis remains largely elusive. Here, we identified a nuclear-encoded pentatricopeptide repeat (PPR) protein LOW PHOTOSYNTHETIC EFFICIENCY 1 (LPE1; encoded by At3g46610) in Arabidopsis, which plays a crucial role in PSII biogenesis. LPE1 is exclusively targeted to chloroplasts and directly binds to the 5' UTR of psbA mRNA which encodes the PSII reaction center protein D1. The loss of LPE1 results in less efficient loading of ribosome on the psbA mRNA and great synthesis defects in D1 protein. We further found that LPE1 interacts with a known regulator of psbA mRNA translation HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) and facilitates the association of HCF173 with psbA mRNA. More interestingly, our results indicate that LPE1 associates with psbA mRNA in a light-dependent manner through a redox-based mechanism. This study enhances our understanding of the mechanism of light-regulated D1 synthesis, providing important insight into PSII biogenesis and the functional maintenance of efficient photosynthesis in higher plants.
Assuntos
Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Proteínas de Membrana Transportadoras/metabolismo , Complexo de Proteína do Fotossistema II/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Iniciação em Eucariotos/genética , Proteínas de Membrana Transportadoras/genética , Complexo de Proteína do Fotossistema II/genéticaRESUMO
Because bladder cancer (BC) is one of the most common malignant cancers of the urinary system, identification of BC cell growth-associated effectors is of great significance. Cyclin-dependent kinase (CDK)6 is a member of the CDK family of cell cycle-related proteins and plays an important role in cancer cell growth. This is borne out by the fact that a CDK6 inhibitor had been approved to treat several types of cancers. Nevertheless, underlying molecular mechanisms concerning how to regulate CDK6 expression in BC remains unclear. In the present study, it was observed that miR-934 was much higher in human BCs and human BC cell lines as well. The results also revealed that miR-934 inhibition dramatically decreased human BC cell monolayer growth in vitro and xenograft tumor growth in vivo; the outcomes were accompanied by CDK6 protein down-regulation and G0-G1 cell cycle arrest. Moreover, overexpression of CDK6 reversed the inhibition of BC cell growth induced by miR-934. Further studies showed that miR-934 binds to a 3'-UTR of ubiquitin-conjugating enzyme 2N (ube2n) mRNA, down-regulated UBE2N protein expression; this, in turn, attenuated CDK6 protein degradation and led to CDK6 protein accumulation as well as the promotion of BC tumor growth. Collectively, this study not only establishes a novel regulatory axis of miR-934/UBE2N of CDK6 but also provides data suggesting that miR-934 and UBE2N may be potentially promising targets for therapeutic strategies against BC.-Yan, H., Ren, S., Lin, Q., Yu, Y., Chen, C., Hua, X., Jin, H., Lu, Y., Zhang, H., Xie, Q., Huang, C., Huang, H. Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.
Assuntos
Proliferação de Células/genética , Quinase 6 Dependente de Ciclina/genética , MicroRNAs/genética , Enzimas de Conjugação de Ubiquitina/genética , Neoplasias da Bexiga Urinária/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Proteólise , Interferência de RNA , Terapêutica com RNAi/métodos , Enzimas de Conjugação de Ubiquitina/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Cyclin E2, a member of the cyclin family, is a key cell cycle-related protein. This protein plays essential roles in cancer progression, and, as such, an inhibitor of cyclin E2 has been approved to treat several types of cancers. Even so, mechanisms underlying how to regulate cyclin E2 expression in cancer remain largely unknown. In the current study, miR-3687 was upregulated in clinical bladder cancer (BC) tumor tissues, The Cancer Genome Atlas (TCGA) database, and human BC cell lines. Inhibition of miR-3687 expression significantly reduced human BC cell proliferation in vitro and tumor growth in vivo, which coincided with the induction of G0/G1 cell cycle arrest and downregulation of cyclin E2 protein expression. Interestingly, overexpression of cyclin E2 reversed the inhibition of BC proliferation induced by miR-3687. Mechanistic studies suggested that miR-3687 binds to the 3' UTR of foxp1 mRNA, downregulates FOXP1 protein expression, and in turn promotes the transcription of cyclin E2, thereby promoting the growth of BC cells. Collectively, the current study not only establishes a novel regulatory axis of miR-3687/FOXP1 regarding regulation of cyclin E2 expression in BC cells, but also provides strong suggestive evidence that miR-3687 and FOXP1 may be promising targets in therapeutic strategies for human BC.
Assuntos
Ciclinas/genética , Fatores de Transcrição Forkhead/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transcrição Gênica , Ativação Transcricional/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
The balance between cellular carbon (C) and nitrogen (N) must be tightly coordinated to sustain optimal growth and development in plants. In chloroplasts, photosynthesis converts inorganic C to organic C, which is important for maintenance of C content in plant cells. However, little is known about the role of chloroplasts in C/N balance. Here, we identified a nuclear-encoded protein LOW PHOTOSYNTHETIC EFFICIENCY2 (LPE2) that it is required for photosynthesis and C/N balance in Arabidopsis. LPE2 is specifically localized in the chloroplast. Both loss-of-function mutants, lpe2-1 and lpe2-2, showed lower photosynthetic activity, characterized by slower electron transport and lower PSII quantum yield than the wild type. Notably, LPE2 is predicted to encode the plastid ribosomal protein S21 (RPS21). Deficiency of LPE2 significantly perturbed the thylakoid membrane composition and plastid protein accumulation, although the transcription of plastid genes is not affected obviously. More interestingly, transcriptome analysis indicated that the loss of LPE2 altered the expression of C and N response related genes in nucleus, which is confirmed by quantitative real-time-polymerase chain reaction. Moreover, deficiency of LPE2 suppressed the response of C/N balance in physiological level. Taken together, our findings suggest that LPE2 plays dual roles in photosynthesis and the response to C/N balance.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carbono/metabolismo , Cloroplastos/metabolismo , Nitrogênio/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Fotossíntese/genética , Fotossíntese/fisiologiaRESUMO
Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiß mRNA stability and expression. Positively regulation of rhogdiß mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiß mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIß axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.
Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , MicroRNAs/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Estabilidade de RNA , Fator de Transcrição Sp1/metabolismo , Ubiquitina Tiolesterase/metabolismo , Neoplasias da Bexiga Urinária/patologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , MicroRNAs/genética , Subunidade p52 de NF-kappa B/genética , Biossíntese de Proteínas , Proteólise , Fator de Transcrição Sp1/genética , Células Tumorais Cultivadas , Ubiquitina Tiolesterase/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/química , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genéticaRESUMO
There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Regulação Neoplásica da Expressão Gênica/fisiologia , Estilbenos/farmacologia , Neoplasias da Bexiga Urinária/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ativação Transcricional/fisiologia , Regulação para Cima , Neoplasias da Bexiga Urinária/genéticaRESUMO
Bladder cancer (BC) is the sixth most common cancer in the United States and is the number one cause of death among patients with urinary system malignancies. This makes the identification of invasive regulator(s)/effector(s) as the potential therapeutic targets for managing BC a high priority. p63 is a member of the p53 family of tumor suppressor genes/proteins, plays a role in the differentiation of epithelial tissues, and is believed to function as a tumor suppressor. However, it remains unclear whether and how p63 functions in BC cell invasion after tumorigenesis. Here, we show that p63α protein levels were much higher in mouse high-invasive BC tissues than in normal tissues. Our results also revealed that p63α is crucial for heat shock protein 70 (Hsp70) expression and subsequently increases the ability of BC invasion. Mechanistic experiments demonstrated that p63α can transcriptionally up-regulate Hsp70 expression, thereby promoting BC cell invasion via the Hsp70/Wasf3/Wave3/MMP-9 axis. We further show that E2F transcription factor 1 (E2F1) mediates p63α overexpression-induced Hsp70 transcription. We also found that p63α overexpression activates E2F1 transcription, which appears to be stimulated by p63α together with E2F1. Collectively, our results demonstrate that p63α is a positive regulator of BC cell invasion after tumorigenesis, providing significant insights into the biological function of p63α in BC and supporting the notion that p63α might be a potential target for invasive BC therapy.
Assuntos
Fator de Transcrição E2F1/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Neoplasias da Bexiga Urinária/patologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Fator de Transcrição E2F1/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.
Assuntos
Proteínas Inibidoras de Apoptose/biossíntese , Neoplasias Pulmonares/secundário , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Neoplasias da Bexiga Urinária/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HCT116 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Invasividade Neoplásica , RNA Mensageiro/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , NucleolinaRESUMO
In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway.
Assuntos
Arabidopsis/enzimologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , NADP/metabolismo , Oxirredução , Folhas de Planta/enzimologia , Folhas de Planta/genética , Isoformas de Proteínas , Plântula/enzimologia , Plântula/genética , Tetrapirróis/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/metabolismoRESUMO
Iron (Fe) is an indispensable micronutrient for plant growth and development. The regulation of Fe homeostasis in plants is complex and involves a number of transcription factors. Here, we demonstrate that a basic helix-loop-helix (bHLH) transcription factor, bHLH104, belonging to the IVc subgroup of bHLH family, acts as a key component positively regulating Fe deficiency responses. Knockout of bHLH104 in Arabidopsis thaliana greatly reduced tolerance to Fe deficiency, whereas overexpression of bHLH104 had the opposite effect and led to accumulation of excess Fe in soil-grown conditions. The activation of Fe deficiency-inducible genes was substantially suppressed by loss of bHLH104. Further investigation showed that bHLH104 interacted with another IVc subgroup bHLH protein, IAA-LEUCINE RESISTANT3 (ILR3), which also plays an important role in Fe homeostasis. Moreover, bHLH104 and ILR3 could bind directly to the promoters of Ib subgroup bHLH genes and POPEYE (PYE) functioning in the regulation of Fe deficiency responses. Interestingly, genetic analysis showed that loss of bHLH104 could decrease the tolerance to Fe deficiency conferred by the lesion of BRUTUS, which encodes an E3 ligase and interacts with bHLH104. Collectively, our data support that bHLH104 and ILR3 play pivotal roles in the regulation of Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE expression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Homeostase/efeitos dos fármacos , Ferro/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Inativação de Genes , Genes de Plantas , Deficiências de Ferro , Modelos Biológicos , Mutação/genética , Motivos de Nucleotídeos/genética , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , SoloRESUMO
KEY MESSAGE: M-type thioredoxins are required to regulate zeaxanthin epoxidase activity and to maintain the steady-state level of the proton motive force, thereby influencing NPQ properties under low-light conditions in Arabidopsis. Non-photochemical quenching (NPQ) helps protect photosynthetic organisms from photooxidative damage via the non-radiative dissipation of energy as heat. Energy-dependent quenching (qE) is a major constituent of NPQ. However, the mechanism underlying the regulation of qE is not well understood. In this study, we demonstrate that the m-type thioredoxins TRX-m1, TRX-m2, and TRX-m4 (TRX-ms) interact with the xanthophyll cycle enzyme zeaxanthin epoxidase (ZE) and are required for maintaining the redox-dependent stabilization of ZE by regulating its intermolecular disulfide bridges. Reduced ZE activity and accumulated zeaxanthin levels were observed under TRX-ms deficiency. Furthermore, concurrent deficiency of TRX-ms resulted in a significant increase in proton motive force (pmf) and acidification of the thylakoid lumen under low irradiance, perhaps due to the significantly reduced ATP synthase activity under TRX-ms deficiency. The increased pmf, combined with acidification of the thylakoid lumen and the accumulation of zeaxanthin, ultimately contribute to the elevated stable qE in VIGS-TRX-m2m4/m1 plants under low-light conditions. Taken together, these results indicate that TRX-ms are involved in regulating NPQ-dependent photoprotection in Arabidopsis.