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1.
Mol Ther Nucleic Acids ; 26: 333-346, 2021 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-34513313

RESUMO

Esophageal cancer is a lethal malignancy with a high mortality rate, while the molecular mechanisms underlying esophageal cancer pathogenesis are still poorly understood. Here, we found that the N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) is significantly upregulated in esophageal squamous cell carcinoma (ESCC) and associated with poor patient prognosis. Depletion of METTL3 results in decreased ESCC growth and progression in vitro and in vivo. We further established ESCC initiation and progression models using Mettl3 conditional knockout mouse and revealed that 3METTL3-mediated m6A modification promotes ESCC initiation and progression in vivo. Moreover, using METTL3 overexpression ESCC cell model and Mettl3 conditional knockin mouse model, we demonstrated the critical function of METTL3 in promoting ESCC tumorigenesis in vitro and in vivo. Mechanistically, METTL3-catalyzed m6A modification promotes NOTCH1 expression and the activation of the Notch signaling pathway. Forced activation of Notch signaling pathway successfully rescues the growth, migration, and invasion capacities of METTL3-depleted ESCC cells. Our data uncovered important mechanistical insights underlying ESCC tumorigenesis and provided molecular basis for the development of novel strategies for ESCC diagnosis and treatment.

2.
Mol Cell ; 81(16): 3339-3355.e8, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352206

RESUMO

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.


Assuntos
Colangiocarcinoma/genética , Proteínas de Ligação ao GTP/genética , Metiltransferases/genética , Biossíntese de Proteínas , Animais , Carcinogênese/genética , Colangiocarcinoma/patologia , Progressão da Doença , Receptores ErbB/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Camundongos , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
3.
Mol Ther ; 2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34371184

RESUMO

Mis-regulated epigenetic modifications in RNAs are associated with human cancers. The transfer RNAs (tRNAs) are the most heavily modified RNA species in cells; however, little is known about the functions of tRNA modifications in cancers. In this study, we uncovered that the expression levels of tRNA N7-methylguanosine (m7G) methyltransferase complex components methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) are significantly elevated in human lung cancer samples and negatively associated with patient prognosis. Impaired m7G tRNA modification upon METTL1/WDR4 depletion resulted in decreased cell proliferation, colony formation, cell invasion, and impaired tumorigenic capacities of lung cancer cells in vitro and in vivo. Moreover, gain-of-function and mutagenesis experiments revealed that METTL1 promoted lung cancer growth and invasion through regulation of m7G tRNA modifications. Profiling of tRNA methylation and mRNA translation revealed that highly translated mRNAs have higher frequencies of m7G tRNA-decoded codons, and knockdown of METTL1 resulted in decreased translation of mRNAs with higher frequencies of m7G tRNA codons, suggesting that tRNA modifications and codon usage play an essential function in mRNA translation regulation. Our data uncovered novel insights on mRNA translation regulation through tRNA modifications and the corresponding mRNA codon compositions in lung cancer, providing a new molecular basis underlying lung cancer progression.

4.
Acta Biomater ; 131: 326-340, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34246802

RESUMO

Besides molecular and phenotypic variations observed in cancer cells, intratumoral heterogeneity also occurs in the tumor microenvironment. Correlative stiffness maps of different intratumor locations in breast tumor biopsies show that stiffness increases from core to periphery. However, how different local ECM stiffness regulates key functions of cancer cells in tumor progression remains unclear. Although increased tissue stiffness is an established driver of breast cancer progression, conclusions from 2D cultures do not correspond with newer data from cancer cells in 3D environments. Many past studies of breast cancer in 3D culture fail to recapitulate the stiffness of a real breast tumor or the various local stiffnesses present in a tumor microenvironment. In this study, we developed a series of collagen/alginate hybrid hydrogels with adjustable stiffness to match the core, middle, and peripheral zones of a breast tumor. We used this hydrogel system to investigate effects of different local stiffness on morphology, proliferation, and migration of breast cancer cells. RNA sequencing of cells in hydrogels with different stiffness revealed changes in multiple cellular processes underlying cancer progression, including angiogenesis and metabolism. We discovered that tumor cells in a soft environment enriched YAP1 and AP1 signaling related genes, whereas tumor cells in a stiff environment became more pro-angiogenic by upregulating fibronectin 1 (FN1) and matrix metalloproteinase 9 (MMP9) expression. This systematic study defines how the range of environmental stiffnesses present in a breast tumor regulates cancer cells, providing new insights into tumorigenesis and disease progression at the tumor-stroma interface. STATEMENT OF SIGNIFICANCE: Applied a well-defined hybrid hydrogel system to mimic the tumor microenvironment with heterogeneous local stiffness. Breast cancer cells tended to proliferate in soft core environment while migrate in stiff peripheral environment. Breast cancer cells shift from glycolysis to OXPHOS and fatty acid metabolism responding to stiff matrix microenvironment. The transcriptomic profile of breast cancer cells altered due to microenvironmental stiffness changes.


Assuntos
Neoplasias da Mama , Linhagem Celular Tumoral , Colágeno , Matriz Extracelular , Feminino , Humanos , Hidrogéis , Microambiente Tumoral
5.
Stem Cells Int ; 2021: 9955691, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093712

RESUMO

The Pax7+ muscle stem cells (MuSCs) are essential for skeletal muscle homeostasis and muscle regeneration upon injury, while the molecular mechanisms underlying muscle stem cell fate determination and muscle regeneration are still not fully understood. N6-methyladenosine (m6A) RNA modification is catalyzed by METTL3 and plays important functions in posttranscriptional gene expression regulation and various biological processes. Here, we generated muscle stem cell-specific METTL3 conditional knockout mouse model and revealed that METTL3 knockout in muscle stem cells significantly inhibits the proliferation of muscle stem cells and blocks the muscle regeneration after injury. Moreover, knockin of METTL3 in muscle stem cells promotes the muscle stem cell proliferation and muscle regeneration in vivo. Mechanistically, METTL3-m6A-YTHDF1 axis regulates the mRNA translation of Notch signaling pathway. Our data demonstrated the important in vivo physiological function of METTL3-mediated m6A modification in muscle stem cells and muscle regeneration, providing molecular basis for the therapy of stem cell-related muscle diseases.

6.
JCI Insight ; 6(7)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33830080

RESUMO

No effective systemic treatment is available for patients with unresectable, recurrent, or metastatic mucoepidermoid carcinoma (MEC), the most common salivary gland malignancy. MEC is frequently associated with a t(11;19)(q14-21;p12-13) translocation that creates a CRTC1-MAML2 fusion gene. The CRTC1-MAML2 fusion exhibited transforming activity in vitro; however, whether it serves as an oncogenic driver for MEC establishment and maintenance in vivo remains unknown. Here, we show that doxycycline-induced CRTC1-MAML2 knockdown blocked the growth of established MEC xenografts, validating CRTC1-MAML2 as a therapeutic target. We further generated a conditional transgenic mouse model and observed that Cre-induced CRTC1-MAML2 expression caused 100% penetrant formation of salivary gland tumors resembling histological and molecular characteristics of human MEC. Molecular analysis of MEC tumors revealed altered p16-CDK4/6-RB pathway activity as a potential cooperating event in promoting CRTC1-MAML2-induced tumorigenesis. Cotargeting of aberrant p16-CDK4/6-RB signaling and CRTC1-MAML2 fusion-activated AREG/EGFR signaling with the respective CDK4/6 inhibitor Palbociclib and EGFR inhibitor Erlotinib produced enhanced antitumor responses in vitro and in vivo. Collectively, this study provides direct evidence for CRTC1-MAML2 as a key driver for MEC development and maintenance and identifies a potentially novel combination therapy with FDA-approved EGFR and CDK4/6 inhibitors as a potential viable strategy for patients with MEC.

7.
J Cell Mol Med ; 25(9): 4220-4234, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33759344

RESUMO

Proliferative vitreoretinopathy (PVR) is a refractory vitreoretinal fibrosis disease, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the key pathological mechanism of PVR. However, few studies focused on the role of METTL3, the dominating methyltransferase for m6A RNA modification in PVR pathogenesis. Immunofluorescence staining and qRT-PCR were used to determine the expression of METTL3 in human tissues. Lentiviral transfection was used to stably overexpress and knockdown METTL3 in ARPE-19 cells. MTT assay was employed to study the effects of METTL3 on cell proliferation. The impact of METTL3 on the EMT of ARPE-19 cells was assessed by migratory assay, morphological observation and expression of EMT markers. Intravitreal injection of cells overexpressing METTL3 was used to assess the impact of METTL3 on the establishment of the PVR model. We found that METTL3 expression was less in human PVR membranes than in the normal RPE layers. In ARPE-19 cells, total m6A abundance and the METTL3 expression were down-regulated after EMT. Additionally, METTL3 overexpression inhibited cell proliferation through inducing cell cycle arrest at G0/G1 phase. Furthermore, METTL3 overexpression weakened the capacity of TGFß1 to trigger EMT by regulating wnt/ß -catenin pathway. Oppositely, knockdown of METTL3 facilitated proliferation and EMT of ARPE-19 cells. In vivo, intravitreal injection of METTL3-overexpressing cells delayed the development of PVR compared with injection of control cells. In summary, this study suggested that METTL3 is involved in the PVR process, and METTL3 overexpression inhibits the EMT of ARPE-19 cells in vitro and suppresses the PVR process in vivo.


Assuntos
Transição Epitelial-Mesenquimal , Metiltransferases/metabolismo , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/prevenção & controle , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Adolescente , Adulto , Idoso , Feminino , Seguimentos , Regulação da Expressão Gênica , Humanos , Masculino , Metiltransferases/genética , Pessoa de Meia-Idade , Prognóstico , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteínas Wnt/genética , Adulto Jovem , beta Catenina/genética
8.
Hepatology ; 74(3): 1339-1356, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33638162

RESUMO

BACKGROUND AND AIMS: The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. APPROACH AND RESULTS: Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. CONCLUSIONS: The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.

9.
J Cell Mol Med ; 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33090698

RESUMO

Retinoblastoma (RB) is a common intraocular malignancy in children. Due to the poor prognosis of RB, it is crucial to search for efficient diagnostic and therapeutic strategies. Studies have shown that methyltransferase-like 3 (METTL3), a major RNA N (6)-adenosine methyltransferase, is closely related to the initiation and development of cancers. Nevertheless, whether METTL3 is associated with RB remains unexplored. Therefore, we investigated the function and mechanisms of METTL3 in the regulation of RB progression. We manipulated METTL3 expression in RB cells. Then, cell proliferation, apoptosis, migration and invasion were analysed. We also analysed the expression of PI3K/AKT/mTOR pathway members. Finally, we incorporated subcutaneous xenograft mouse models into our studies. The results showed that METTL3 is highly expressed in RB patients and RB cells. We found that METTL3 knockdown decreases cell proliferation, migration and invasion of RB cells, while METTL3 overexpression promotes RB progression in vitro and in vivo. Moreover, two downstream members of the PI3K/AKT/mTOR pathway, P70S6K and 4EBP1, were affected by METTL3. Our study revealed that METTL3 promotes the progression of RB through PI3K/AKT/mTOR pathways in vitro and in vivo. Targeting the METTL3/PI3K/AKT/mTOR signalling axis could be a promising therapeutic strategy for the treatment of RB.

10.
Stem Cell Res Ther ; 11(1): 306, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32698871

RESUMO

BACKGROUND: 7-Methylguanosine (m7G) is one of the most conserved modifications in nucleosides within tRNAs and rRNAs. It plays essential roles in the regulation of mRNA export, splicing, and translation. Recent studies highlighted the importance of METTL1-mediated m7G tRNA methylome in the self-renewal of mouse embryonic stem cells (mESCs) through its ability to regulate mRNA translation. However, the exact mechanisms by which METTL1 regulates pluripotency and differentiation in human induced pluripotent stem cells (hiPSCs) remain unknown. In this study, we evaluated the functions and underlying molecular mechanisms of METTL1 in regulating hiPSC self-renewal and differentiation in vivo and in vitro. METHODS: By establishing METTL1 knockdown (KD) hiPSCs, gene expression profiling was performed by RNA sequencing followed by pathway analyses. Anti-m7G northwestern assay was used to identify m7G modifications in tRNAs and mRNAs. Polysome profiling was used to assess the translation efficiency of the major pluripotent transcription factors. Moreover, the in vitro and in vivo differentiation capacities of METTL1-KD hiPSCs were assessed in embryoid body (EB) formation and teratoma formation assays. RESULTS: METTL1 silencing resulted in alterations in the global m7G profile in hiPSCs and reduced the translational efficiency of stem cell marker genes. METTL1-KD hiPSCs exhibited reduced pluripotency with slower cell cycling. Moreover, METTL1 silencing accelerates hiPSC differentiation into EBs and promotes the expression of mesoderm-related genes. Similarly, METTL1 knockdown enhances teratoma formation and mesoderm differentiation in vivo by promoting cell proliferation and angiogenesis in nude mice. CONCLUSION: Our findings provided novel insight into the critical role of METTL1-mediated m7G modification in the regulation of hiPSC pluripotency and differentiation, as well as its potential roles in vascular development and the treatment of vascular diseases.


Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesoderma/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Nus
11.
Nat Commun ; 11(1): 2578, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444598

RESUMO

Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have sprung up in recent years. We find m6A can positively regulate the glycolysis of cancer cells. Specifically, m6A-sequencing and functional studies confirm that pyruvate dehydrogenase kinase 4 (PDK4) is involved in m6A regulated glycolysis and ATP generation. The m6A modified 5'UTR of PDK4 positively regulates its translation elongation and mRNA stability via binding with YTHDF1/eEF-2 complex and IGF2BP3, respectively. Targeted specific demethylation of PDK4 m6A by dm6ACRISPR system can significantly decrease the expression of PDK4 and glycolysis of cancer cells. Further, TATA-binding protein (TBP) can transcriptionally increase the expression of Mettl3 in cervical cancer cells via binding to its promoter. In vivo and clinical data confirm the positive roles of m6A/PDK4 in tumor growth and progression of cervical and liver cancer. Our study reveals that m6A regulates glycolysis of cancer cells through PDK4.


Assuntos
Adenosina/análogos & derivados , Glicólise/fisiologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Neoplasias do Colo do Útero/genética , Regiões 5' não Traduzidas , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Glicólise/genética , Células HeLa , Humanos , Estimativa de Kaplan-Meier , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Neoplasias do Colo do Útero/mortalidade , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Biochem Biophys Res Commun ; 527(3): 791-798, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32430183

RESUMO

Transplantation of endothelial progenitor cells (EPCs) has high therapeutic potential for ischemia-related ailments like heart attacks and claudication. Due to limited EPC sources, direct reprogramming is a fast-developing way to convert human-induced pluripotent stem cells (hiPSCs) into EPCs fit for transplantation. However, the procedural efficacy was affected by multiple factors, including epigenetic modifications. Recent studies have shown that m7G methylation mediated by Methyltransferase like 1 (METTL1) is required for mouse embryonic stem cells (mESCs) to differentiate normally. Yet, its contributions to EPC differentiation still require elucidation. Here, using immunofluorescence microscopy and Fluorescence-activated Cell Sorting (FACS), we found that the typical EPC markers were significantly increased in METTL1 knockdown (METTL1-KD) hiPSCs-derived EPCs compared to those of control types. In addition, we found that METTL1 knockdown activates the MAPK/ERK signaling pathway during EPCs differentiation from hiPSCs. Furthermore, functional properties of METTL1-KD EPCs were significantly raised above those of control hiPSCs-derived EPCs. Moreover, we proved that METTL1-KD hiPSCs-derived EPCs significantly accelerate vascular smooth muscle cell proliferation and 'phenotype switching' through a co-culture system. To sum up, our results demonstrate that METTL1-KD significantly promotes the differentiation of EPCs along with their in vitro functions, and this effect may be achieved through activation of the MAPK/ERK signaling pathway. This enhances current knowledge of EPC generation from hiPSCs and presents a new therapeutic target of vascular diseases.


Assuntos
Células Progenitoras Endoteliais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Sistema de Sinalização das MAP Quinases , Metiltransferases/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Progenitoras Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Metiltransferases/genética
13.
ACS Appl Bio Mater ; 3(5): 2838-2849, 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-33681722

RESUMO

The past decade has witnessed the blossom of two fields: nucleic acid therapeutics and cancer immunotherapy. Unlike traditional small molecule medicines or protein biologics, nucleic acid therapeutics have characteristic features such as storing genetic information, immunomodulation, and easy conformational recovery. Immunotherapy uses the patients' own immune system to treat cancer. A variety of strategies have been developed for cancer immunotherapy including immune checkpoint blockade, adoptive cell transfer therapy, therapeutic vaccines, and oncolytic virotherapy. Interestingly, nucleic acid therapeutics have emerged as a pivotal class of regimen for cancer immunotherapy. Examples of such nucleic acid immunotherapeutics include immunostimulatory DNA/RNA, mRNA/plasmids that can be translated into immunotherapeutic proteins/peptides, and genome-editing nucleic acids. Like many other therapeutic nucleic acids, nucleic acid immunotherapeutics often require chemical modifications to protect them from enzymatic degradation and need drug delivery systems for optimal delivery to target tissues and cells and subcellular locations. In this review, we attempted to summarize recent advancement in the interfacial field of nucleic acid immunotherapeutics for cancer treatment.

14.
Adv Ther (Weinh) ; 3(9)2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34337143

RESUMO

Cyclic dinucleotides (CDNs), such as c-di-GMP (CDG), are agonists for stimulator of interferon genes (STING) and are promising for cancer immunotherapy. Yet, the therapeutic efficacy of CDNs has been limited by poor delivery and biostability. Here, STING-activating DNA nanovaccines (STING-NVs) are developed, which biostabilize, deliver, and conditionally release CDG in the endosome of immune cells, elicit potent antitumor immune responses in murine and human immune cells, ameliorate immunosuppression in vitro and in the tumor microenvironment, and mediate potent cancer immunotherapy in a murine melanoma model. STING-NVs have PLA-b-PEG in the core and cytosine (C)-rich i-motif DNA on the surface. i-Motif DNA undergoes characteristic pH-responsive conformational switch, allowing efficient CDG loading via C:G base pairing at physiological pH, and CDG release in sensitive response to acidic environment such as cell endosome. STING-NVs protect CDG from enzymatic degradation. STING-NVs facilitate cell delivery. Remarkably, STING-NVs promote the endosome escape of CDG by ninefold, and potentiate antitumor immunity. STING-NVs repolarize immunosuppressive M2-like macrophages into antitumor M1-like macrophages in vitro and in the tumor microenvironment of melanoma. In a poorly immunogenic murine melanoma model, intralesional STING-NVs outperform liposomal CDG and fluoride-CDG for melanoma immunotherapy. These results suggest the great potential of STING-NVs for cancer immunotherapy.

15.
Theranostics ; 9(25): 7759-7771, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695799

RESUMO

Cancer immunotherapy modulates and leverages the host immune system to treat cancer. The past decade has witnessed historical advancement of cancer immunotherapy. A myriad of approaches have been explored to elicit or augment anticancer innate immunity and/or adaptive immunity. Recently, activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor residing in the endoplasmic reticulum, has shown great potential to enhance antitumor immunity through the induction of a variety of pro-inflammatory cytokines and chemokines, including type I IFNs. A number of natural and synthetic STING agonists have been discovered or developed, and tested in preclinical models and in the clinic for the immunotherapy of diseases such as cancer and infectious diseases. Cyclic dinucleotides (CDNs), such as cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic GMP-AMP (cGAMP), are a class of STING agonists that can elicit immune responses. However, natural CDNs are hydrophilic small molecules with negative charges and are susceptible to enzymatic degradation, leading to low bioavailability in target tissues yet unwanted toxicities and narrow therapeutic windows. Drug delivery systems, coupled with nucleic acid chemistry, have been exploited to address these challenges. Here, we will discuss the underlying immunological mechanisms and approaches to STING activation, with a focus on the delivery of STING agonists, for cancer immunotherapy.


Assuntos
Proteínas de Membrana/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Humanos , Imunidade Inata/imunologia , Imunoterapia/métodos , Ácidos Nucleicos/imunologia , Nucleotídeos Cíclicos/imunologia
16.
Nat Protoc ; 14(11): 3220-3242, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31619810

RESUMO

Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased global mapping of 7-methylguansine (m7G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m7G TRAC-Seq involves the treatment of size-selected (<200 nt) RNAs with the demethylase AlkB to remove major tRNA modifications, followed by sodium borohydride (NaBH4) reduction of m7G sites and subsequent aniline-mediated cleavage of the RNA chain at the resulting abasic sites. The cleaved sites are subsequently ligated with adaptors for the construction of libraries for high-throughput sequencing. The m7G modification sites are identified using a bioinformatic pipeline that calculates the cleavage scores at individual sites on all tRNAs. Unlike antibody-based methods, such as methylated RNA immunoprecipitation and sequencing (meRIP-Seq) for enrichment of methylated RNA sequences, chemically based approaches, including TRAC-Seq, can provide nucleotide-level resolution of modification sites. Compared to the related method AlkAniline-Seq (alkaline hydrolysis and aniline cleavage sequencing), TRAC-Seq incorporates small RNA selection, AlkB demethylation, and sodium borohydride reduction steps to achieve specific and efficient single-nucleotide resolution profiling of m7G sites in tRNAs. The m7G TRAC-Seq protocol could be adapted to chemical cleavage-mediated detection of other RNA modifications. The protocol can be completed within ~9 d for four biological replicates of input and treated samples.


Assuntos
Guanosina/análogos & derivados , RNA de Transferência/química , Análise de Sequência de RNA/métodos , Animais , Genômica , Guanosina/análise , Guanosina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metilação , Camundongos , RNA de Transferência/genética , Software , Transcriptoma
17.
EBioMedicine ; 47: 195-207, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31409574

RESUMO

BACKGROUND: Accumulating evidence has revealed the critical roles of N6-methyladenosine (m6A) modification of mRNA in various cancers. However, the biological function and regulation of m6A in bladder cancer (BC) are not yet fully understood. METHODS: We performed cell phenotype analysis and established in vivo mouse xenograft models to assess the effects of m6A-modified ITGA6 on BC growth and progression. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation and luciferase reporter and mutagenesis assays were used to define the mechanism of m6A-modified ITGA6. Immunohistochemical analysis was performed to assess the correlation between METTL3 and ITGA6 expression in bladder cancer patients. FINDINGS: We show that the m6A writer METTL3 and eraser ALKBH5 altered cell adhesion by regulating ITGA6 expression in bladder cancer cells. Moreover, upregulation of ITGA6 is correlated with the increase in METTL3 expression in human BC tissues, and higher expression of ITGA6 in patients indicates a lower survival rate. Mechanistically, m6A is highly enriched within the ITGA6 transcripts, and increased m6A methylations of the ITGA6 mRNA 3'UTR promotes the translation of ITGA6 mRNA via binding of the m6A readers YTHDF1 and YTHDF3. Inhibition of ITGA6 results in decreased growth and progression of bladder cancer cells in vitro and in vivo. Furthermore, overexpression of ITGA6 in METTL3-depleted cells partially restores the BC adhesion, migration and invasion phenotypes. INTERPRETATION: Our results demonstrate an oncogenic role of m6A-modified ITGA6 and show its regulatory mechanisms in BC development and progression, thus identifying a potential therapeutic target for BC. FUND: This work was supported by National Natural Science Foundation of China (81772699, 81472999).


Assuntos
Adenosina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina alfa6/genética , RNA Mensageiro/genética , Neoplasias da Bexiga Urinária/genética , Adenosina/farmacologia , Adulto , Idoso , Homólogo AlkB 5 da RNA Desmetilase/genética , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Integrina alfa6/metabolismo , Masculino , Metiltransferases/genética , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
18.
Mol Cell Biol ; 39(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31285272

RESUMO

The long noncoding RNA HOXA-AS3 has recently been reported to act as a critical regulator in inflammation-linked lung adenocarcinoma. However, the roles of HOXA-AS3 in endothelium inflammation and related vascular disorders remain poorly defined. In the current study, we identified HOXA-AS3 to be a critical activator to promote NF-κB-mediated endothelium inflammation. HOXA-AS3, a chromatin-associated regulator which colocalizes with NF-κB at specific gene promoters, was found to interact with NF-κB and positively regulate its activity through control of the expression of the NF-κB inhibitor protein IκBα and the acetylation status at the K310 site of p65. More importantly, clinicopathological analysis showed that HOXA-AS3 expression has a significant positive correlation with atherosclerosis. Thus, we conclude that HOXA-AS3 may serve as a crucial biomarker for the clinical diagnosis of atherosclerosis, as well as a promising therapeutic target for the treatment of multiple inflammatory vascular diseases. In addition, this study suggests the functional importance of HOXA-AS3 in the regulation of inflammatory disorders.


Assuntos
Aterosclerose/genética , Marcadores Genéticos/genética , RNA Longo não Codificante/genética , Transdução de Sinais , Regulação para Cima , Acetilação , Idoso , Diagnóstico Precoce , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas
19.
J Cancer ; 10(16): 3778-3788, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31333795

RESUMO

Selective covalent CDK7 inhibitor THZ1 is a promising potential anti-tumor drug in many kinds of cancers. Epithelial-mesenchymal Transition (EMT) is highly related to cancer initiation, development, invasion and metastasis and other pathogenesis processes. We treated cancer cell line Hela229 and three retinoblastoma cell lines so-RB50, WERI-Rb-1, Y79 with gradient concentration of THZ1, and found that THZ1 could inhibit cell viability and EMT, suggesting that THZ1 may be a promising drug for human cervical cancer and retinoblastoma treatment. Our results verified the role of THZ1 in EMT for the first time, however, the mechanism needs further study. Here we report that THZ1 suppresses the TGFß2 induced EMT in human SRA01/04 lens epithelial cells (LECs), rabbit primary lens epithelial cells, and whole rat lens culture semi-in vivo model. RNA-sequencing and KEGG analysis revealed that the THZ1 inhibits EMT by down-regulating phosphorylate Smad2 and Notch signaling pathway. On the other hand, we found that THZ1 could strongly inhibit LECs proliferation through G2/M phase arrest as well as attenuating of MAPK, PI3K/AKT signaling pathway. Our results uncovered the function and underlying mechanism of THZ1 in regulation of EMT, which provides a new perspective of the anti-tumor effect by THZ1 and may offer a novel treatment for PCO.

20.
Artigo em Inglês | MEDLINE | ID: mdl-31172659

RESUMO

The past few decades have witnessed the booming field of cancer immunotherapy. Cancer therapeutic vaccines, either alone or in combination with other immunotherapies such as adoptive cell therapy or immune checkpoint blockade therapy, are an attractive class of cancer immunotherapeutics. However, cancer vaccines have thus far shown suboptimal efficacy in the clinic. Nanomedicines offer unique opportunities to improve the efficacy of these vaccines. A variety of nanoplatforms have been investigated to deliver molecular or cellular or subcellular vaccines to target lymphoid tissues and cells, thereby promoting the potency and durability of anti-tumor immunity while reducing adverse side effects. In this article, we reviewed the key parameters and features of nanovaccines for cancer immunotherapy; we highlighted recent advances in the development of cancer nanovaccines based on synthetic nanocarriers, biogenic nanocarriers, as well as semi-biogenic nanocarriers; and we summarized newly emerging types of nanovaccines, such as those based on stimulator of interferon genes agonists, cancer neoantigens, mRNA vaccines, as well as artificial antigen-presenting cells. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.


Assuntos
Vacinas Anticâncer/imunologia , Imunoterapia , Nanomedicina , Neoplasias/imunologia , Neoplasias/terapia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos
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