RESUMO
MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti-miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2-amino-6-vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross-linked with AVP-conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross-linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7-propynyl group - which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP - a deoxy analog of ADpVP - through a simplified synthesis protocol. Evaluation of the cross-linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross-linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross-linking nucleosides for indirect miRNA function inhibition.
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2'-Amino-LNA has the potential to acquire various functions through chemical modification at the 2'-nitrogen atom. This study focused on 2'-N-alkylaminocarbonyl 2'-amino-LNA, which is a derivative of 2'-amino-LNA. We evaluated its practical usefulness as a chemical modification of anti-miRNA oligonucleotide. The synthesis of phosphoramidites of 2'-N-alkylaminocarbonyl substituted 2'-amino-LNA bearing thymine and 5-methylcytosine proceeded in good yields. Incorporating the 2'-N-alkylaminocarbonyl-2'-amino-LNA monomers into oligonucleotides improved the duplex stability for complementary RNA strands and robust nuclease resistance. Moreover, 2'-N-alkylaminocarbonyl-2'-amino-LNA is a promising scaffold that significantly increases the potency of anti-miRNA oligonucleotides.
Assuntos
MicroRNAs , Oligonucleotídeos/farmacologia , Alcanos/químicaRESUMO
The interstrand crosslinking of nucleic acids is one of the strategies to create the stable complex between an oligonucleotide and RNA by covalent bond formation. We previously reported that fully 2'-O-methylated (2'-OMe) RNAs having the 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. In this study, we established a chemical method to efficiently synthesize the crosslinked 2'-OMe RNA duplexes using AVP and prepared the anti-miRNA oligonucleotides (AMOs) containing the antisense targeting miR-21 and crosslinked duplex at the terminal sequences. These AMOs showed a markedly higher anti miRNA activity than that of the commercially-available miR-21 inhibitor which has locked nucleic acid (LNA) residues.
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MicroRNAs/antagonistas & inibidores , RNA/farmacologia , Relação Dose-Resposta a Droga , Humanos , Metilação , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Purinas/química , Purinas/farmacologia , RNA/síntese química , RNA/química , Relação Estrutura-Atividade , Compostos de Vinila/química , Compostos de Vinila/farmacologiaRESUMO
The Ras family of GTPases is involved in cell proliferation, cell survival, and angiogenesis. It is upregulated in several cancers, including pancreatic cancer (PC) and leads to uncontrolled growth and aggressiveness. PC is well known to be a lethal disease with poor prognosis, plagued by limited therapeutic modalities. MicroRNAs (miRNAs), which are short non-coding RNA molecules, have recently emerged as regulators of signaling networks and have shown potential to target pathway components for therapeutic use in several malignancies. K-Ras mutations are widespread in PC cases (90%), with mutations detectable as early as pancreatic intraepithelial neoplasias and in later metastatic stages alike; therefore, these mutations in K-Ras are obvious drivers and potential targets for PC therapy. Several K-Ras targeting miRNAs have lately been discovered, and many of them have shown promise in combating pancreatic tumor growth in vitro and in mouse models. However, the field of miRNA therapy is still in its infancy, and miRNA mimics or anti-miRNA oligonucleotides that target Ras pathway have thus far not been evaluated in PC patients. In this review, we summarize the role of several miRNAs that regulate oncogenic K-Ras signaling in PC, with their prospective roles as therapeutic agents for targeting K-Ras pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Animais , Epistasia Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Terapia de Alvo Molecular , Mutação , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Triple-negative breast cancer (TNBC) is an aggressive disease with a short median time from relapse to death. The increased aggressiveness, drug resistance, disease relapse, and metastasis are associated with the presence of stem cells within tumors. Several stem cell markers, such as CD24, CD44, CD133, ALDH1, and ABCG2, have been reported, but their roles in breast cancer tumorigenesis remain unclear. Herein, we apply RNA nanotechnology to deliver anti-microRNA (miRNA) for TNBC therapy. The thermodynamically and chemically stable three-way junction (3WJ) motif was utilized as the scaffold to carry an RNA aptamer binding to CD133 receptor and a locked nuclei acid (LNA) sequence for miRNA21 inhibition. Binding assays revealed the specific uptake of the nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. Functional assays showed that cancer cell migration was reduced, miR21 expression was inhibited, and downstream tumor suppressor PTEN and PDCD4 expressions were upregulated. In vitro and in vivo studies revealed that these therapeutic RNA nanoparticles did not induce cytokine secretion. Systemic injection of these RNA nanoparticles in animal trial demonstrated high specificity in TNBC tumor targeting and high efficacy for tumor growth inhibition. These results revealed the clinical translation potential of these RNA nanoparticles for TNBC therapy.
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Antígeno AC133/metabolismo , Sistemas de Liberação de Medicamentos/métodos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Nanopartículas/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Células RAW 264.7 , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses-miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide "shoulders" of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5-7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5'- and 3'-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.
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MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , Peptídeos/metabolismo , Ribonucleases/metabolismo , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Sequência de Bases , Biocatálise , Domínio Catalítico , Humanos , Concentração de Íons de Hidrogênio , Oligonucleotídeos/síntese química , Peptídeos/síntese química , Estabilidade de RNA , Ribonucleases/síntese químicaRESUMO
BACKGROUND: miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents. RESULTS: The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents. CONCLUSION: In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.
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Diterpenos do Tipo Caurano/biossíntese , Glucosídeos/biossíntese , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , Stevia/metabolismo , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Inativação Gênica , Glucosídeos/química , Glucosídeos/genética , MicroRNAs/genética , Folhas de Planta/química , Regiões Promotoras Genéticas , RNA de Plantas/genética , Stevia/genética , Edulcorantes/químicaRESUMO
MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. As a consequence of their function towards mRNA, miRNAs are widely associated with the pathogenesis of several human diseases, making miRNAs a target for new therapeutic strategies based on the control of their expression. Indeed, numerous works were published in the past decades showing the potential use of antisense oligonucleotides to target aberrant miRNAs (AMOs) involved in several human pathologies. New classes of chemical-modified-AMOs, including locked nucleic acid oligonucleotides, have recently proved their worth in silencing miRNAs. A correct design of a specific AMOs can help to improve their performance and potency towards the target miRNA by increasing for instance nuclease resistance and target affinity. This review outlines the technologies involved to suppress aberrant miRNAs. From the design strategies used in AMOs to its application in novel miRNA-based therapeutics and detection methodologies.
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Antineoplásicos/química , MicroRNAs/antagonistas & inibidores , Neoplasias/genética , Oligonucleotídeos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Desenho de Fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Oligonucleotídeos/farmacologia , Oligonucleotídeos/uso terapêuticoRESUMO
The practical use of solid lipid nanoparticles (SLNs) in research has been highlighted in the literature, but few reports have combined SLNs with miRNA-based therapy and chemotherapy. We aimed to prepare cationic SLNs (cSLNs) to load anti-miR-21 oligonucleotide and pemetrexed for glioblastoma therapy in vitro. cSLNs were employed to encapsulate both pemetrexed and anti-miR-21 by a high-pressure homogenization method, and then the properties of cSLNs were characterized. We studied cellular uptake and cytotoxicity properties of cSLNs in U87MG cells. cSLNs were 124.9 ± 1.6 nm in size and 27.3 ± 1.6 mV in zeta potential with spherical morphology in the TEM image. cSLNs uptake by U87MG cells was increased significantly higher and more effective than free pemetrexed. These findings suggest that cSLNs represent a potential new approach for carrying both pemetrexed and anti-miR-21 for glioblastoma therapy.
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Glioblastoma/tratamento farmacológico , Nanopartículas/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Pemetrexede/administração & dosagem , Pemetrexede/farmacologia , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Humanos , Lipídeos/química , MicroRNAs , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
MicroRNAs (miRNAs) are short RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play an important role in the pathogenesis of diseases. Numerous studies aimed at developing novel miRNA-based drugs or determining miRNA functions have been conducted by inhibiting miRNAs using anti-miRNA oligonucleotides (AMOs), which inhibit the function by hybridizing with miRNA. To increase the binding affinity and specificity to target miRNA, AMOs with various chemical modifications have been developed. Evaluating the potency of these various types of AMOs is an essential step in their development. In this study, we developed a capillary electrophoresis with laser-induced fluorescence (CE-LIF) method to evaluate the potency of AMOs by measuring changes in miRNA levels with fluorescence-labeled ssDNA probes using AMO-miR-23a, which inhibits miR-23a related to lung cancer. In order to eliminate interference by excess AMOs during hybridization of the ssDNA probe with the miR-23a, the concentration of the ssDNA probe was optimized. This newly developed method was used to compare the potency of two different modified AMOs. The data were supported by the results of a luciferase assay. This study demonstrated that CE-LIF analysis could be used to accurately evaluate AMO potency in biological samples.
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Antagomirs/administração & dosagem , Antagomirs/genética , Regulação para Baixo/genética , Eletroforese Capilar/métodos , Regulação da Expressão Gênica/genética , MicroRNAs/biossíntese , Espectrometria de Fluorescência/métodos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
MiR-21 acts as a ubiquitous oncogene in major classes of human cancers and is a potential target for therapeutic intervention. However, the relative expression of miR-21 in retinoblastoma is poorly understood. Here we detected miR-21 expression in HXO-RB44 cell line human normal retinal tissues and retinoblastoma (Rb) tissue specimens, and studied its function using an 8-mer tiny seed-targeting anti-miR-21 (t-anti-miR-21). RT-PCR revealed that miR-21 was highly overexpressed in HXO-RB44 cells and Rb tissue specimens compared with normal human retinal tissues. The localization and transfection efficiency of t-anti-miR-21 and the cell cycle distribution were detected by confocal microscopy and flow cytometry. In addition, we found that t-anti-miR-21 led to a significant inhibition of retinoblastoma cell proliferation, migration and colony formation in vitro, with a similar effect to anti-miR-21. Anti-miR-21 down-regulated the miR-21 level, whereas both 8-mer t-anti-miR-21 and 15-mer m-anti-miR-21 had no impact on miR-21 expression levels. Finally, the phosphorylation signaling pathway, down-regulated by t-anti-miR-21, was integrated by KEGG assay, which elucidated the potential mechanisms of inhibition of miR-21 in retinoblastoma. Taken together, knockdown of miR-21 in the HXO-RB44 cell is capable of inhibiting cancer progression in retinoblastoma. Seed-targeting t-anti-miR-21 was a novel strategy for mir-21-based therapeutics and drug discovery.
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MicroRNAs/genética , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/genética , Neoplasias da Retina/prevenção & controle , Retinoblastoma/prevenção & controle , Transdução de Sinais/fisiologia , Apoptose , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/metabolismo , Microscopia Confocal , Proteínas de Neoplasias/metabolismo , Fosforilação , Análise Serial de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Retina/metabolismo , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: MicroRNAs (miRNA) are small noncoding RNAs that play a significant role in the regulation of gene expression. The literature has explored the key involvement of miRNAs in the diagnosis, prognosis, and treatment of various neurodegenerative diseases (NDD), such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The miRNA regulates various signalling pathways; its dysregulation is involved in the pathogenesis of NDD. OBJECTIVE: The present review is focused on the involvement of miRNAs in the pathogenesis of NDD and their role in the treatment or management of NDD. The literature provides comprehensive and cutting-edge knowledge for students studying neurology, researchers, clinical psychologists, practitioners, pathologists, and drug development agencies to comprehend the role of miRNAs in the NDD's pathogenesis, regulation of various genes/signalling pathways, such as α-synuclein, P53, amyloid-ß, high mobility group protein (HMGB1), and IL-1ß, NMDA receptor signalling, cholinergic signalling, etc. Methods: The issues associated with using anti-miRNA therapy are also summarized in this review. The data for this literature were extracted and summarized using various search engines, such as Google Scholar, Pubmed, Scopus, and NCBI using different terms, such as NDD, PD, AD, HD, nanoformulations of mRNA, and role of miRNA in diagnosis and treatment. RESULTS: The miRNAs control various biological actions, such as neuronal differentiation, synaptic plasticity, cytoprotection, neuroinflammation, oxidative stress, apoptosis and chaperone-mediated autophagy, and neurite growth in the central nervous system and diagnosis. Various miRNAs are involved in the regulation of protein aggregation in PD and modulating ß-secretase activity in AD. In HD, mutation in the huntingtin (Htt) protein interferes with Ago1 and Ago2, thus affecting the miRNA biogenesis. Currently, many anti-sense technologies are in the research phase for either inhibiting or promoting the activity of miRNA. CONCLUSION: This review provides new therapeutic approaches and novel biomarkers for the diagnosis and prognosis of NDDs by using miRNA.
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Doença de Alzheimer , Doença de Huntington , MicroRNAs , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Doença de Parkinson/terapia , Doença de Huntington/genéticaRESUMO
Rationale: Growing evidence has demonstrated that miRNA-21 (miR-21) upregulation is closely associated with tumor pathogenesis. However, the mechanisms by which miR-21 inhibition modulates the immunosuppressive tumor microenvironment (TME) and improves tumor sensitivity to immune checkpoint blockade therapies remain largely unexplored. In this study, we demonstrate the precise delivery of anti-miR-21 using a PD-L1-targeting peptide conjugate (P21) to the PD-L1high TME. Methods: Investigating miR-21 inhibition mechanisms involved conducting quantitative real-time PCR, western blot, flow cytometry, and confocal microscopy analyses. The antitumor efficacy and immune profile of P21 monotherapy, or combined with anti-PD-L1 immune checkpoint inhibitors, were assessed in mouse models bearing CT26.CL25 tumors and 4T1 breast cancer. Results Inhibition of oncogenic miR-21 in cancer cells by P21 efficiently activates tumor suppressor genes, inducing autophagy and endoplasmic reticulum stress. Subsequent cell-death-associated immune activation (immunogenic cell death) is initiated via the release of damage-associated molecular patterns. The in vivo results also illustrated that the immunogenic cell death triggered by P21 could effectively sensitize the immunosuppressive TME. That is, P21 enhances CD8+ T cell infiltration in tumor tissues by conferring immunogenicity to dying cancer cells and promoting dendritic cell maturation. Meanwhile, combining P21 with an anti-PD-L1 immune checkpoint inhibitor elicits a highly potent antitumor effect in a CT26.CL25 tumor-bearing mouse model and 4T1 metastatic tumor model. Conclusions: Collectively, we have clarified a miR-21-related immunogenic cell death mechanism through the precise delivery of anti-miR-21 to the PD-L1high TME. These findings highlight the potential of miR-21 as a target for immunotherapeutic interventions.
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Antígeno B7-H1 , Morte Celular Imunogênica , Imunoterapia , MicroRNAs , Microambiente Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Camundongos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Morte Celular Imunogênica/efeitos dos fármacos , Linhagem Celular Tumoral , Imunoterapia/métodos , Feminino , Camundongos Endogâmicos BALB C , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genéticaRESUMO
Non-coding RNAs (ncRNAs) have emerged as key regulators of gene expression, with growing evidence implicating their involvement in cancer development and progression. The potential of ncRNAs as diagnostic and prognostic biomarkers for cancer is promising, with emphasis on their use in liquid biopsy and tissue-based diagnostics. In a nutshell, the review comprehensively summarizes the diverse classes of ncRNAs implicated in cancer, including microRNAs, long non-coding RNAs, and circular RNAs, and their functions and mechanisms of action. Furthermore, we describe the potential therapeutic applications of ncRNAs, including anti-miRNA oligonucleotides, siRNAs, and other RNA-based therapeutics in cancer treatment. However, significant challenges remain in developing effective ncRNA-based diagnostics and therapeutics, including the lack of specificity, limited understanding of mechanisms, and delivery challenges. This review also covers the current state-of-the-art non-coding RNA research technologies and bioinformatic analysis tools. Lastly, we outline future research directions in non-coding RNA research in cancer, including developing novel biomarkers, therapeutic targets, and modalities. In summary, this review provides a comprehensive understanding of non-coding RNAs in cancer and their potential clinical applications, highlighting both the opportunities and challenges in this rapidly evolving field.
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Postmenopausal bone loss often leads to osteoporosis and fragility fractures. Bone mass can be increased by the first 34 amino acids of human parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), or by a monoclonal antibody against sclerostin (Scl-Ab). Here, we show that PTH and Scl-Ab reduce the expression of microRNA-19a and microRNA-19b (miR-19a/b) in bone. In bones from patients with lower bone mass and from osteoporotic mice, miR-19a/b expression is elevated, suggesting an inhibitory function in bone remodeling. Indeed, antagonizing miR-19a/b in vivo increased bone mass without overt cytotoxic effects. We identified TG-interacting factor 1 (Tgif1) as the target of miR-19a/b in osteoblasts and essential for the increase in bone mass following miR-19a/b inhibition. Furthermore, antagonizing miR-19a/b augments the gain in bone mass by PTH and restores bone loss in mouse models of osteoporosis in a dual mode of action by supporting bone formation and decreasing receptor activator of NF-κB ligand (RANKL)-dependent bone resorption. Thus, this study identifies novel mechanisms regulating bone remodeling, which opens opportunities for new therapeutic concepts to treat bone fragility.
Assuntos
MicroRNAs , Osteoporose , Humanos , Camundongos , Animais , Densidade Óssea , Osteoporose/tratamento farmacológico , Osso e Ossos , Osteoblastos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Homeodomínio/metabolismoRESUMO
Crosslinking reactions to nucleic acids are an effective way to prepare stable complexes formed by covalent bonding. We demonstrated that fully 2'-O-methylated (2'-OMe) RNAs having a 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. Recently, we reported the preparation of crosslinked 2'-OMe RNA duplexes using AVP and the anti-miRNA oligonucleotides (AMOs) containing crosslinked duplexes at the terminal positions. These AMOs exhibited efficient microRNA (miRNA) inhibition at very low concentrations. In this article, we describe the chemical synthesis of 2'-OMe oligonucleotides containing AVP and preparation of the AMOs bearing crosslinked 2'-OMe RNA duplexes using AVP. In addition, we describe in detail the miRNA inhibition assay using these AMOs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphoramidite of 2-amino-6-vinylguanosine derivative Basic Protocol 2: Synthesis of AVP-2'-OMe RNA Basic Protocol 3: Evaluation of the crosslink reactivity of CFO containing AVP to the 2'-OMe RNA and preparation of AMOs containing crosslinked duplex Basic Protocol 4: miRNA inhibition assays.
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MicroRNAs , Oligonucleotídeos/química , Purinas/química , Compostos de Vinila/químicaRESUMO
Upregulation of oncogenic miRNA21 (miR-21) plays a pivotal role in proliferation, migration and invasion of cancer cells. In addition to cancer cells, tumor-associated macrophages (TAMs) also have high abundance of miR-21, which accelerates malignant progression of tumors in the late stages of carcinogenesis. Despite of the pro-tumorigenic functions of miR-21 in TAMs and cancer cells, reliable therapeutic strategies to simultaneously inhibit miR-21 activity in both types of cell have not yet been developed. In this study, we designed a dual-targeting drug delivery system of miR-21 inhibitors that could bind to both tumor cells and macrophages with overexpressed PD-L1 receptors. This peptide-oligonucleotide conjugate (Pep-21) consists of a PDL1-binding peptide covalently linked with an anti-miR-21 inhibitor via click chemistry. Pep-21 was preferentially internalized in both cell types, consequently depleting endogenous miR-21. Our studies found that Pep-21 treatment reduced tumor cell migration, reprogrammed immunosuppressive M2-type TAMs into M1-type macrophages, and restrained tumor progression. Collectively, neutralization of miR-21 activity in both cancer cells and TAMs can be a promising strategy for effective antitumor responses.
Assuntos
MicroRNAs , Neoplasias , Antígeno B7-H1/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Peptídeos , Microambiente Tumoral , Macrófagos Associados a TumorRESUMO
Rational combinations of sequence-specific inhibitors of pro-oncogenic miRNAs can efficiently interfere with specific tumor survival pathways, offering great promise for targeted therapy of oncological diseases. Herein, we uncovered the potential of multicomponent therapy by double or triple combinations of highly potent mesyl phosphoramidate (µ) antisense oligodeoxynucleotides targeted to three proven pro-oncogenic microRNAs-miR-17, miR-21, and miR-155. A strong synergism in the inhibition of proliferation and migration of B16 melanoma cells was demonstrated in vitro for pairs of µ-oligonucleotides, which resulted in vivo in profound inhibition (up to 85%) of lung metastases development after intravenous injection of µ-oligonucleotide-transfected B16 cells in mice. A clear benefit of µ-21-ON/µ-17-ON and µ-17-ON/µ-155-ON/µ-21-ON combination antitumor therapy was shown for the lymphosarcoma RLS40 solid tumor model. In vivo administration of the µ-17-ON/µ-155-ON/µ-21-ON cocktail into RLS40-bearing mice elicited fourfold delay of tumor growth as a result of strong inhibition of tumor mitotic activity. It was discovered that the cocktail of µ-21-ON/µ-17-ON/µ-155-ON led to a twofold decrease in total destructive changes in murine liver, which indicates both the reduction in toxic tumor burden and the absence of specific toxicity of the proposed therapy.
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Antisense imaging uses radionuclide labeled antisense oligonucleotides to hybridize with nucleic acids in vivo, display the expression of target genes, and directly quantify biological processes at the cellular and subcellular levels. The anti-miRNA oligonucleotides (AMOs) are a series of single-stranded DNA oligonucleotides that are widely used in gene imaging and gene therapy. However, due to the negative charge and high molecular weight, the permeability through the membrane of AMOs is generally low so that most AMOs cannot enter the cells. Based on the 99mTc-labeled AMOs imaging in previous studies, this study developed a novel tetrapeptide Glycine-Alanine-Glycine-Lysine (Gly-Ala-Gly-Lys, GAGK) for one-step labeling AMO with 99mTc. The labeling conditions were optimized by changing the number of stannous ions, the reaction time, and the temperature, respectively. The labeled products were identified by gel electrophoresis and their serum stability was evaluated. The optimal labeling condition in this study was using 1 mg/mL SnCl2·2H2O and heating for 30 min at 100°C. Gel electrophoresis confirmed the verification of successful labeling of 99mTc-GAGK-AMO. After being incubated with human fresh serum for 12 h, 99mTc-GAGK-AMO showed good stability and no obvious degradation. Therefore, this labeling method has high labeling efficiency and stable labeling, which provides an effective method for the application of miRNA-targeted imaging.
RESUMO
At present, treating of triple negative breast cancer (TNBC) mainly depends on chemotherapy with more toxic side effects, but the effect is limited and it is highly prone to drug resistance. Gene therapy using anti-microRNAs maybe one of alternative therapeutic strategies. Due to the poor cell permeability and significant in vivo decomposition rate of anti-microRNAs, which limits their clinical application, we developed a core-shell supramolecular nanovector of "chitosome" that were self-assembled from the synthetic amphiphilic chitosan derivatives. The constructed chitosomes could co-load hydrophilic anti-miR-21 and hydrophobic docetaxel (DTX) into one combo nanocarrier with entrapment efficiency of more than 80%, as well as spherical morphology and average particle size of 90 nm. In comparison with the naked ones, anti-miR-21 encapsulated with chitosomes showed significantly increased cellular transfection and stability against degradation by nuclease in serum. Compared with DTX or anti-miR-21 formulations used alone, the co-delivery of the two drugs with the combo chitosome obtained improved chemosensitivity of TNBC cells to DTX treatment through their synergistic mechanisms. Taken together, the developed chitosome could be a promising candidate for simultaneous delivery of insoluble chemotherapeutic drugs and gene agents for TNBC therapy. Graphical abstract.