RESUMO
MicroRNAs (miRNAs) are recognized as an essential component of the RNA family, exerting multiple and intricate biological functions, particularly in the process of tumorigenesis, proliferation, and metastatic progression. MiRNAs are altered in gastric cancer (GC), showing activity as both tumor suppressors and oncogenes, although their true roles have not been fully understood. This review will focus upon the recent advances of miRNA studies related to the regulatory mechanisms of gastric tumor cell proliferation, apoptosis, and cell cycle. We hope to provide an in-depth insight into the mechanistic role of miRNAs in GC development and progression. In particular, we summarize the latest studies relevant to miRNAs' impact upon the epithelial-mesenchymal transition, tumor microenvironment, and chemoresistance in GC cells. We expect to elucidate the molecular mechanisms involving miRNAs for better understanding the etiology of GC, and facilitating the development of new treatment regimens for the treatment of GC.
Assuntos
MicroRNAs/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Transdução de Sinais/fisiologia , Neoplasias Gástricas/fisiopatologia , Microambiente Tumoral/fisiologiaRESUMO
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system is an RNA-guided, DNA editing method that has been widely used for gene editing, including human viruses. Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), following latent infection in human cells, can cause a variety of malignancies, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), with a high prevalence in immunocompromised patients. Of significant concern, the latent infection with KSHV has been shown to lead to increased resistance to antiviral therapies. MicroRNAs (miRNAs) are a set of non-coding, small RNA molecules that regulate protein-coding genes at the post-transcriptional and translational levels. KSHV has its miRNAs, most of which are expressed in latently infected cells and play a crucial role in maintaining KSHV latency. Notably, by regulating the expression of the downstream target genes in host cells, KSHV miRNAs can interact with the host environment to promote the development of KSHV-related diseases. Although CRISPR/Cas9 has been reported to edit KSHV protein-coding genes, there is no published literature on whether the CRISPR/Cas9 system can regulate the expression of KSHV miRNAs. In this study, we used CRISPR/Cas9 to inhibit the expression of KSHV miRNAs by directly editing the DNA sequences of individual KSHV miRNAs, or the promoter of clustered KHSV miRNAs, in latent KSHV-infected PEL cells. Our results show that CRISPR/Cas9 can ablate KSHV miRNAs expression, which in turn leads to the upregulation of viral lytic genes and alteration of host cellular gene expression. To the best of our knowledge, our study is the first reported demonstration of the CRISPR/Cas9 system editing KSHV miRNAs, further expanding the application of CRISPR/Cas9 as a novel antiviral strategy targeting KSHV latency.
Assuntos
Sistemas CRISPR-Cas , Herpesvirus Humano 8/genética , Infecção Latente/genética , Linfoma de Efusão Primária/virologia , Edição de Genes , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , MicroRNAs , Oxigênio/metabolismo , Medicina de Precisão/métodos , Sarcoma de Kaposi/virologiaRESUMO
The immune system plays an essential and central role in tumor cell differentiation, proliferation, angiogenesis, apoptosis, invasion, and metastasis. Over the past decade, cancer therapy has rapidly evolved from traditional approaches, such as surgery, chemotherapy, and radiotherapy, to revolutionary new treatment options with immunotherapy. This new era of cancer treatment options has now been clinically tested and applied to many forms of human malignancies, often with quite dramatic results. As we develop more effective combinations of cancer treatment, several agents have been recently investigated, putatively identified as anticancer agents, or immunostimulatory molecules. One such agent is metformin, originally developed as a fairly standard first-line therapy for patients with type-2 diabetes mellitus (T2DM). Given the underlying mechanisms of action, researchers began to examine the alternative functions and possible utility of metformin, finding that the cancer risk in patients with T2DM was reduced. It appears that metformin, at least in part, has an antitumor effect through activation of the 5' adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Moreover, numerous studies have demonstrated that metformin interferes with key immunopathological mechanisms that are involved in the pathological processes or associated with malignant progression. Such insights may shed light on further analyzing whether metformin enhances the effectiveness of the immunotherapy and overcomes the immunotherapy resistance in the patients. Herein, we provide a comprehensive review of the literature examining the impact of metformin upon the host immune system and cancer immunity.
Assuntos
Antineoplásicos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Metformina/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antineoplásicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores Imunológicos/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Metformina/farmacologia , Células Mieloides/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
LncRNAs represent a large class of noncoding RNA molecules that have important functions and play key roles in a variety of human diseases. There is an urgent need to develop bioinformatics tools as to gain insight into lncRNAs. This study developed a sequence-based bioinformatics method, LncDisease, to predict the lncRNA-disease associations based on the crosstalk between lncRNAs and miRNAs. Using LncDisease, we predicted the lncRNAs associated with breast cancer and hypertension. The breast-cancer-associated lncRNAs were studied in two breast tumor cell lines, MCF-7 and MDA-MB-231. The qRT-PCR results showed that 11 (91.7%) of the 12 predicted lncRNAs could be validated in both breast cancer cell lines. The hypertension-associated lncRNAs were further evaluated in human vascular smooth muscle cells (VSMCs) stimulated with angiotensin II (Ang II). The qRT-PCR results showed that 3 (75.0%) of the 4 predicted lncRNAs could be validated in Ang II-treated human VSMCs. In addition, we predicted 6 diseases associated with the lncRNA GAS5 and validated 4 (66.7%) of them by literature mining. These results greatly support the specificity and efficacy of LncDisease in the study of lncRNAs in human diseases. The LncDisease software is freely available on the Software Page: http://www.cuilab.cn/.
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Neoplasias da Mama/genética , Biologia Computacional/métodos , Hipertensão/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Angiotensina II/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , SoftwareRESUMO
AIM: We have reported novel anticancer bioactive peptides (ACBPs) that show tumor-suppressive activities in human gastric cancer, leukemia, nasopharyngeal cancer, and gallbladder cancer. In this study, we investigated the effects of ACBPs on human colorectal cancer and the underlying mechanisms. METHODS: Cell growth and apoptosis of human colorectal tumor cell line HCT116 were measured using cell proliferation assay and flow cytometry, respectively. The expression levels of PARP, p53 and Mcl1A were assessed with Western blotting and immunohistochemistry. For evaluation of the in vivo antitumor activity of ACBPs, HCT116 xenograft nude mice were treated with ACBPs (35 µg/mL, ip) for 10 days. RESULTS: Treatment of HCT116 cells with ACBPs (35 µg/mL) for 4-6 days significantly inhibited the cell growth. Furthermore, treatment of HCT116 cells with ACBPs (35 µg/mL) for 6-12 h significantly enhanced UV-induced apoptosis, increased the expression of PARP and p53, and decreased the expression of Mcl-1. Administration of ACBPs did not change the body weight of HCT116 xenograft nude mice, but decreased the tumor growth by approximately 43%, and increased the expression of PARP and p53, and decreased the expression of Mcl-1 in xenograft mouse tumor tissues. CONCLUSION: Administration of ACBPs inhibits human colorectal tumor cell growth and induces apoptosis in vitro and in vivo through modulating the PARP-p53-Mcl-1 signaling pathway.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Peptídeos/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Masculino , Camundongos Nus , Reto/efeitos dos fármacos , Reto/metabolismo , Reto/patologiaRESUMO
The thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC) is responsible for the major sodium chloride reabsorption pathway, which is located in the apical membrane of the epithelial cells of the distal convoluted tubule. TSC is involved in several physiological activities including transepithelial ion absorption and secretion, cell volume regulation, and setting intracellular Cl(-) concentration below or above its electrochemical potential equilibrium. In addition, TSC serves as the target of thiazide-type diuretics that are the first line of therapy for the treatment of hypertension in the clinic, and its mutants are also reported to be associated with the hereditary disease, Gitelman's syndrome. This review aims to summarize the publications with regard to the TSC by focusing on the association between TSC mutants and human hypertension as well as Gitelman's syndrome.
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Polimorfismo Genético , Genótipo , Síndrome de Gitelman/genética , Humanos , Hipertensão/genética , Mutação de Sentido Incorreto , Fenótipo , Simportadores de Cloreto de Sódio/genética , Simportadores de Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Tiazidas/farmacologiaRESUMO
MicroRNAs (miRNAs) are a class of small, non-coding, and endogenous RNA molecules, which are evolutionarily conserved but play a significant role in regulation of protein-coding gene expression at posttranscriptional and translational levels. Strikingly, a single miRNA is able to trigger hundreds of putative target genes by incomplete or complete complementary binding to their 3' untranslated regions. Given their appearance in almost all types of tissues, miRNAs have been demonstrated to be intensively involved in normal and pathological processes of human cells. Aside from the role as invaluable biomarkers in indication of tumorigenesis and tumor progression, numerous studies have revealed the potential of miRNAs as novel targets of anticancer drugs in cancer therapy. In this review article, we focus on the summary of the latest publications on the topic of miRNA and anticancer drugs, and expect to shed light on understanding the molecular mechanisms of chemoresistance involving miRNA regulation. These pieces of evidence will eventually provide insight into the development of novel and more efficacious anticancer drugs in the future.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Regiões 3' não Traduzidas/genética , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , HumanosRESUMO
Clinical and preclinical studies provide strong evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) can prevent numerous types of cancers, especially colorectal cancer. Unfortunately, the depletion of physiologically important prostaglandins due to cyclooxygenase (COX) inhibition results in potentially fatal toxicities that preclude the long-term use of NSAIDs for cancer chemoprevention. While studies have shown an involvement of COX-2 in colorectal tumorigenesis, other studies suggest that a COX-independent target may be at least partially responsible for the antineoplastic activity of NSAIDs. For example, certain NSAID derivatives have been identified that do not inhibit COX-2 but have demonstrated efficacy to suppress carcinogenesis with potential for reduced toxicity. A number of alternative targets have also been reported to account for the tumor cell growth inhibitory activity of NSAIDs, including the inhibition of cyclic guanosine monophosphate phosphodiesterases (cGMP PDEs), generation of reactive oxygen species (ROS), the suppression of the apoptosis inhibitor protein, survivin, and others. Here, we review several promising mechanisms that are being targeted to develop safer and more efficacious NSAID derivatives for colon cancer chemoprevention.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Anticarcinógenos/uso terapêutico , Neoplasias Colorretais/prevenção & controle , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , SurvivinaRESUMO
Hypoxia plays an important role in the tumor microenvironment by allowing the development and maintenance of cancer cells, but the regulatory mechanisms by which tumor cells adapt to hypoxic conditions are not yet well understood. MicroRNAs are recognized as a new class of master regulators that control gene expression and are responsible for many normal and pathological cellular processes. Studies have shown that hypoxia inducible factor 1 (HIF1) regulates a panel of microRNAs, whereas some of microRNAs target HIF1. The interaction between microRNAs and HIF1 can account for many vital events relevant to tumorigenesis, such as angiogenesis, metabolism, apoptosis, cell cycle regulation, proliferation, metastasis, and resistance to anticancer therapy. This review will summarize recent findings on the roles of hypoxia and microRNAs in human cancer and illustrate the machinery by which microRNAs interact with hypoxia in tumor cells. It is expected to update our knowledge about the regulatory roles of microRNAs in regulating tumor microenvironments and thus benefit the development of new anticancer drugs.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Neoplasias , Hipóxia Celular/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/metabolismo , Neovascularização Patológica/genética , Regulação para CimaRESUMO
MicroRNAs (miRNAs) are small non-coding RNA molecules, whose primary function is to regulate gene expression at the post-transcriptional/translational levels. MiRNAs play crucial roles in normal biological processes and are commonly dys-regulated in human diseases. Stem cells are regarded as the "mother" cells of all types of differentiated cells that comprise tissues and organs of the body. A novel hypothesis proposes that tumors are composed of heterogeneous cells derived from cancer stem cells, which have self-renewal and differentiation capabilities similar to those of normal stem cells. Cancer stem cells have been isolated and characterized from various tumors. Given recent studies supporting the critical regulatory roles of miRNAs in the self-renewal and differentiation of cancer stem cells, better understanding the functions of miRNAs will provide invaluable insights into the prevention of tumorigenesis and tumor progression. In this review, we will summarize the research progress in the study of miRNAs involved in the self-renewal and differentiation of cancer stem cells.
Assuntos
MicroRNAs/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Diferenciação Celular , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Processamento Pós-Transcricional do RNARESUMO
SnoRNAs are frequently processed into snoRNA-derived RNAs (sdRNAs) that function much like traditional microRNAs (miRNAs). That said, our analyses suggest a global switch from DICER-dependent (predominately miRNA) to DICER-independent (predominately sdRNA) biogenesis/gene regulation in colon cancer. Whereas the expressions of 259 of 288 appreciably expressed miRNAs are significantly decreased (avg. 6.4% of WT) in human colon cancer DICER-KOs, 95 of 103 sdRNAs are conversely, significantly increased (avg. 679.3%) in DICER-KOs as compared to WT. As many diseases are characterized by DICER deficiency, this putative global switch to DICER-independent sdRNA regulations may contribute to an array of human diseases.
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Introduction: Triple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs. Methods: We investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo. Results: We confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo. Discussion: Our data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.
Assuntos
Sulindaco , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Sulindaco/farmacologia , Sulindaco/uso terapêutico , Secretases da Proteína Precursora do Amiloide , Neoplasias de Mama Triplo Negativas/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Modelos Animais de DoençasRESUMO
MOTIVATION: Compared with complementary DNA (cDNA) or messenger RNA (mRNA) microarray data, microRNA (miRNA) microarray data are harder to normalize due to the facts that the total number of miRNAs is small, and that the majority of miRNAs usually have low expression levels. In bead-based microarrays, the hybridization is completed in several pools. As a result, the number of miRNAs tested in each pool is even smaller, which poses extra difficulty to intrasample normalization and ultimately affects the quality of the final profiles assembled from various pools. In this article, we consider a measurement error model-based method for bead-based microarray intrasample normalization. RESULTS: In this study, results from quantitative real-time PCR (qRT-PCR) assays are used as 'gold standards' for validation. The performance of the proposed measurement error model-based method is evaluated via a simulation study and real bead-based miRNA expression data. Simulation results show that the new method performs well to assemble complete profiles from subprofiles from various pools. Compared with two intrasample normalization methods recommended by the manufacturer, the proposed approach produces more robust final complete profiles and results in better agreement with the qRT-PCR results in identifying differentially expressed miRNAs, and hence improves the reproducibility between the two microarray platforms. Meaningful results are obtained by the proposed intrasample normalization method, together with quantile normalization as a subsequent complemental intersample normalization method. AVAILABILITY: Datasets and R package are available at http://gauss.usouthal.edu/publ/beadsme/.
Assuntos
MicroRNAs/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Humanos , Funções Verossimilhança , MicroRNAs/metabolismo , Modelos Biológicos , Reação em Cadeia da PolimeraseRESUMO
We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biossíntese de Proteínas , RNA Mensageiro/isolamento & purificação , Anticorpos , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Fluoruracila/farmacologia , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/isolamento & purificação , Humanos , Imunoprecipitação , Polirribossomos/metabolismoRESUMO
Subsequently to the publication of the above article, an interested reader drew to the authors' attention that a pair of data panels presented in each of Figs. 3 and 4 appeared to be overlapping, such that these data may have been derived from the same original sources where they were intended to have shown the results from experiments performed under different experimental conditions. The authors realised that these figures had inadvertently been assembled incorrectly; however, as they had retained their access to the raw data, the authors were able to make the appropriate corrections required for these figures. The corrected versions of Figs. 3 and 4, showing the correct wound healing assay result for the DU1450siSPAG9 experiment at 24 h in Fig. 3F and the correct Matrigel cell invasion assay result for PC3siSPAG9 in Fig. 4C, are shown on the subsequent pages. Note that these errors did not adversely affect the major conclusions reported in the study. The authors all agree with these corrections and thank the Editor of Oncology Reports for allowing them the opportunity to publish this corrigendum. The authors also apologize for any inconvenience caused, and agree to address any additional questions regarding their results. All raw data are available from the authors upon request. [Oncology Reports 32: 25332540, 2014; DOI: 10.3892/or.2014.3539].
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We have identified 38 specifically excised, differentially expressed snoRNA fragments (sdRNAs) in TCGA prostate cancer (PCa) patient samples as compared to normal prostate controls. SnoRNA-derived fragments sdRNA-D19b and -A24 emerged among the most differentially expressed and were selected for further experimentation. We found that the overexpression of either sdRNA significantly increased PC3 (a well-established model of castration-resistant prostate cancer (CRPC)) cell proliferation, and that sdRNA-D19b overexpression also markedly increased the rate of PC3 cell migration. In addition, both sdRNAs provided drug-specific resistances with sdRNA-D19b levels correlating with paclitaxel resistance and sdRNA-24A conferring dasatinib resistance. In silico and in vitro analyses revealed that two established PCa tumor suppressor genes, CD44 and CDK12, represent targets for sdRNA-D19b and sdRNA-A24, respectively. This outlines a biologically coherent mechanism by which sdRNAs downregulate tumor suppressors in AR-PCa to enhance proliferative and metastatic capabilities and to encourage chemotherapeutic resistance. Aggressive proliferation, rampant metastasis, and recalcitrance to chemotherapy are core characteristics of CRPC that synergize to produce a pathology that ranks second in cancer-related deaths for men. This study defines sdRNA-D19b and -A24 as contributors to AR-PCa, potentially providing novel biomarkers and therapeutic targets of use in PCa clinical intervention.
Assuntos
MicroRNAs , Neoplasias de Próstata Resistentes à Castração , Proliferação de Células/genética , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/uso terapêutico , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/metabolismo , RNA Nucleolar Pequeno/genéticaRESUMO
Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Apoptose , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MOTIVATION: MicroRNA (miRNA) is a set of newly discovered non-coding small RNA molecules. Its significant effects have contributed to a number of critical biological events including cell proliferation, apoptosis development, as well as tumorigenesis. High-dimensional genomic discovery platforms (e.g. microarray) have been employed to evaluate the important roles of miRNAs by analyzing their expression profiling. However, because of the small total number of miRNAs and the absence of well-known endogenous controls, the traditional normalization methods for messenger RNA (mRNA) profiling analysis could not offer a suitable solution for miRNA analysis. The need for the establishment of new adaptive methods has come to the forefront. RESULTS: Locked nucleic acid (LNA)-based miRNA array was employed to profile miRNAs using colorectal cancer cell lines under different treatments. The expression pattern of overall miRNA profiling was pre-evaluated by a panel of miRNAs using Taqman-based quantitative real-time polymerase chain reaction (qRT-PCR) miRNA assays. A logistic regression model was built based on qRT-PCR results and then applied to the normalization of miRNA array data. The expression levels of 20 additional miRNAs selected from the normalized list were post-validated. Compared with other popularly used normalization methods, the logistic regression model efficiently calibrates the variance across arrays and improves miRNA microarray discovery accuracy. AVAILABILITY: Datasets and R package are available at http://gauss.usouthal.edu/publ/logit/.
Assuntos
Modelos Logísticos , MicroRNAs/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células HCT116 , Humanos , MicroRNAs/metabolismo , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricosRESUMO
Long noncoding RNAs (lncRNAs) are a class of RNA transcripts greater than 200 nucleotides in length and makeup a considerable part of the human genome. LncRNAs are well established as crucial players in a myriad of physiological and pathological processes; however, despite their abundance and versatility, the functional characteristics of lncRNAs remain largely unknown predominantly due to the lack of suitable genetic editing strategies. The complexity of their genetic structure and regulation combined with their unique functionality poses several limitations in the application of classic genetic manipulation methods in lncRNA functional studies. Several reports have demonstrated the successful implementation of CRISPR/Cas9 technology in screening and identifying the function of specific lncRNAs. Here, we describe a detailed protocol utilizing CRISPR/Cas9 genetic editing technology for knocking down lncRNAs in vitro.
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Edição de Genes , Sistemas CRISPR-Cas/genética , Humanos , Interferência de RNA , RNA Longo não Codificante/genética , TecnologiaRESUMO
MicroRNAs (miRNAs) are a class of small noncoding single-stranded RNA molecules containing 18-22 nucleotides that play an important role in the regulation of gene expression at the post-transcriptional and translational levels. Loss-of-function studies are the fundamental strategy to examine miRNA function and target genes in cellular and molecular biology. Traditional methods for miRNA loss-of-function studies include miRNA-specific antisense inhibitors, miRNA sponges, and genetic knockout. However, efficiency, specificity, and stability of these methods are not adequate. Our study suggests that CRISPR/Cas9 is an economic, convenient, and innovative strategy with high efficiency, specificity, and stability for the modulation of miRNA expression. Herein, we describe a detailed protocol for knocking out miRNA genes in vitro and in vivo with the CRISPR/Cas9 system.