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1.
PLoS Genet ; 15(12): e1008261, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31860668

RESUMO

Germline genome defense evolves to recognize and suppress retrotransposons. One of defensive mechanisms is the PIWI-associated RNA (piRNA) pathway, which employs small RNAs for sequence-specific repression. The loss of the piRNA pathway in mice causes male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes posses also RNA interference (RNAi), another small RNA pathway capable of retrotransposon suppression. To examine whether RNAi compensates the loss of the piRNA pathway, we produced a new RNAi pathway mutant DicerSOM and crossed it with a catalytically-dead mutant of Mili, an essential piRNA gene. Normal follicular and oocyte development in double mutants showed that RNAi does not suppress a strong ovarian piRNA knock-out phenotype. However, we observed redundant and non-redundant targeting of specific retrotransposon families illustrating stochasticity of recognition and targeting of invading retrotransposons. Intracisternal A Particle retrotransposon was mainly targeted by the piRNA pathway, MaLR and RLTR10 retrotransposons were targeted mainly by RNAi. Double mutants showed accumulations of LINE-1 retrotransposon transcripts. However, we did not find strong evidence for transcriptional activation and mobilization of retrotransposition competent LINE-1 elements suggesting that while both defense pathways are simultaneously expendable for ovarian oocyte development, yet another transcriptional silencing mechanism prevents mobilization of LINE-1 elements.


Assuntos
Oócitos/crescimento & desenvolvimento , Interferência de RNA , RNA Interferente Pequeno/genética , Retroelementos , Animais , Proteínas Argonauta/genética , RNA Helicases DEAD-box/genética , Feminino , Camundongos , Mutação , Oócitos/química , Ribonuclease III/genética , Transdução de Sinais
2.
BMC Plant Biol ; 19(1): 503, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729948

RESUMO

BACKGROUND: Dicer-like proteins (DCLs) are essential players in RNA-silencing mechanisms, acting in gene regulation via miRNAs and in antiviral protection in plants and have also been associated to other biotic and abiotic stresses. To the best of our knowledge, despite being identified in some crops, cotton DCLs haven't been characterized until now. In this work, we characterized the DCLs of three cotton species and analyzed their expression profiles during biotic stress. RESULTS: As main results, 11 DCLs in the allotetraploid cotton Gossypium hirsutum, 7 and 6 in the diploid G. arboreum and G. raimondii, were identified, respectively. Among some DCLs duplications observed in these genomes, the presence of an extra DCL3 in the three cotton species were detected, which haven't been found in others eudicots. All the DCL types identified by in silico analysis in the allotetraploid cotton genome were able to generate transcripts, as observed by gene expression analysis in distinct tissues. Based on the importance of DCLs for plant defense against virus, responses of cotton DCLs to virus infection and/or herbivore attack using two commercial cotton cultivars (cv.), one susceptible (FM966) and another resistant (DO) to polerovirus CLRDV infection, were analyzed. Both cvs. Responded differently to virus infection. At the inoculation site, the resistant cv. showed strong induction of DCL2a and b, while the susceptible cv. showed a down-regulation of these genes, wherever DCL4 expression was highly induced. A time course of DCL expression in aerial parts far from inoculation site along infection showed that DCL2b and DCL4 were repressed 24 h after infection in the susceptible cotton. As CLRDV is aphid-transmitted, herbivore attack was also checked. Opposite expression pattern of DCL2a and b and DCL4 was observed for R and S cottons, showing that aphid feeding alone may induce DCL modulation. CONCLUSIONS: Almost all the DCLs of the allotetraploide G. hirsutum cotton were found in their relative diploids. Duplications of DCL2 and DCL3 were found in the three species. All four classes of DCL responded to aphid attack and virus infection in G. hirsutum. DCLs initial responses against the virus itself and/or herbivore attack may be contributing towards virus resistance.


Assuntos
Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Gossypium/genética , Ribonuclease III/genética , Estresse Fisiológico , Diploide , Perfilação da Expressão Gênica , Gossypium/fisiologia , MicroRNAs/genética , Proteínas de Plantas/genética , Poliploidia , RNA de Plantas/genética
3.
Bull Cancer ; 106(12): 1177-1189, 2019 Dec.
Artigo em Francês | MEDLINE | ID: mdl-31610911

RESUMO

Inactivating germline pathogenic variants of the DICER1 gene are responsible for a spectrum of rare diseases, which expanded a lot in recent years. The constitution of an U.S. registry with these patients and their families as well as the registration of patients in European databases of rare tumors helped to better identify diseases encountered in this syndrome but also to study its pathophysiology (major role in miRNA maturation and recently discovered functions, e.g. in genome integrity maintenance). Most encountered disorders are pediatric malignancies, mainly the pulmonary pneumoblastoma and Sertoli-Leydig tumours. However, benign pathologies such as thyroid goiters, cystic nephromas or pulmonary cystic lesions are also frequently reported. Homogeneous guidelines regimens written by the European groups working on very rare pediatric tumors are proposed but it is important to underscore that they rely on rare scientific data; therefore overall consensus remains precarious. The genetic counseling to families is still difficult due to the large observed spectrum of tumors and the incomplete penetrance. In this article, the authors update current knowledge on the DICER1 syndrome.


Assuntos
RNA Helicases DEAD-box/genética , Neoplasias/genética , Doenças Raras/genética , Ribonuclease III/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Aconselhamento Genético , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Doenças Raras/diagnóstico , Doenças Raras/metabolismo , Ribonuclease III/metabolismo , Síndrome
4.
J Exp Clin Cancer Res ; 38(1): 413, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590696

RESUMO

BACKGROUND: CyclinD1 is crucial for cell cycling and can regulate the expression of Dicer, a crucial regulator of microRNA maturation. However, little is known on how CyclinD1 regulates Dicer and miRNA expression, and the progression of intrahepatic cholangiocarcinoma (ICC). METHODS: The expression of CyclinD1 and Dicer in non-tumor cholangiocytes, ICC cells and tissues as well as their association with clinicopathological characteristics and survival were examined. The potential mechanisms by which CyclinD1 regulates Dicer and relative miRNA expression were determined by immunoprecipitation, ChIP sequence, BSP and luciferase reporter assays following induction of CyclinD1 over-expression or silencing and Dicer silencing. The impact of CyclinD1 and/or Dicer silencing on the growth of ICC was tested in vivo. RESULTS: Up-regulated CyclinD1 was associated with down-regulated Dicer expression in ICC tissues and poorer overall survival in patients with ICC. CyclinD1 interacted with the nuclear H3K9me3 and SUV39H1 and bound to the Dicer promoter to increase its CpG island methylation in ICC cells. Functionally, CyclinD1 silencing inhibited the malignancy of ICC cells, which were mitigated partially by Dicer silencing in ICC cells. Dicer silencing down-regulated miR-1914-5p and miR-541-5p expression, which targeted and promoted CyclinD1 and CDK6 expression in ICC cells. CONCLUSIONS: Our findings uncover that CyclinD1 inhibits Dicer expression by chromatin modification to reduce miR-1914-5p/miR-541-5p expression, which positively-feedback enhances CyclinD1 and CDK6 expression and progression of ICC.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Cromatina/genética , Ciclina D1/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Cromatina/metabolismo , Ciclina D1/genética , RNA Helicases DEAD-box/genética , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Ribonuclease III/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Physiol Biochem ; 53(5): 760-773, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31647206

RESUMO

BACKGROUND/AIMS: Perturbations in the expression of microRNAs (miRNAs) and their maturing machinery components such as Dicer have been previously described for basal cell carcinoma (BCC). However, the mutational status of Dicer in BCC is unclear. Further, the sclerodermiform subtype of BCC (sBCC) has not been previously investigated regarding its methylation profile or its smallRNA expression profile via RNA sequencing. We conducted this study to investigate the mutational status of Dicer in BCC. METHODS: Dicer sequencing was performed on the Illumina MiSeq System in a total of 16 BCC samples (8 nodular BCCs, 8 sBCCs) and mapped against the human reference genome (i.e., hg19). Dicer sequencing was performed in all 16 BCC samples. We performed whole genome methylation profiling with Infinium MethylationEPIC BeadChips as well as mRNA and smallRNA sequencing in 5 sBCCs with the Illumina NextSeq500 next-generation sequencing system. RESULTS: Compared to the wildtype Dicer sequence, we found 5 to 7 variants per sBCC sample including insertion, deletion, and multiple nucleotide variants. Global methylation profiles were highly similar between groups. mRNA sequencing revealed S100A9, KRT14, KRT10, S100A8, S100A7, COX1, KRT1, COX3, and smallRNA sequencing analysis miR-21, miR-99a, miR26-a-2, let-7f, let-7g, let-7i, miR-100, and miR-205 were the most strongly expressed in sBCCs. CONCLUSION: We identified a variety of Dicer mutations that could play a role in aberrant miRNA expression in BCC. The noted RNA sequences should be further evaluated in functional studies to explore their potential pathogenetic role in sBCC.


Assuntos
RNA Helicases DEAD-box/genética , Metilação de DNA , MicroRNAs/química , RNA Mensageiro/química , Ribonuclease III/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Basocelular , Linhagem Celular Tumoral , Feminino , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
6.
Mo Med ; 116(3): 206-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31527943

RESUMO

Pleuropulmonary blastoma (PPB), the most common primary malignant neoplasm of the lung in childhood, occurs in the same early age group (0-6 years) as the other more common solid tumors such as neuroblastoma and Wilms tumor. The tumor begins as a cystic lung lesion with the potential over a period of 3-5 years to progress to a high grade multipatterned primitive sarcoma in the absence of a malignant epithelial component. Several years after its initial description as a unique clinicopathologic entity, this and other tumors appeared to have a familial predilection which was later confirmed with the discovery of a heterozygous germline mutation in DICER1 whose protein is a member of ribonuclease III family of enzymes. It is estimated that 75%-80% of children with a PPB have the germline mutation. The other notable finding from our studies is the identification of a family of extrapulmonary neoplasms, including cystic nephroma and Sertoli-Leydig cell tumor of the ovary as two examples, also with DICER1 mutations.


Assuntos
RNA Helicases DEAD-box/genética , Mutação em Linhagem Germinativa , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Blastoma Pulmonar/genética , Blastoma Pulmonar/patologia , Ribonuclease III/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Neoplasias Pulmonares/diagnóstico , Masculino , Blastoma Pulmonar/diagnóstico
7.
Brain Tumor Pathol ; 36(4): 174-182, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31487013

RESUMO

Rhabdomyosarcoma is the most common soft-tissue sarcoma affecting children and adolescents. It is defined as a malignant neoplasm characterized by morphologic, immunohistochemical, ultrastructural, or molecular genetic evidence of primary skeletal muscle differentiation, usually in the absence of any other pattern of differentiation. Primary intracranial rhabdomyosarcoma (PIRMS) is an extremely rare neoplasm, with only 60 cases reported in the literature, and generally has poor prognosis with an overall survival of only 9.1 months. The DICER1 gene encodes an RNA endoribonuclease that plays a key role in gene expression regulation through the production of small RNAs. Herein, we report two cases of PIRMS with somatic DICER1 mutation showing morphological and immunohistochemical evidence of primary skeletal muscle differentiation; the two cases share common clinical features, including young age, supratentorial tumor, and onset of intratumoral bleeding. Although methylation profiling was not performed, both cases shared clinical and pathological characteristics in common with recently proposed methylation entity "spindle cell sarcoma with rhabdomyosarcoma-like features, DICER1 mutant (SCS-RMSlike-DICER1)''. Our cases provide further evidence of the link between primary intracranial sarcoma and DICER1 mutation which may form a distinct entity.


Assuntos
RNA Helicases DEAD-box/genética , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Ribonuclease III/genética , Adulto , Biomarcadores Tumorais/genética , Criança , RNA Helicases DEAD-box/metabolismo , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Mutação , Ribonuclease III/metabolismo
8.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510013

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Each step of their production and maturation has to be strictly regulated, as any disruption of control mechanisms may lead to cancer. Thus, we have measured the expression of 19 genes involved in miRNAs biogenesis pathway in tumor tissues of 239 colorectal cancer (CRC) patients, 17 CRC patients with liver metastases and 239 adjacent tissues using real-time PCR. Subsequently, the expression of analyzed genes was correlated with the clinical-pathological features as well as with the survival of patients. In total, significant over-expression of all analyzed genes was observed in tumor tissues as well as in liver metastases except for LIN28A/B. Furthermore, it was shown that the deregulated levels of some of the analyzed genes significantly correlate with tumor stage, grade, location, size and lymph node positivity. Finally, high levels of DROSHA and TARBP2 were associated with shorter disease-free survival, while the over-expression of XPO5, TNRC6A and DDX17 was detected in tissues of patients with shorter overall survival and poor prognosis. Our data indicate that changed levels of miRNA biogenesis genes may contribute to origin as well as progression of CRC; thus, these molecules could serve as potential therapeutic targets.


Assuntos
Vias Biossintéticas/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , MicroRNAs/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Carioferinas/genética , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Prognóstico , Ribonuclease III/genética
9.
Mol Cell ; 75(3): 576-589.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398324

RESUMO

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.


Assuntos
Proteínas de Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Replicase/genética , Ribonuclease III/genética , Transcrição Genética , Arabidopsis/genética , Proteínas Argonauta/genética , Metilação de DNA/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Transdução de Sinais
10.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374978

RESUMO

Venous thromboembolism (VTE) involves the formation of a blood clot, typically in the deep veins of the leg or arm (deep vein thrombosis), which then travels via the circulatory system and ultimately lodges in the lungs, resulting in pulmonary embolism. A number of microRNAs (miRNAs) are well-known regulators of thrombosis and thrombolysis, and mutations in miRNA biogenesis genes, such as DICER1, DROSHA have been implicated in miRNA synthesis and function. We investigated the genetic association between polymorphisms in four miRNA biogenesis genes, DICER1 rs3742330A > G, DROSHA rs10719T > C, RAN rs14035C > T and XPO5 rs11077A > C, and VTE in 503 Koreans: 300 controls and 203 patients. Genotyping was assessed with polymerase chain reaction-restriction fragment length polymorphism assays. We detected associations between polymorphisms in RAN and XPO5 and VTE prevalence (RAN rs14035CC + CT versus TT: p = 0.018; XPO5 rs11077AA + AC versus CC: p < 0.001). Analysis of allele combinations of all four polymorphisms (DICER1, DROSHA, RAN, XPO5) revealed that A-T-T-A was associated with decreased VTE prevalence (p = 0.0002), and A-T-C-C was associated with increased VTE prevalence (p = 0.027). Moreover, in subjects with provoked VTE, the DROSHA rs10719T > C, polymorphism was associated with increased disease prevalence (TT versus TC + CC: p < 0.039). Our study demonstrates that RAN and XPO5 polymorphisms are associated with risk for VTE in Korean subjects.


Assuntos
MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Tromboembolia Venosa/genética , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/genética , RNA Helicases DEAD-box/genética , Feminino , Predisposição Genética para Doença , Humanos , Carioferinas/genética , Masculino , Pessoa de Meia-Idade , República da Coreia/epidemiologia , Ribonuclease III/genética , Tromboembolia Venosa/epidemiologia , Proteína ran de Ligação ao GTP/genética
11.
Nat Commun ; 10(1): 3682, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417090

RESUMO

Somatic mutations in the RNase IIIb domain of DICER1 arise in cancer and disrupt the cleavage of 5' pre-miRNA arms. Here, we characterize an unstudied, recurrent, mutation (S1344L) in the DICER1 RNase IIIa domain in tumors from The Cancer Genome Atlas (TCGA) project and MSK-IMPACT profiling. RNase IIIa/b hotspots are absent from most cancers, but are notably enriched in uterine cancers. Systematic analysis of TCGA small RNA datasets show that DICER1 RNase IIIa-S1344L tumors deplete 5p-miRNAs, analogous to RNase IIIb hotspot samples. Structural and evolutionary coupling analyses reveal constrained proximity of RNase IIIa-S1344 to the RNase IIIb catalytic site, rationalizing why mutation of this site phenocopies known hotspot alterations. Finally, examination of DICER1 hotspot endometrial tumors reveals derepression of specific miRNA target signatures. In summary, comprehensive analyses of DICER1 somatic mutations and small RNA data reveal a mechanistic aspect of pre-miRNA processing that manifests in specific cancer settings.


Assuntos
RNA Helicases DEAD-box/genética , Neoplasias do Endométrio/genética , MicroRNAs/biossíntese , Ribonuclease III/genética , Bases de Dados Genéticas , Feminino , Humanos , MicroRNAs/genética , Mutação
12.
Klin Onkol ; 32(Supplementum2): 123-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409088

RESUMO

DICER1 syndrome is an inherited disorder that increases the risk of different types of malignant and benign tumors. The syndrome is caused by mutations in the DICER1 gene, which is located on the long arm of chromosome 14, region q32.13. Patients with DICER1 syndrome commonly develop pleuropulmonary blastoma (PPB), multinodular goiter, ovarian Sertoli-Leydig cell tumors, and/or other types of tumors. In approximately 35% of families with children manifesting PPB, further (and rather rare) malignancies may be observed, including cystic nephroma, nodular dysplasia of the thyroid gland, medulloepithelioma of the iris, embryonal rhabdomyosarcoma botryoid type, nasal epithelial hamartoma, pituitary blastoma, and/or pineoblastoma. Large studies report a high variability of tumors associated with DICER1. DICER1 syndrome, which is associated with an inherited predisposition to tumors, is inherited in an autosomal dominant pattern. Symptoms of DICER1 syndrome may vary, even within families. Preventive screening of carriers with causative mutations is complicated. Follow-up is undertaken as recommended by the 2016 International PPB Register. This work was supported by grant of Ministry of Health of the Czech Republic AZV 16-3329A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 4. 6. 2019 Accepted: 6. 6. 2019.


Assuntos
RNA Helicases DEAD-box/genética , Síndromes Neoplásicas Hereditárias/genética , Ribonuclease III/genética , Predisposição Genética para Doença , Humanos , Mutação
13.
PLoS Comput Biol ; 15(8): e1007278, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31449515

RESUMO

Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model's parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/fisiopatologia , Adaptação Fisiológica/genética , Sistemas CRISPR-Cas , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/fisiologia , Técnicas de Cocultura , Biologia Computacional , Simulação por Computador , RNA Helicases DEAD-box/genética , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/genética , Modelos Biológicos , Mutação , Ribonuclease III/genética
14.
PLoS Genet ; 15(7): e1008273, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31329584

RESUMO

5' ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5' monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3' end of tRNAHis. Moreover, we were able to generate this 'miRNA' in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5'P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3'-fragment/'miR-4454' levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3'-fragment processing without negatively affecting tRNAHis's canonical function of aminoacylation.


Assuntos
RNA Helicases DEAD-box/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metiltransferases/genética , RNA de Transferência de Histidina/metabolismo , Ribonuclease III/genética , Linhagem Celular , Humanos , MicroRNAs/genética , Análise de Sequência de RNA , Aminoacilação de RNA de Transferência
15.
Mediators Inflamm ; 2019: 6768504, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275058

RESUMO

Dysregulation of multiple microRNAs widely takes place during rheumatoid arthritis (RA) and experimental arthritides. This study is performed to explore the possible mechanism underlying DICER1 deficiency-mediated inflammation in human synoviocytes SW982. Firstly, RNAi of DICER1 led to increased COX2, MMP3, and MMP13 protein production, while DICER1 overexpression could reduce MMP13 expression. Secondly, the increase of IL-8 and decrease of TGF-ß1 and TIMP1 were determined in the supernatant derived from DICER1 siRNA-treated cells, while DICER1 overexpression was found capable to reverse this effect. Ingenuity pathway analysis (IPA) software predicted that the Dicer1 deficiency-induced dysregulated cytokines in synoviocytes could possibly lead to the inflammatory disorders in the synovial tissue. Moreover, DICER1 deficiency could also reduce apoptosis, while DICER1 overexpression was found to decrease the proliferation and enhance apoptosis. In addition, DICER1 deficiency could lower the expression of multiple RA-related miRNAs such as miR-155. Meanwhile, DICER1 overexpression could rescue their low expression levels. And then, gain or loss of miR-155 function could regulate the protein levels of MMP3 and MMP13. These results indicated that DICER1 might play its role through regulating its downstream RA-related miRNAs. Our data demonstrated that DICER1 deficiency could cause multiple proinflammatory events in human synoviocytes SW982. This mechanism study might provide the possible target molecule to modify the inflammatory destruction and overproliferation in synoviocytes.


Assuntos
RNA Helicases DEAD-box/metabolismo , Inflamação/metabolismo , Ribonuclease III/metabolismo , Sinoviócitos/metabolismo , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Inflamação/genética , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Interferência de RNA , Ribonuclease III/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
16.
Life Sci ; 232: 116656, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31306658

RESUMO

AIMS: Tamoxifen-induced liver-specific Dicer1 deletion (iDicer1-/-) in mature mice may provide clues demonstrating the genuine effects of acute loss of Dicer1 and miRNAs in the liver regeneration process. MAIN METHODS: In this study, mice with tamoxifen-induced Dicer1 deletion through the Cre/LoxP system were constructed and then underwent classic 70% partial hepatectomy or CCl4-induced liver injury. To rescue the inhibitory effect of Dicer1 ablation on liver regeneration, miR-21 agomir was injected into the tail vein of iDicer1-/- mice. KEY FINDINGS: Unlike constitutive embryonic deletion of Dicer1, tamoxifen-induced Dicer1 deletion did not result in severe liver injury or lesions, providing an ideal model for investigating acute loss of Dicer1 and miRNAs in liver regeneration. Dicer1 deletion led to impaired liver regeneration through the inhibitory effect of miR-21 on PTEN and Rhob expression. SIGNIFICANCE: In our previous study, we found that embryonic loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation, while the role of Dicer1 in liver regeneration remains largely unknown. We clearly identified the promotion effect of Dicer1 on liver regeneration by increasing miR-21 expression, which inhibits the expression of two negative cell proliferation regulators, Pten and Rhob.


Assuntos
RNA Helicases DEAD-box/fisiologia , Regeneração Hepática/fisiologia , MicroRNAs/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Ribonuclease III/fisiologia , Proteína rhoB de Ligação ao GTP/metabolismo , Animais , RNA Helicases DEAD-box/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Camundongos , Camundongos Knockout , Ribonuclease III/genética , Tamoxifeno/administração & dosagem
17.
Nucleic Acids Res ; 47(15): 7886-7900, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31216029

RESUMO

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Genoma de Planta , MicroRNAs/genética , Proteínas Periplásmicas de Ligação/genética , Processamento de RNA , Proteínas Repressoras/metabolismo , Spliceossomos/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Éxons , Regulação da Expressão Gênica de Plantas , Íntrons , MicroRNAs/biossíntese , Proteínas Periplásmicas de Ligação/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Spliceossomos/metabolismo
18.
Thromb Res ; 180: 32-36, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31185329

RESUMO

INTRODUCTION: Single-nucleotide polymorphisms (SNPs) in microRNA (miRNA) machinery genes may affect the regulatory capacity of miRNAs by impacting their biogenesis. The aim of the study was to analyze the association between SNPs in two key genes (DICER rs1057035T>C and XPO5 rs11077A>C) and coronary artery disease (CAD) risk as well as to examine their effects on circulating levels of vascular miRNAs. MATERIALS AND METHODS: Within the Italian GENOCOR cohort, we studied a cohort of 557 patients (502 males, 57 ±â€¯9 years) with angiographically documented CAD. A total of 443 healthy controls (262 males, 56 ±â€¯12 years) was also enrolled. Genotyping was determined by using a TaqMan®SNP genotyping assay. Analysis of miR-132 and miR-140-3p was assessed in a subset of 70 CAD patients by using qRT-PCR. RESULTS: There were statistically significant differences between CAD patients and healthy controls in the distribution of both DICER and XPO5 genotypes (p = 0.03 and p = 0.02, respectively). Multivariate analysis showed a significantly decreased risk of CAD by 50% in patients with DICER rs105703CC genotype as compared to TC heterozygote and TT homozygote patients (ORadjusted = 0.50; CI: 0.30-0.83, p = 0.007). In a recessive model, the XPO5 rs11077CC genotype was associated with a 32% reduced risk of CAD (ORadjusted = 0.68; CI: 0.30-0.99 p = 0.047). XPO5 rs11077CC genotype was significantly associated with higher levels of both miRNA-132 (p = 0.04) and miRNA-140-3p (p = 0.03). CONCLUSIONS: Genetic polymorphisms in DICER and XPO5 genes are associated with a decreased risk of CAD, probably by impacting expression levels of vascular and cardiac-specific miRNAs. Further studies are needed to better elucidate the biological relevance of both variants in CAD development.


Assuntos
Doença da Artéria Coronariana/genética , RNA Helicases DEAD-box/genética , Carioferinas/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Ribonuclease III/genética , Adulto , Idoso , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Feminino , Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade
19.
Genes (Basel) ; 10(6)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208028

RESUMO

Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria Escherichia coli and Sinorhizobium meliloti were largely processed into shorter dsRNA fragments with no or few full-length molecules being present. A loss-of-function mutation in the dsRNA-processing enzyme RNase III, in the widely used E. coli HT115 strain, did not prevent the processing of hpRNA. Consistent with previous observations in plants, the loop sequence of long hpRNA expressed in Agrobacterium-infiltrated Nicotiana benthamiana leaves was excised, leaving no detectable levels of full-length hpRNA molecule. In contrast to bacteria and plants, long hpRNAs expressed in the budding yeast Saccharomyces cerevisiae accumulated as intact, full-length molecules. RNA extracted from hpRNA-expressing yeast cells was shown to be capable of inducing RNAi against a ß-glucuronidase (GUS) reporter gene in tobacco leaves when applied topically on leaf surfaces. Our results indicate that yeast can potentially be used to express full-length hpRNA molecules for RNAi and perhaps other structured RNAs that are important in biological applications.


Assuntos
Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , Saccharomyces cerevisiae/química , Agrobacterium/química , Agrobacterium/genética , Escherichia coli/química , Escherichia coli/genética , Mutação com Perda de Função , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Ribonuclease III/química , Ribonuclease III/genética , Saccharomyces cerevisiae/genética , Tabaco/química , Tabaco/genética
20.
RNA ; 25(9): 1078-1090, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31164480

RESUMO

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.


Assuntos
Arabidopsis/genética , Indenos/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Interferência de RNA/efeitos dos fármacos , Tionas/farmacologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Argonauta/genética , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética
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