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1.
Zhonghua Wai Ke Za Zhi ; 57(9): 691-697, 2019 Sep 01.
Artigo em Chinês | MEDLINE | ID: mdl-31474062

RESUMO

Objectives: To examine the expression of the long coding RNA GSTM3TV2 in pancreatic cancer tissues and to examine its role and mechanism in chemoresistance of pancreatic cancer cells. Methods: The expression of lncRNA GSTM3TV2 in 15 pancreatic cancer specimens and corresponding adjacent to cancer tissue samples diagnosed by Department of Pathology, Peking Union Medical College Hospital was detected by real-time PCR.And the expressions of GSTM3TV2 in pancreatic cancer cell AsPC-1, BxPC-3, MIAPaCa-2, PanC-1, SU86.86, T3M4, and chemoresistant cells AsPC-1/GR and MIAPaCa-2/GR, and human pancreatic nestin-expressing cells hTERT-HPNE were detected. Pancreatic cancer cell lines were transfected with GSTM3TV2-pcDNA3.1(+)in order to get cells with GSTM3TV2 overexpression.GSTM3TV2-siRNA was transfected into pancreatic cancer cells to knock down GSTM3TV2. The cell chemoresistance was measured by CCK-8 and flow cytometry assay when incubated with nab-paclitaxel. At the same time, subcutaneous xenograft tumor models were established in nude mice to observe the effect of GSTM3TV2 on chemoresistance of tumor growth in nude mice.Western blot assay was also performed to detect the molecular mechanism of chemoresistance of GSTM3TV2. Results: Comparing toadjacent tissues(0.084±0.019), GSTM3TV2 expression was significantly upregulated in the pancreatic cancer tissues(0.493±0.084) (t=5.146, P<0.05). GSTM3TV2 expression were higher in the chemotherapy resistance pancreatic cancer cells AsPC-1/GR(210.799±19.788) and MIAPaCa-2/GR(122.408±23.419) than that in the AsPC-1(3.793±0.615) and the MIAPaCa-2(5.179±1.095)(t=21.800,P<0.05;t=-18.490,P<0.05). The results of in vivo experiments showed that the volume of subcutaneously transplanted tumors in the overexpressing GSTM3TV2 group ((1 059.609±102.498)mm(3)) was significantly larger than that in the control group((566.414±81.087) mm(3)) by treated with nab-paclitaxel(t=4.230,P<0.05).Meanwhile, GSTM3TV2 could promote the expression of Cyclin D1, CDK6, Cyclin E1, Vimentin, N-cadherin, ZEB1, Snail and Slug; but decrease cleaved caspase-3, cleaved PARP in pancreatic cancer cells. Conclusions: The expression level of GSTM3TV2 in pancreatic canceris higher than that in paired adjacent tissues. GSTM3TV2 may act as an oncogene to promote chemoresistance in pancreatic cancer through regulation of cell proliferation, apoptosis, and epithelial-mesenchymal transition.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Glutationa Transferase/genética , Oncogenes/genética , Neoplasias Pancreáticas/genética , RNA não Traduzido/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
2.
BMC Evol Biol ; 19(1): 124, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31215393

RESUMO

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.


Assuntos
Genoma Bacteriano , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/genética , RNA Bacteriano/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Aminoacil-tRNA Sintetases/genética , Bacteriófagos/genética , Tamanho do Genoma , Genômica , Anotação de Sequência Molecular , Mycobacterium/classificação , Filogenia , Plasmídeos/genética , RNA não Traduzido/química , Ribonuclease P/genética , Inversão de Sequência
3.
Life Sci ; 231: 116509, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31152812

RESUMO

Non-coding RNAs (NcRNAs), a family of functional RNA molecules that cannot translate into proteins but control specific gene expression programs, have been shown to be implicated in various biological processes, including fatty acid metabolism. Fast-growing tumor cells rewire their fatty acid metabolic circuitry in order to meet the needs of energy storage, membrane proliferation, and the generation of signaling molecules, which is achieved by regulating a variety of key enzymes along with related signaling pathways in fatty acid metabolism. This review presents an update of our knowledge about the regulatory network of ncRNAs-specifically, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs)-in this metabolic shift and discusses the possibility of ncRNA-based therapeutics being applied to the restoration of cancer-related fatty acid metabolism.


Assuntos
Ácidos Graxos/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reprogramação Celular/fisiologia , Ácidos Graxos/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , RNA/genética , RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais
4.
Cancer Sci ; 110(8): 2328-2336, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31228211

RESUMO

Changes of nuclear localization of lineage-specific genes from a transcriptionally inert to permissive environment are a crucial step in establishing the identity of a cell. Noncoding RNA transcription-mediated genome folding and activation of target gene expression have been found in a variety of cell types. Noncoding RNA ThymoD (thymocyte differentiation factor) transcription at superenhancers is essential for mouse T-cell lineage commitment. The cessation of ThymoD transcription abolishes transcription-mediated demethylation, recruiting looping factors such as the cohesin complex, CCCTC-binding factor (CTCF), ultimately leading to the phenotype of severe combined immunodeficiency and T-cell leukemia/lymphoma. In this review, we describe the functional role of RNA polymerase II-mediated transcription at enhancers and in genome folding. We also highlight the involvement of faulty activation or suppression of enhancer transcription and enhancer-promoter interaction in cancer development.


Assuntos
Elementos Facilitadores Genéticos/genética , Genoma/genética , Neoplasias/genética , RNA não Traduzido/genética , Transcrição Genética/genética , Animais , Humanos , Regiões Promotoras Genéticas/genética
5.
Zhonghua Zhong Liu Za Zhi ; 41(5): 331-337, 2019 May 23.
Artigo em Chinês | MEDLINE | ID: mdl-31137165

RESUMO

Objective: To investigate the differential expression profiles of circular RNA (circRNA) in human epidermal growth factor receptor 2 (HER-2) positive breast cancer cells and normal mammary epithelial cells, and to develop novel diagnostic and therapeutic markers for HER-2 positive breast cancer. Methods: Total RNA were extracted from HER-2 positive breast cancer cell SK-BR-3 and normal mammary epithelial cell MCF10A. RNA quality was detected using NanoDrop ND-1000. Rnase R was applied to remove linear RNA and enrich circRNAs. After amplification and reverse transcription into fluorescent complementary RNA (cRNA) using random primer, the labeled cRNAs were hybridized onto the Arraystar Human circRNA Arrays. The raw data were extracted and the acquired array images were subjected to quantile normalization followed by heat map and volcano plot analysis. The expression of circRNAs with large fold change was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in the differentially expressed circRNAs and circRNA-microRNA (miRNA) network was constructed. Results: The total RNA extracted from SK-BR-3 and MCF10A had high integrity and quality. The expression profiles of circRNA in SK-BR-3 and MCF10A cells were significantly different shown by fluorescent expression signals. Compared with MCF10A cells, there were 6 584 up-regulated circRNAs and 6254 down-regulated circRNAs in SK-BR-3 cells. There were 348 circRNAs with |log(2)FC|≥2, of which 153 were up-regulated and 195 were down-regulated. Moreover, 8 circRNAs with |log(2)FC|>5. Among them, 5 were up-regulated in SK-BR-3 cells, including hsa_circRNA_074595 (|log(2)FC|=7.84), hsa_circRNA_074598 (|log(2)FC|=6.50), hsa_circRNA_085362 (|log(2)FC|=5.86), hsa_circRNA_101379 (|log(2)FC|=5.71) and hsa_circRNA_406683 (|log(2)FC|=5.34); as well as 3 were down-regulated, including hsa_circRNA_021714 (|log(2)FC|=5.46), hsa_circRNA_100777 (|log(2)FC|=5.40), and hsa_circRNA_100796 (|log(2)FC|=5.03). The expression levels of hsa_circRNA_074595, hsa_circRNA_074598 and hsa_circRNA_100777 were further validated by RT-qPCR in consistent with the results of microarray. GO analysis showed that differentially expressed circRNAs were significantly enriched in the biological process of heart development (P<0.001), cellular component in the cell adhesion-related components (P<0.001), molecular function in protein serine/threonine kinase activity (P<0.001). KEGG analysis revealed that differentially expressed circRNAs were significantly enriched in the PI3K-Akt signaling pathway. Conclusions: The expression profile of circRNA in HER-2 positive breast cancer cells is significantly different from that in normal mammary epithelial cells. The differentially expressed circRNAs may be served as potential diagnostic or therapeutic targets for HER-2 positive breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Mama/metabolismo , Células Epiteliais/metabolismo , RNA não Traduzido/biossíntese , Receptor ErbB-2/metabolismo , Mama/citologia , Células Epiteliais/citologia , Feminino , Perfilação da Expressão Gênica , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , RNA não Traduzido/genética , Receptor ErbB-2/genética
6.
Gene ; 710: 59-65, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31039434

RESUMO

Mitochondrial DNA is typically passed to offspring through maternal inheritance. However, in mussels, two kinds of mitochondrial DNA exist: F and M type, which are referred to as doubly uniparental inheritance (DUI). Studies have shown that DUI may be related to gender determination. In this study, we obtained the first complete F-type mitochondrial genome of Lamprotula scripta and Lamprotula caveata which were 16,250 bp and 16,641 bp in length, respectively, and had 13 protein coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs and 27 non-coding (NC) regions. The largest NC region of L. scripta was 639 bp and located between ND5 and tRNAGln. The largest NC of L. caveata was 1046 bp and also located between ND5 and tRNAGln. The overall AT content of L. scripta and L. caveata was 58.95% and 58.66%, respectively, which were lower than Lamprotula leai, Lamprotula gottschei and Lamprotula tortuosa. We next compared F and M mitochondrial genomic data on freshwater mussels and established a phylogenetic tree based on amino acid sequences of 13 PCGs and COII gene. Our results showed that F- and M-type mitochondria were significantly separated into two branches, and the basic structure of phylogenetic trees were divided into four distinct groups: Unioninae, Anodontini, Gonideinae and Ambleminae. Relatives of Gonideinae and Ambleminae were more closely related than Unioninae and Anodontini, indicating significant differences in mtDNA between the two mitogenome types. Moreover, we revealed that L. scripta and L. caveata are closely relatives, suggesting that they are both subordinates of the Gonideinae subfamily. Consequently, we speculate that the formation of DUI hinders their disappearance, which provides a basis for further studies into the mechanisms and genetic diversities of DUI formation.


Assuntos
Genoma Mitocondrial , Unionidae/classificação , Unionidae/genética , Animais , Evolução Molecular , Tamanho do Genoma , Herança Materna , Mitocôndrias/genética , Fases de Leitura Aberta , Filogenia , RNA de Transferência/genética , RNA não Traduzido/genética
7.
Retrovirology ; 16(1): 13, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036006

RESUMO

BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo. RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state. CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/genética , RNA não Traduzido/genética , Transcrição Genética , Antirretrovirais/farmacologia , Biomimética , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , Inativação Gênica , HIV-1/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , RNA Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
8.
Zoolog Sci ; 36(1): 23-30, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31116535

RESUMO

In the agouti signaling gene protein (Asip) of the house mouse (Mus musculus), inverted repeat (IR) arrays are known to exist in a non-coding region adjacent to the ventral-specific promoter region and the accompanying two exons (exons 1A and 1A'), which are around 100 kb upstream from the amino acid coding regions of exons 2, 3, and 4. To determine the gene structure of mammalian Asip and to elucidate trends in its evolution, non-coding sequences of six rodent (mouse, rat, Chinese hamster, squirrel, guinea pig, and naked mole rat) and three non-rodent (rabbit, human, and cow) species were retrieved from databases and compared. Our homology search analyses revealed the presence of three to five highly conserved non-coding elements (CNE). These CNEs were found to form IRs in rodents and lagomorphs. Combinations of IRs were further shown to build symmetric, long IR arrays. Intra- and inter-specific comparisons of the sequences of three universal CNEs showed homogeneity between CNE pairs within species. This implies that certain evolutionary constraints maintained the IR structure in the rodent and rabbit species.


Assuntos
Proteína Agouti Sinalizadora/genética , Sequências Repetidas Invertidas/genética , Mamíferos/genética , Animais , Evolução Molecular , Humanos , RNA não Traduzido/genética
9.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052302

RESUMO

Non-coding RNAs (ncRNAs) are essential regulators of gene expression. In recent years, it has become more and more evident that the different classes of ncRNAs, such as micro RNAs, long non-coding RNAs and circular RNAs are organized in tightly controlled networks. It has been suggested that deregulation of these networks can lead to disease. Several studies show a contribution of these so-called competing-endogenous RNA networks in various cancer entities. In this review, we highlight the involvement of ncRNA networks in anaplastic-large cell lymphoma (ALCL), a T-cell neoplasia. A majority of ALCL cases harbor the molecular hallmark of this disease, a fusion of the anaplastic lymphoma kinase (ALK) gene with the nucleophosmin (NPM, NPM1) gene leading to a permanently active kinase that promotes the malignant phenotype. We have focused especially on ncRNAs that are regulated by the NPM-ALK fusion gene and illustrate how their deregulation contributes to the pathogenesis of ALCL. Lastly, we summarize the findings and point out potential therapeutic implications.


Assuntos
Quinase do Linfoma Anaplásico/genética , Redes Reguladoras de Genes , Linfoma Anaplásico de Células Grandes/genética , RNA não Traduzido/genética , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Anaplásico de Células Grandes/metabolismo , RNA não Traduzido/metabolismo
10.
Nucleic Acids Res ; 47(7): 3739-3751, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30993347

RESUMO

Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.


Assuntos
Endorribonucleases/química , HIV-1/química , RNA Catalítico/química , Proteínas de Ligação a RNA/química , RNA/química , Sítios de Ligação , Endorribonucleases/genética , Genes de RNAr/genética , HIV-1/genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/genética , RNA Catalítico/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Termodinâmica
11.
Plant Genome ; 12(1)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30951085

RESUMO

Epigenetic regulations in the form of changes in differential expression of noncoding RNAs (ncRNAs) are an essential mechanism of stress response in plants. Previously we showed that heat treatment in L. results in the differential processing and accumulation of ncRNA fragments (ncRFs) stemming from transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs). In this work, we analyzed whether ncRFs are differentially expressed in the progeny of heat-stressed plants. We found significant changes in the size of tRF reads and a significant decrease in the percentage of tRFs mapping to tRNA-Ala, tRNA-Arg, and tRNA-Tyr and an increase in tRFs mapping to tRNA-Asp. The enrichment analysis showed significant differences in processing of tRFs from tRNA, tRNA, tRNA, tRNA, tRNA, and tRNA isoacceptors. Analysis of potential targets of tRFs showed that they regulate brassinosteroid metabolism, the proton pump ATPase activity, the antiporter activity, the mRNA decay activity as well as nucleosome positioning and the epigenetic regulation of transgenerational response. Gene ontology term analysis of potential targets demonstrated a significant enrichment in tRFs that potentially targeted a cellular component endoplasmic reticulum (ER) and in small nucleolar RNA fragments (snoRFs), the molecular function protein binding. To summarize, our work demonstrated that the progeny of heat-stressed plants exhibit changes in the expression of tRFs and snoRFs but not of small nuclear RNA fragments (snRFs) or ribosomal RNA fragments (rRFs) and these changes likely better prepare the progeny of stressed plants to future stress encounters.


Assuntos
Brassica rapa/genética , Resposta ao Choque Térmico/genética , RNA de Plantas/biossíntese , RNA não Traduzido/biossíntese , Brassica rapa/fisiologia , Ontologia Genética , Genes de Plantas , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA não Traduzido/genética , Reprodução
12.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940699

RESUMO

After an adaptive immune response is mounted, gammaherpesviruses achieve persistence through the utilization of viral noncoding RNAs to craft a suitable host cell environment in an immunologically transparent manner. While gammaherpesvirus long noncoding RNAs (lncRNAs) and microRNAs have been recognized for some time and have been actively investigated, a recent spate of reports have now identified repertoires of the circular RNA (circRNA) class of noncoding RNAs in both the lymphocryptovirus and rhadinovirus genera of gammaherpesviruses. Despite the recent nature of these findings, the detection of circRNAs across viruses and viral gene expression programs, the conservation of some viral circRNAs, and their detection in the clinical setting already raises the spectrum of functional importance in gammaherpesvirus biology and associated malignancies. Here, we provide an overview of currently known gammaherpesvirus circular RNAs and discuss reported physical and contextual properties that may be germane to future functional studies. With the Epstein-Barr virus (EBV) circRNAome being the most extensively studied to date, our discussions will be weighted toward EBV circRNAs while also addressing circRNAs discovered in the rhesus macaque lymphocryptovirus (rLCV), the Kaposi's sarcoma herpesvirus (KSHV), and the murid gammaherpesvirus 68 (MHV68). We hope that this will help set the stage for future investigations into the functions and relevance of this new class of viral noncoding RNAs in infection and disease.


Assuntos
Gammaherpesvirinae/fisiologia , RNA Viral/genética , RNA/genética , Latência Viral , Animais , Infecções por Herpesviridae/virologia , Humanos , RNA não Traduzido/genética
13.
Nat Protoc ; 14(5): 1489-1508, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962605

RESUMO

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.


Assuntos
Dano ao DNA , Reparo do DNA , RNA não Traduzido , Ribonuclease Pancreático/metabolismo , Animais , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Técnicas Genéticas , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , RNA/análise , RNA/genética , RNA/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/fisiologia
14.
Biomed Res Int ; 2019: 8690592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30931332

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is increasing in prevalence globally, but little is known about its specific molecular mechanisms. During the past decade, noncoding RNAs (ncRNAs) have been linked to NAFLD initiation and progression. They are a class of RNAs that play an important role in regulating gene expression despite not encoding proteins. This review summarizes recent research on the relationship between ncRNAs and NAFLD. We discussed the potential applicability of ncRNAs as a biomarker for early NAFLD diagnosis and assessment of disease severity. With further study, ncRNAs should prove to be valuable new targets for NAFLD treatment and benefit the development of noninvasive diagnostic methods.


Assuntos
Biomarcadores , Hepatopatia Gordurosa não Alcoólica/diagnóstico , RNA não Traduzido/genética , Regulação da Expressão Gênica/genética , Humanos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia
15.
BMC Mol Biol ; 20(1): 11, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961536

RESUMO

BACKGROUND: Camels possess the characteristics of salt- and drought-resistances, due to the long-time adaption to the living environment in desert. The camel resistance research on transcriptome is rare and deficient, especially reabsorption in renal cortex. Non-coding RNAs are normally considered as the RNA molecules that are not translated into proteins, their current roles remain mostly in regulation of information flux from DNA to protein, further on normal life activities and diseases. In order to reveal the mysterious veil of the post-transcriptional regulation of ncRNAs in renal cortex for the first time as far as we know, we designed and carried out the experiment of salt stress and water-deprivation stress in camel. RESULTS: By means of RNA-seq in renal cortex of Alxa Bactrian Camel (Camelus bactrianus), we identified certain significantly differential RNAs, including 4 novel lncRNAs, 11 miRNAs and 13 mRNAs under salt stress, 0 lncRNAs, 18 miRNAs and 14 mRNAs under water-deprivation stress. By data analysis, the response pathway of post-transcriptional regulation concerning salt and water-deprivation stresses was put forward, involving preventing sodium from entering the cell, purifying of water and compensating neutral amino acids by miR-193b, miR-542-5p interaction with SLC6A19 mRNA. CONCLUSION: Based on the resistance-related lncRNAs, miRNAs, and mRNAs, we proposed the post-transcriptional regulation pathway to explain how camels respond to salt and water-deprivation stresses in the ncRNAs regulation level of renal cortex for the first time, thus hoping to provide a theoretical basis for therapy of disease that is similar to high blood pressure in humans.


Assuntos
Camelus/genética , Camelus/fisiologia , Córtex Renal/fisiologia , RNA não Traduzido/genética , Estresse Salino/genética , Transcriptoma , Privação de Água , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Regulação da Expressão Gênica , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/genética , RNA Mensageiro/genética
16.
Planta ; 250(1): 23-40, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30993403

RESUMO

MAIN CONCLUSION: This review will provide evidence for the indispensable function of these elements in regulating plant development and resistance to biotic and abiotic stresses, as well as their evolutionary role in facilitating plant adaptation. Over millions of years of evolution, plant genomes have acquired a complex constitution. Plant genomes consist not only of protein coding sequences, but also contain large proportions of non-coding sequences. These include introns of protein-coding genes, and intergenic sequences such as non-coding RNA, repeat sequences and transposable elements. These non-coding sequences help to regulate gene expression, and are increasingly being recognized as playing an important role in genome organization and function. In this review, we summarize the known molecular mechanisms by which gene expression is regulated by several species of non-coding RNAs (microRNAs, long non-coding RNAs, and circular RNAs) and by transposable elements. We further discuss how these non-coding RNAs and transposable elements evolve and emerge in the genome, and the potential influence and importance of these non-coding RNAs and transposable elements in plant development and in stress responses.


Assuntos
Elementos de DNA Transponíveis/genética , Genoma de Planta/genética , Desenvolvimento Vegetal , Fenômenos Fisiológicos Vegetais , Plantas/genética , RNA não Traduzido/genética , Íntrons/genética , MicroRNAs/genética , RNA/genética , RNA Longo não Codificante/genética , RNA de Plantas/genética , Estresse Fisiológico
17.
Methods Mol Biol ; 1962: 15-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31020552

RESUMO

Noncoding RNA (ncRNA) research is already a routine in every genomics or transcriptomics initiatives. According to their functions, ncRNAs can be grouped into several different RNA families, which can be represented by conserved primary sequences, secondary structures, or covariance models (CMs). CMs are very sensitive in predicting RNA families in nucleotide sequences and have been widely used in characterizing the repertoire of ncRNAs in organisms from all domains of life. However, the large-scale prediction and annotation of ncRNAs require multiple tools along the process, imposing a great obstacle for researchers with lesser computational or bioinformatics background. StructRNAfinder emerged as an automated tool to avoid these bottlenecks, by performing the automatic identification and complete annotation of regulatory RNA families derived directly from nucleotide sequences. In this chapter, we provide a complete tutorial for both stand-alone and web server versions of StructRNAfinder. This will help users to install the tool and to perform predictions of RNA families in any genome or transcriptome sequences dataset.


Assuntos
Biologia Computacional/métodos , RNA não Traduzido/genética , Software , Classificação , Apresentação de Dados , Genoma , Internet , Anotação de Sequência Molecular
18.
Mol Cancer ; 18(1): 74, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940133

RESUMO

tRNA-derived small RNA (tsRNA) is a novel regulatory small non-coding RNA and participates in diverse physiological and pathological processes. However, the presence of tsRNAs in exosome and their diagnostic potential remain unclear. In this study, we took advantage of small RNA-seq technology to profile exosomal tsRNAs from cell culture medium and plasma, and found ubiquitous presence of tsRNAs in exosome. To explore the potential value of tsRNA for cancer diagnosis, we compared exosomal tsRNA levels between liver cancer patients and healthy donors, revealing that tsRNAs were dramatically increased in plasma exosomes of liver cancer patients. Importantly, patients with liver cancer exhibited significantly higher levels of four tsRNAs (tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5 and tRNA-GluCTC-5) in plasma exosome, demonstrating that plasma exosomal tsRNA could serve as a novel diagnostic biomarker. Taken together, our results not only expand non-coding RNA species in exosome, but also highlight the potential of tsRNAs as a promising biomarker for cancer diagnosis.


Assuntos
Exossomos/genética , Neoplasias/diagnóstico , RNA de Transferência/genética , RNA não Traduzido/genética , Biomarcadores Tumorais/genética , Técnicas de Cultura de Células , Detecção Precoce de Câncer , Humanos , Neoplasias/genética , Análise de Sequência de RNA , Células Tumorais Cultivadas
19.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018516

RESUMO

Fibrosis, or tissue scarring, is defined as the excessive, persistent and destructive accumulation of extracellular matrix components in response to chronic tissue injury. Renal fibrosis represents the final stage of most chronic kidney diseases and contributes to the progressive and irreversible decline in kidney function. Limited therapeutic options are available and the molecular mechanisms governing the renal fibrosis process are complex and remain poorly understood. Recently, the role of non-coding RNAs, and in particular microRNAs (miRNAs), has been described in kidney fibrosis. Seminal studies have highlighted their potential importance as new therapeutic targets and innovative diagnostic and/or prognostic biomarkers. This review will summarize recent scientific advances and will discuss potential clinical applications as well as future research directions.


Assuntos
Nefropatias/genética , Nefropatias/patologia , Rim/patologia , RNA não Traduzido/genética , Animais , Fibrose , Terapia Genética , Humanos , Rim/metabolismo , Nefropatias/diagnóstico , Nefropatias/terapia , Terapia de Alvo Molecular , RNA não Traduzido/análise
20.
Mol Cell ; 74(3): 521-533.e6, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30952514

RESUMO

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.


Assuntos
MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Grânulos Citoplasmáticos/genética , Inativação Gênica , Células HeLa , Humanos , Processamento Pós-Transcricional do RNA/genética , RNA não Traduzido/genética , Imagem Individual de Molécula
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