RESUMO

Weeping lovegrass (Eragrostis curvula [Shrad.] Nees) is a perennial grass typically established in semi-arid regions, with good adaptability to dry conditions and sandy soils. This polymorphic complex includes both sexual and apomictic cytotypes, with different ploidy levels (2x-8x). Diploids are known to be sexual, while most polyploids are facultative apomicts, and full apomicts have also been reported. Plant breeding studies throughout the years have focused on achieving the introgression of apomixis into species of agricultural relevance, but, given the complexity of the trait, a deeper understanding of the molecular basis of regulatory mechanisms of apomixis is still required. Apomixis is thought to be associated with silencing or disruption of the sexual pathway, and studies have shown it is influenced by epigenetic mechanisms. In a previous study, we explored the role of miRNA-mRNA interactions using two contrasting E. curvula phenotypes. Here, the sexual OTA-S, the facultative Don Walter and the obligate apomictic Tanganyika cDNA and sRNA libraries were inquired, searching for miRNA discovery and miRNA expression regulation of genes related to the reproductive mode. This allowed for the characterization of seven miRNAs and the validation of their miRNA-target interactions. Interestingly, a kinesin gene was found to be repressed in the apomictic cultivar Tanganyika, targeted by a novel miRNA that was found to be overexpressed in this genotype, suggestive of an involvement in the reproductive mode expression. Our work provided additional evidence of the contribution of the epigenetic regulation of the apomictic pathway.

RESUMO

BACKGROUND: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. METHODS: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. RESULTS: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. CONCLUSIONS: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

Assuntos

Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Biomarcadores Tumorais/análise , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Biologia Computacional , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Ciclina D1/genética , Feminino , Proteínas Ligadas por GPI/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/análise , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Membro 10c de Receptores do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/genética

RESUMO

Aire is a transcriptional controller in medullary thymic epithelial cells (mTECs) modulating a set of peripheral tissue antigens (PTAs) and non-PTA mRNAs as well as miRNAs. Even miRNAs exerting posttranscriptional control of mRNAs in mTECs, the composition of miRNA-mRNA networks may differ. Under reduction in Aire expression, networks exhibited greater miRNA diversity controlling mRNAs. Variations in the number of 3'UTR binding sites of Aire-dependent mRNAs may represent a crucial factor that influence the miRNA interaction. To test this hypothesis, we analyzed through bioinformatics the length of 3'UTRs of a large set of Aire-dependent mRNAs. The data were obtained from existing RNA-seq of mTECs of wild type or Aire-knockout (KO) mice. We used computational algorithms as FASTQC, STAR and HTSEQ for sequence alignment and counting reads, DESEQ2 for the differential expression, 3USS for the alternative 3'UTRs and TAPAS for the alternative polyadenylation sites. We identified 152 differentially expressed mRNAs between these samples comprising those that encode PTAs as well as transcription regulators. In Aire KO mTECs, most of these mRNAs featured an increase in the length of their 3'UTRs originating additional miRNA binding sites and new miRNA controllers. Results from the in silico analysis were statistically significant and the predicted miRNA-mRNA interactions were thermodynamically stable. Even with no in vivo or in vitro experiments, they were adequate to show that lack of Aire in mTECs might favor the downregulation of PTA mRNAs and transcription regulators via miRNA control. This could unbalance the overall transcriptional activity in mTECs and thus the self-representation.

Assuntos

Regiões 3' não Traduzidas , RNA Mensageiro/genética , Timo/metabolismo , Fatores de Transcrição/genética , Algoritmos , Animais , Antígenos/genética , Sítios de Ligação/genética , Simulação por Computador , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , MicroRNAs/genética , Poliadenilação/genética , Poliendocrinopatias Autoimunes/genética , RNA-Seq , Alinhamento de Sequência , Timo/citologia , Timo/imunologia , Fatores de Transcrição/deficiência , Proteína AIRE

RESUMO

During tumor progression, cancer cells rewire their metabolism to face their bioenergetic demands. In recent years, microRNAs (miRNAs) have emerged as regulatory elements that inhibit the translation and stability of crucial mRNAs, some of them causing direct metabolic alterations in cancer. In this study, we investigated the relationship between miRNAs and their targets mRNAs that control metabolism, and how this fine-tuned regulation is diversified depending on the tumor stage. To do so, we implemented a paired analysis of RNA-seq and small RNA-seq in a breast cancer cell line (MCF7). The cell line was cultured in multicellular tumor spheroid (MCTS) and monoculture conditions. For MCTS, we selected two-time points during their development to recapitulate a proliferative and quiescent stage and contrast their miRNA and mRNA expression patterns associated with metabolism. As a result, we identified a set of new direct putative regulatory interactions between miRNAs and metabolic mRNAs representative for proliferative and quiescent stages. Notably, our study allows us to suggest that miR-3143 regulates the carbon metabolism by targeting hexokinase-2. Also, we found that the overexpression of several miRNAs could directly overturn the expression of mRNAs that control glycerophospholipid and N-Glycan metabolism. While this set of miRNAs downregulates their expression in the quiescent stage, the same set is upregulated in proliferative stages. This last finding suggests an additional metabolic switch of the above mentioned metabolic pathways between the quiescent and proliferative stages. Our results contribute to a better understanding of how miRNAs modulate the metabolic landscape in breast cancer MCTS, which eventually will help to design new strategies to mitigate cancer phenotype.

RESUMO

Autoimmune regulator (Aire) is a transcriptional regulator of peripheral tissue antigens (PTAs) and microRNAs (miRNAs) in medullary thymic epithelial cells (mTECs). In this study, we tested the hypothesis that Aire also played a role as an upstream posttranscriptional controller in these cells and that variation in its expression might be associated with changes in the interactions between miRNAs and the mRNAs encoding PTAs. We demonstrated that downregulation of Aire in vivo in the thymuses of BALB/c mice imbalanced the large-scale expression of these two RNA species and consequently their interactions. The expression profiles of a large set of mTEC miRNAs and mRNAs isolated from the thymuses of mice subjected (or not) to small-interfering-induced Aire gene knockdown revealed that 87 miRNAs and 4,558 mRNAs were differentially expressed. The reconstruction of the miRNA-mRNA interaction networks demonstrated that interactions between these RNAs were under Aire influence and therefore changed when this gene was downregulated. Prior to Aire-knockdown, only members of the miR-let-7 family interacted with a set of PTA mRNAs. Under Aire-knockdown conditions, a larger set of miRNA families and their members established this type of interaction. Notably, no previously described Aire-dependent PTA interacted with the miRNAs, indicating that these PTAs were somehow refractory. The miRNA-mRNA interactions were validated by calculating the minimal free energy of the pairings between the miRNA seed regions and the mRNA 3' UTRs and within the cellular milieu using the luciferase reporter gene assay. These results suggest the existence of a link between transcriptional and posttranscriptional control because Aire downregulation alters the miRNA-mRNA network controlling PTAs in mTEC cells.