Fragments derived from non-coding RNAs: how complex is genome regulation?

The human genome is highly dynamic and only a small fraction of it codes for proteins, but most of the genome is transcribed, highlighting the importance of non-coding RNAs on cellular functions. In addition, it is now known the generation of non-coding RNA fragments under particular cellular conditions and their functions have revealed unexpected mechanisms of action, converging, in some cases, with the biogenic pathways and action machineries of microRNAs or Piwi-interacting RNAs. This led us to the question why the cell produces so many apparently redundant molecules to exert similar functions and regulate apparently convergent processes? However, non-coding RNAs fragments can also function similarly to aptamers, with secondary and tertiary conformations determining their functions. In the present work, it was reviewed and analyzed the current information about the non-coding RNAs fragments, describing their structure and biogenic pathways, with special emphasis on their cellular functions.

Streptozotocin-Induced Diabetic Rats Showed a Differential Glycine Receptor Expression in the Spinal Cord: A GlyR Role in Diabetic Neuropathy.

In the spinal cord, attenuation of the inhibitory action of glycine is related to an increase in both inflammatory and diabetic neuropathic pain; however, the glycine receptor involvement in diabetic neuropathy has not been reported. We determined the expression of the glycine receptor subunits (α1-α3 and ß) in streptozotocin-induced diabetic Long-Evans rats by qPCR and Western blot. The total mRNA and protein expression (whole spinal cord homogenate) of the α1, α3, and ß subunits did not change during diabetes; however, the α2 subunit mRNA, but not the protein, was overexpressed 45 days after diabetes induction. By contrast, the synaptic expression of the α1 and α2 subunits decreased in all the studied stages of diabetes, but that of the α3 subunit increased on day 45 after diabetes induction. Intradermal capsaicin produced higher paw-licking behavior in the streptozotocin-induced diabetic rats than in the control animals. In addition, the nocifensive response was higher at 45 days than at 20 days. During diabetes, the expression of the glycine receptor was altered in the spinal cord, which strongly suggests its involvement in diabetic neuropathy.

Circular RNAs: the next level of gene regulation.

Gene regulation is a highly complex process involving the presence and participation of many molecules and complexes that regulate gene expression in the genome, which occurs in a precise and coordinated way. Among all these regulatory molecules, the circular RNAs (circRNAs) are the most novel and peculiar family of noncoding RNAs (ncRNAs) as they have a circular structure, are very specific on their expression, highly conserved, and highly resistant to degradation. These molecules have been described in recent years as excellent disease markers and as potential therapeutic targets. In this review, we focused on general characteristics and on the evolution of the circRNAs, as well as on their biological functions, emphasizing on their participation in the formation of brain tumors.

Long Interspersed Nuclear Elements 1 (LINE1): The chimeric transcript L1-MET and its involvement in cancer.

Long interspersed nuclear elements 1 (LINE1) are non-LTR retrotransposons that represent the greatest remodeling force of the human genome during evolution. Genomically, LINE1 are constituted by a 5´ untranslated region (UTR), where the promoter regions are located, three open reading frames (ORF0, ORF1, and ORF2) and one 3´UTR, which has a poly(A) tail that harbors the short interspersed nuclear elements (SINEs) Alu and SVA. Although the intrinsic nature of LINE1 is to be copied and inserted into the genome, an increase in their mobility produces genomic instability. In response to this, the cell has "designed" many mechanisms controlling the retrotransposition levels of LINE1; however, alterations in these regulation systems can increase LINE1 mobility and the formation of chimeric genes. Evidence indicates that 988 human genes have LINE1 inserted in their sequence, resulting in the transcriptional control of genes by their own promoters, as well as by the LINE1 antisense promoter (ASP). To date, very little is known about the biologic impact of this and the L1-MET chimera is a more or less studied case. ASP hypomethylation has been observed in all studied cancer types, leading to increased L1-MET expression. In specific types of cancer, this L1-MET increase controls both low and high MET protein levels. It remains to be clarified if this protein product is a chimeric protein.

Glycine receptor is differentially expressed in the rat retina at early stages of streptozotocin-induced diabetes.

Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Neuronal abnormalities have been reported to appear before microvasculature alterations. We analyzed the expression levels of GlyR subunits in the retina at 7, 20, and 45 days after streptozotocin-induced diabetes to gain insight into the pathogenesis of diabetic retinopathy. We determined the mRNA and protein expression by qPCR and western blot, respectively. The mRNA and protein expression of the α1 subunit was not altered over the study period; however, they were slightly reduced in α2 yet statistically significant. While protein expression of α3 subunit was only reduced at 45 days diabetes. The mRNA and protein expression of the α4 subunit was remarkably decreased since day 7 of diabetes, remaining only ∼20% on day 45 of diabetes. Surprisingly, the mRNA of the ß subunit was highly increased, while its protein levels were not changed. The decrease in GlyR α subunits expression in the retina from diabetic animals suggest a perturbation in the inhibitory glycine signaling pathway, which might be related to the visual alterations observed in diabetes.

Glycine receptor subunits expression in the developing rat retina.

BACKGROUND AND METHODS: Glycine receptor (GlyR) consists of two α (1-4) and three ß subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. RESULTS: mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and ß GlyR subunits during postnatal ages tested. The mRNA ß subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. CONCLUSIONS: These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina.

Differentially Expressed Long Non-Coding RNAs Were Predicted to Be Involved in the Control of Signaling Pathways in Pediatric Astrocytoma.

Expression changes for long non-coding RNAs (lncRNAs) have been identified in adult glioblastoma multiforme (GBM) and in a mixture of adult and pediatric astrocytoma. Since adult and pediatric astrocytomas are molecularly different, the mixture of both could mask specific features in each. We determined the global expression patterns of lncRNAs and messenger RNA (mRNAs) in pediatric astrocytoma of different histological grades. Transcript expression changes were determined with an HTA 2.0 array. lncRNA interactions with microRNAs and mRNAs were predicted by using an algorithm and the LncTar tool, respectively. Interactomes were constructed with the HIPPIE database and visualized with the Cytoscape platform. The array showed expression changes in 156 and 207 lncRNAs in tumors (versus the control) and in pediatric GBM (versus low-grade astrocytoma), respectively. Predictions identified lncRNAs that have putative microRNA binding sites, which might suggest that they function as sponges in these tumors. Also, lncRNAs were shown to interact with many mRNAs, such as Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and sulfatase 2 (SULF2). For example, qPCR found long intergenic non-coding RNA regulator of reprogramming (linc-RoR) expression levels upregulated in pediatric GBM when they were compared with control tissues or with low-grade tumors. Meanwhile, PHLDA1 and ELAV-like RNA binding protein 1 (ELAV1) showed expression changes in tumors relative to the control. Our data showed many lncRNAs with expression changes in pediatric astrocytoma, which might be involved in the regulation of different signaling pathways.

Breast cancer cell line MDA-MB-231 miRNA profile expression after BIK interference: BIK involvement in autophagy.

B-cell lymphoma 2 (BCL2)-interacting killer (apoptosis inducing) (BIK) has been proposed as a tumor suppressor in diverse types of cancers. However, BIK's overexpression in breast cancer (BC) and in non-small lung cancer cells (NSCLCs), associated with a poor prognosis, suggests its participation in tumor progression. In this study, we evaluated the global expression pattern of microRNAs (miRNAs), messenger RNA (mRNA) expression changes in autophagy, and autophagic flux after BIK interference. BIK gene expression was silenced by small interfering RNA (siRNA) in BC cell MDA-MB-231, and BIK interference efficiency was tested by real-time PCR and by Western blotting. BIK expression levels decreased by 75 ± 18 % in the presence of 600 nM siRNA, resulting in the abolishment of BIK expression by 94 ± 30 %. BIK interference resulted in the overexpression of 17 miRNAs that, according to the DIANA-miRPath v3.0 database, are mainly implied in the control of cell signaling, gene expression, and autophagy. The autophagy array revealed downregulation of transcripts which participate in autophagy, and their interactome revealed a complex network, where hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), α-synuclein (SNCA), unc-51-like autophagy activating kinase 1/2 (ULK1/2), and mitogen-activated protein kinase 3 (MAPK3) were shown to be signaling hubs. LC3-II expression-an autophagy marker-was increased by 169 ± 25 % after BIK interference, which indicates the involvement of BIK in autophagy. Altogether, our results indicate-for the first time-that BIK controls the expression of miRNAs, as well as the autophagic flux in MDA-MB-231 cells.

Involvement of multiple cellular pathways in regulating resistance to tamoxifen in BIK-suppressed MCF-7 cells.

Majority of women with estrogen receptor (ER)-positive breast cancers initially respond to hormone therapies such as tamoxifen (TAM; antagonist of estrogen). However, many tumors eventually become resistant to TAM. Therefore, understanding the various cellular components involved in causing resistance to TAM is of paramount importance in designing novel entities for efficacious hormone therapy. Previously, we found that suppression of BIK gene expression induced TAM resistance in MCF-7 breast cancer cells. In order to understand the response of these cells to TAM and its association with resistance, a microarray analysis of gene expression was performed in the BIK-suppressed MCF-7 cells and compared it to the TAM-only-treated cells (controls). Several genes participating in various cellular pathways were identified. Molecules identified in the drug resistance pathway were 14-3-3z or YWHAZ, WEE1, PRKACA, NADK, and HSP90AA 1. Further, genes involved in cell cycle control, apoptosis, and cell proliferation were also found differentially expressed in these cells. Transcriptional and translational analysis of key molecules such as STAT2, AKT 3, and 14-3-3z revealed similar changes at the messenger RNA (mRNA) as well as at the protein level. Importantly, there was no cytotoxic effect of TAM on BIK-suppressed MCF-7 cells. Further, these cells were not arrested at the G0-G1 phase of the cell cycle although 30 % of BIK-suppressed cells were arrested at the G2 phase of the cycle on TAM treatment. Furthermore, we found a relevant interaction between 14-3-3z and WEE1, suggesting that the cytotoxic effect of TAM was prevented in BIK-suppressed cells because this interaction leads to transitory arrest in the G2 phase leading to the repair of damaged DNA and allowing the cells to proliferate.

[Genomics in medicine]. / La genómica en la medicina.

The development of new fields of study in genetics, as the -omic sciences (transcriptomics, proteomics, metabolomics), has allowed the study of the regulation and expression of genomes. Therefore, nowadays it is possible to study global alterations--in the whole genome--and their effect at the protein and metabolic levels. Importantly, this new way of studying genetics has opened new areas of knowledge, and new cellular mechanisms that regulate the functioning of biological systems have been elucidated. In the clinical field, in the last years new molecular tools have been implemented. These tools are favorable to a better classification, diagnosis and prognosis of several human diseases. Additionally, in some cases best treatments, which improve the quality of life of patients, have been established. Due to the previous assertion, it is important to review and divulge changes in the study of genetics as a result of the development of the -omic sciences, which is the aim of this review.

El desarrollo de nuevas áreas de estudio dentro de la genética, como las ciencias ómicas (transcriptómica, proteómica, metabolómica), ha permitido estudiar al genoma a diferentes niveles de regulación y expresión. Gracias a esto, actualmente se pueden estudiar las alteraciones génicas de un organismo de forma global ("genoma") y se puede identificar el efecto que tienen estas alteraciones a nivel de proteína y de la producción de metabolitos. De manera importante, esta nueva forma de estudiar la genética ha abierto nuevos campos de conocimiento y ha dilucidado nuevos mecanismos celulares que rigen el funcionamiento de los sistemas biológicos. A nivel clínico, en los últimos años se han implementado nuevas herramientas moleculares que permiten hacer una mejor clasificación, un mejor diagnóstico, así como un pronóstico más acertado de diversas enfermedades. Asimismo, en algunos casos se han establecido mejores tratamientos que favorecen la calidad de vida de los pacientes. Debido a todo lo anterior, es importante revisar y divulgar el cambio que ha tenido el estudio de la genética gracias al desarrollo de las ciencias ómicas, el cual es el objetivo de esta revisión.

Mechanisms associated with resistance to tamoxifen in estrogen receptor-positive breast cancer (review).

Anti-estrogens such as tamoxifen are widely used in the clinic to treat estrogen receptor-positive breast tumors. Patients with estrogen receptor-positive breast cancer initially respond to treatment with anti-hormonal agents such as tamoxifen, but remissions are often followed by the acquisition of resistance and, ultimately, disease relapse. The development of a rationale for the effective treatment of tamoxifen-resistant breast cancer requires an understanding of the complex signal transduction mechanisms. In the present study, we explored some mechanisms associated with resistance to tamoxifen, such as pharmacologic mechanisms, loss or modification in estrogen receptor expression, alterations in co-regulatory proteins and the regulation of the different signaling pathways that participate in different cellular processes such as survival, proliferation, stress, cell cycle, inhibition of apoptosis regulated by the Bcl-2 family, autophagy, altered expression of microRNA, and signaling pathways that regulate the epithelial-mesenchymal transition in the tumor microenvironment. Delineation of the molecular mechanisms underlying the development of resistance may aid in the development of treatment strategies to enhance response and compromise resistance.

Glycine receptor internalization by protein kinases activation.

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.