Identical repeated backbone of the human genome

<p>Abstract</p> <p>Background</p> <p>Identical sequences with a minimal length of about 300 base pairs (bp) have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR) events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB) of the reference human genome.</p> <p>Results</p> <p>The IRB sequences are distributed all over the genome in 66,600 regions, which correspond to ~2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs). By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes.</p> <p>Conclusions</p> <p>In general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.</p

Adipose tissue IL‐18 production is independent of caspase‐1 and caspase‐11

Abstract Background Inflammation in adipose tissue, resulting from imbalanced caloric intake and energy expenditure, contributes to the metabolic dysregulation observed in obesity. The production of inflammatory cytokines, such as IL‐1β and IL‐18, plays a key role in this process. While IL‐1β promotes insulin resistance and diabetes, IL‐18 regulates energy expenditure and food intake. Previous studies have suggested that caspase‐1, activated by the Nlrp3 inflammasome in response to lipid excess, mediates IL‐1β production, whereas activated by the Nlrp1b inflammasome in response to energy excess, mediates IL‐18 production. However, this has not been formally tested. Methods Wild‐type and caspase‐1‐deficient Balb/c mice, carrying the Nlrp1b1 allele, were fed with regular chow or a high‐fat diet for twelve weeks. Food intake and mass gain were recorded weekly. At the end of the twelve weeks, glucose tolerance and insulin resistance were evaluated. Mature IL‐18 protein levels and the inflammatory process in the adipose tissue were determined. Fasting lipid and cytokine levels were quantified in the sera of the different experimental groups. Results We found that IL‐18 production in adipose tissue is independent of caspase‐1 activity, regardless of the metabolic state, while Nlrp3‐mediated IL‐1β production remains caspase‐1 dependent. Additionally, caspase‐1 null Balb/c mice did not develop metabolic abnormalities in response to energy excess from the high‐fat diet. Conclusion Our findings suggest that IL‐18 production in the adipose tissue is independent of Nlrp3 inflammasome and caspase‐1 activation, regardless of caloric food intake. In contrast, Nlrp3‐mediated IL‐1β production is caspase‐1 dependent. These results provide new insights into the mechanisms underlying cytokine production in the adipose tissue during both homeostatic conditions and metabolic stress, highlighting the distinct roles of caspase‐1 and the Nlrp inflammasomes in regulating inflammatory responses

Use of microRNAs as Diagnostic, Prognostic, and Therapeutic Tools for Glioblastoma

Glioblastoma (GB) is the most aggressive and common type of cancer within the central nervous system (CNS). Despite the vast knowledge of its physiopathology and histology, its etiology at the molecular level has not been completely understood. Thus, attaining a cure has not been possible yet and it remains one of the deadliest types of cancer. Usually, GB is diagnosed when some symptoms have already been presented by the patient. This diagnosis is commonly based on a physical exam and imaging studies, such as computed tomography (CT) and magnetic resonance imaging (MRI), together with or followed by a surgical biopsy. As these diagnostic procedures are very invasive and often result only in the confirmation of GB presence, it is necessary to develop less invasive diagnostic and prognostic tools that lead to earlier treatment to increase GB patients’ quality of life. Therefore, blood-based biomarkers (BBBs) represent excellent candidates in this context. microRNAs (miRNAs) are small, non-coding RNAs that have been demonstrated to be very stable in almost all body fluids, including saliva, serum, plasma, urine, cerebrospinal fluid (CFS), semen, and breast milk. In addition, serum-circulating and exosome-contained miRNAs have been successfully used to better classify subtypes of cancer at the molecular level and make better choices regarding the best treatment for specific cases. Moreover, as miRNAs regulate multiple target genes and can also act as tumor suppressors and oncogenes, they are involved in the appearance, progression, and even chemoresistance of most tumors. Thus, in this review, we discuss how dysregulated miRNAs in GB can be used as early diagnosis and prognosis biomarkers as well as molecular markers to subclassify GB cases and provide more personalized treatments, which may have a better response against GB. In addition, we discuss the therapeutic potential of miRNAs, the current challenges to their clinical application, and future directions in the field

MiR-7 Promotes Epithelial Cell Transformation by Targeting the Tumor Suppressor KLF4

<div><p>MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have a pivotal role in the post-transcriptional regulation of gene expression and their misregulation is common in different types of cancer. Although it has been shown that miR-7 plays an oncogenic role in different cellular contexts, the molecular mechanisms by which miR-7 promotes cell transformation are not well understood. Here we show that the transcription factor KLF4 is a direct target of miR-7 and present experimental evidence indicating that the regulation of KLF4 by miR-7 has functional implications in epithelial cell transformation. Stable overexpression of miR-7 into lung and skin epithelial cells enhanced cell proliferation, cell migration and tumor formation. Alteration of these cellular functions by miR-7 resulted from misregulation of KLF4 target genes involved in cell cycle control. miR-7-induced tumors showed decreased p21 and increased Cyclin D levels. Taken together, these findings indicate that miR-7 acts as an oncomiR in epithelial cells in part by directly regulating KLF4 expression. Thus, we conclude that miR-7 acts as an oncomiR in the epithelial cellular context, where through the negative regulation of KLF4-dependent signaling pathways, miR-7 promotes cellular transformation and tumor growth.</p></div

miR-7 overexpression promotes HaCaT cell proliferation and entry to the S phase of the cell cycle.

<p>(A) 5×10³ cells of independent miR-7 or pcDNA overexpressing clones were plated onto 24 well culture plates and counted every 24 hours during four consecutive days. The percentage of cells entering to S phase of the cell cycle was determined by BrdU incorporation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. BrdU+ cells were visualized under UV microscopy (B), cells were counted and the results plotted (C). Results represent the mean of three independent experiments performed with five or six pcDNA, miR-7 or miR-7+KLF4 overexpressing independent clones. ***<i>p</i><0.001 and **<i>p</i><0.01 <i>vs.</i> pcDNA, ###<i>p</i><0.001 <i>vs.</i> miR-7+KLF4.</p

KLF4 3′ UTR contains two evolutionary conserved binding sites that mediate the interaction with miR-7.

<p>(A) Schematic representation of the human KLF4 mRNA indicating the relative positions of the two identified miR-7 binding sites denoted as Seed 1 (S1) and Seed 2 (S2) located respectively, at positions 66–72 nt and 574–580 nt relative to the beginning (1915 nt) of the human KLF4 3′ UTR. Both S1 and S2 are phylogenetically conserved as shown in sequence alignments of different organisms. S1 is conserved in mammals and presents a thermodynamic stability (ΔΔG) of −5.72 kcal/mol while; S2 is conserved in all vertebrates and possesses a ΔΔG of −11.47 kcal/mol. (B) HEK-293 and A549 cells were co-transfected with 200 ng of pc/miR7, pc/miR145, pc/miR881 or empty vector (pcDNA) together with 100 ng of the wt KLF4 3′ UTR (KLF4), the mutated version of KLF4 3′ UTR for the second miR-7 binding site (KLF4-Mut) or empty vector (CHECK2). Luciferase activity was determined 48 hours post-transfection and relative luciferase units were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. Results represent the mean of at least six independent experiments. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 <i>vs.</i> pcDNA.</p

miR-7 overexpression promotes colony formation <i>in vitro</i> and tumor formation <i>in vivo</i> through KLF4 inhibition and regulating Cyclin D and p21 protein levels.

<p>(A) 1×10⁵ cells of three independent clones of A549 cells stably expressing pcDNA, miR-7 or miR-7+KLF4 were plated in a soft agar matrix and formed colonies were counted. (B) 3×10⁶ cells of three independent pcDNA, miR-7 or miR-7+KLF4 overexpressing A549 clones were subcutaneously injected into nude mice and after one-month post-implantation, mice were sacrificed. (C) Tumors were dissected and their mass was determined. (D) The relative expression of miR-7 on the tumors was determined by qPCR. (E) Tumors were analyzed for protein levels of KLF4, Cyclin D and p21 by Western blot assays using specific antibodies. ERK2 protein was used as loading control. The relative expression of each protein was calculated by dividing its densitometric signal by the ERK2 signal. Data were normalized considering the value of pcDNA transfected cells-derived tumors as 100%. Data represent the mean of three independent experiments using three (7 tumors), four (12 tumors) or three (8 tumors) independent miR-7, pcDNA or miR-7+KLF4 overexpressing clones, respectively. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 <i>vs.</i> pcDNA.</p

miR-7 overexpression promotes cell cycle progression.

<p>Values are means ± s.d.; h, hours. n = 3,</p><p>**<i>p</i><0.01,</p><p>*<i>p</i><0.05 <i>vs.</i> pcDNA values.</p><p>miR-7 overexpression promotes cell cycle progression.</p

KLF4 downregulation in A549 cells enhances cell proliferation.

<p>(A) A549 cells were transfected with the KLF4 specific siRNAs (si-KLF4) or unspecific siRNAs (si-ctrl). 48 hours post-transfection total cell extracts were prepared and KLF4, Cyclin D and p21 protein levels were evaluated by Western blot. ERK2 protein was used as loading control. Numbers denote the protein levels as the percentage in si-KLF4 transfected cells relative to si-ctrl transfected cells. (B) The proliferative potential of A549 cells transfected with either si-KLF4 or si-ctrl was evaluated by counting cell number at the indicated time points. Data represent the mean of three independent experiments. *<i>p</i><0.05 <i>vs.</i> si-ctrl at 48 hours.</p

miR-7 overexpression induces wound-healing and migration capacities of HaCaT and A549 cells.

<p>Wound-healing assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. HaCaT (A, left panel) or A549 cells (C, left panel) transfected with the pcDNA vector, expressing miR-7 only or both miR-7 and KLF4 (miR-7+KLF4) were photographed at the indicated times. The percentage of the wound-healed area at these time points was quantified using the TScratch software and plotted as the average of all the analyzed clones (A, C right panel). Migration assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. Sixteen hours after plating, cells were photographed (B, D upper panel). The number of HaCaT (B lower panel) or A549 cells (D lower panel) transfected with the pcDNA vector, expressing miR-7 only or both miR-7 and KLF4 (miR-7+KLF4) was quantified and plotted. Data represents the average of three independent experiments performed with at least three independent clones of each analyzed condition. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 <i>vs.</i> pcDNA.</p