Search results for: non-coding RNAs

Authors: Shu-Jun Li, Cheng-Cao Sun, De-Jia Li

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Recently, long noncoding RNAs (lncRNAs) have been emerged as crucial regulators of human diseases and prognostic markers in numerous of cancers, including colorectal carcinoma (CRC). Here, we identified an oncogenetic lncRNA HULC, which may promote colorectal tumorigenesis. HULC has been found to be up-regulated and acts as oncogene in gastric cancer and hepatocellular carcinoma, but its expression pattern, biological function and underlying mechanism in CRC is still undetermined. Here, we reported that HULC expression is also over-expressed in CRC, and its increased level is associated with poor prognosis and shorter survival. Knockdown of HULC impaired CRC cells proliferation, migration and invasion, facilitated cell apoptosis in vitro, and inhibited tumorigenicity of CRC cells in vivo. Mechanistically, RNA immunoprecipitation (RIP) and RNA pull-down experiment demonstrated that HULC could simultaneously interact with EZH2 to repress underlying targets NKD2 transcription. In addition, rescue experiments determined that HULC oncogenic function is partly dependent on repressing NKD2. Taken together, our findings expound how HULC over-expression endows an oncogenic function in CRC.

Keywords: long noncoding RNA, HULC, NKD2, colorectal carcinoma, proliferation, apoptosis

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Authors: Ipsita Biswas, Arnab Sarkar, Ashikur Rahaman, Gopeswar Mukherjee, Subhrangsu Chatterjee, Shamee Bhattacharjee, Deba Prasad Mandal

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One of the major reasons for the high mortality rate of lung cancer is the substantial delays in disease detection at late metastatic stages. It is of utmost importance to understand the detailed molecular signaling and detect the molecular markers that can be used for the early diagnosis of cancer. Several studies explored the emerging roles of long noncoding RNAs (lncRNAs) in various cancers as well as lung cancer. A long non-coding RNA LINC00273 was recently discovered to promote cancer cell migration and invasion, and its positive correlation with the pathological stages of metastasis may prove it to be a potential target for inhibiting cancer cell metastasis. Comparing real-time expression of LINC00273 in various human clinical cancer tissue samples with normal tissue samples revealed significantly higher expression in cancer tissues. This long intergenic noncoding RNA was found to be highly expressed in human liver tumor-initiating cells, human gastric adenocarcinoma AGS cell line, as well as human non-small cell lung cancer A549 cell line. SiRNA and shRNA-induced knockdown of LINC00273 in both in vitro and in vivo nude mice significantly subsided AGS and A549 cancer cell migration and invasion. LINC00273 knockdown also reduced TGF-β induced SNAIL, SLUG, VIMENTIN, ZEB1 expression, and metastasis in A549 cells. Plenty of reports have suggested the role of microRNAs of the miR200 family in reversing epithelial to mesenchymal transition (EMT) by inhibiting ZEB transcription factors. In this study, hsa-miR-200a-3p was predicted via IntaRNA-Freiburg RNA tools to be a potential target of LINC00273 with a negative free binding energy of −8.793 kcal/mol, and this interaction was verified as a confirmed target of LINC00273 by RNA pulldown, real-time PCR and luciferase assay. Mechanistically, LINC00273 accelerated TGF-β induced EMT by sponging hsa-miR-200a-3p which in turn liberated ZEB1 and promoted prometastatic functions in A549 cells in vitro as verified by real-time PCR and western blotting. The similar expression patterns of these EMT regulatory pathway molecules, viz. LINC00273, hsa-miR-200a-3p, ZEB1 and TGF-β, were also detected in various clinical samples like breast cancer tissues, oral cancer tissues, lung cancer tissues, etc. Overall, this LINC00273 mediated EMT regulatory signaling can serve as a potential therapeutic target for the prevention of lung cancer metastasis.

Keywords: epithelial to mesenchymal transition, long noncoding RNA, microRNA, non-small-cell lung carcinoma

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Authors: Anjan Hazra, Nirjhar Dasgupta, Chandan Sengupta, Sauren Das

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Tea (Camellia sinensis) has received substantial attention from the scientific world time to time, not only for its commercial importance, but also for its demand to the health-conscious people across the world for its extensive use as potential sources of antioxidant supplement. These health-benefit traits primarily rely on some regulatory networks of different metabolic pathways. Development of microsatellite markers from the conserved genomic regions is being worthwhile for studying the genetic diversity of closely related species or self-pollinated species. Although several SSR markers have been reported, in tea the trait-specific Simple Sequence Repeats (SSRs) are yet to be identified, which can be used for marker assisted breeding technique. MicroRNAs are endogenous, noncoding, short RNAs directly involved in regulating gene expressions at the post-transcriptional level. It has been found that diversity in miRNA gene interferes the formation of its characteristic hair pin structure and the subsequent function. In the present study, the precursors of small regulatory RNAs (microRNAs) has been fished out from tea Expressed Sequence Tag (EST) database. Furthermore, the simple sequence repeat motifs within the putative miRNA precursor genes are also identified in order to experimentally validate their existence and function. It is already known that genic-SSR markers are very adept and breeder-friendly source for genetic diversity analysis. So, the potential outcome of this in-silico study would provide some novel clues in understanding the miRNA-triggered polymorphic genic expression controlling specific metabolic pathways, accountable for tea quality.

Keywords: micro RNA, simple sequence repeats, tea quality, trait specific marker

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Authors: Zohar Manber, Ran Elkon

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Accumulation of somatic mutations (SMs) in the genome is a major driving force of cancer development. Most SMs in the tumor's genome are functionally neutral; however, some cause damage to critical processes and provide the tumor with a selective growth advantage (termed cancer driver mutations). Current research on functional significance of SMs is mainly focused on finding alterations in protein coding sequences. However, the exome comprises only 3% of the human genome, and thus, SMs in the noncoding genome significantly outnumber those that map to protein-coding regions. Although our understanding of noncoding driver SMs is very rudimentary, it is likely that disruption of regulatory elements in the genome is an important, yet largely underexplored mechanism by which somatic mutations contribute to cancer development. The expression of most human genes is controlled by multiple enhancers, and therefore, it is conceivable that regulatory SMs are distributed across different enhancers of the same target gene. Yet, to date, most statistical searches for regulatory SMs have considered each regulatory element individually, which may reduce statistical power. The first challenge in considering the cumulative activity of all the enhancers of a gene as a single unit is to map enhancers to their target promoters. Such mapping defines for each gene its set of regulating enhancers (termed "set of regulatory elements" (SRE)). Considering multiple enhancers of each gene as one unit holds great promise for enhancing the identification of driver regulatory SMs. However, the success of this approach is greatly dependent on the availability of comprehensive and accurate enhancer-promoter (E-P) maps. To date, the discovery of driver regulatory SMs has been hindered by insufficient sample sizes and statistical analyses that often considered each regulatory element separately. In this study, we analyzed more than 2,500 whole-genome sequence (WGS) samples provided by The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC) in order to identify such driver regulatory SMs. Our analyses took into account the combinatorial aspect of gene regulation by considering all the enhancers that control the same target gene as one unit, based on E-P maps from three genomics resources. The identification of candidate driver noncoding SMs is based on their recurrence. We searched for SREs of genes that are "hotspots" for SMs (that is, they accumulate SMs at a significantly elevated rate). To test the statistical significance of recurrence of SMs within a gene's SRE, we used both global and local background mutation rates. Using this approach, we detected - in seven different cancer types - numerous "hotspots" for SMs. To support the functional significance of these recurrent noncoding SMs, we further examined their association with the expression level of their target gene (using gene expression data provided by the ICGC and TCGA for samples that were also analyzed by WGS).

Keywords: cancer genomics, enhancers, noncoding genome, regulatory elements

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Authors: Ali Oztuna, Meral Sarper, Deniz Torun, Fatma Bayrakdar, Selcuk Kilic, Mehmet Baysallar

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Bacillus anthracis is one of the biological agents most likely to be used in case of bioterrorist attack as well as being the cause of anthrax. The bacterium's major virulence factors are the anthrax toxins and an antiphagocytic polyglutamic capsule. TEM8 (ANTXR1) and CMG2 (ANTXR2) are ubiquitously expressed type I transmembrane proteins, and ANTXR2 is the major receptor for anthrax toxins. MicroRNAs are 21-24 bp small noncoding RNAs that regulate gene expression by base pairing with the 3' UTR (untranslated regions) of their target mRNAs resulting in mRNA degradation and/or translational repression. MicroRNAs contribute to regulation of most biological processes and influence numerous pathological states like infectious disease. In this study, post-exposure (toxins) protective effect of the hsa-miR-124-3p against Bacillus anthracis was examined. In this context, i) THP-1 and U937 cells were differentiated to MΦ macrophage, ii) miRNA transfection efficiencies were evaluated by flow cytometry and qPCR, iii) protection against Bacillus anthracis toxins were investigated by XTT, cAMP ELISA and MEK2 cleavage assays. Acknowledgements: This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under Grant SBAG-218S467.

Keywords: ANTXR2, hsa-miR-124-3p, MΦ macrophage, THP-1, U937

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Authors: Bruno Baptista, Andreia S. R. Oliveira, Patricia V. Mendonca, Jorge F. J. Coelho, Fani Sousa

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Drug delivery systems are designed to allow adequate protection and controlled delivery of drugs to specific locations. These systems aim to reduce side effects and control the biodistribution profile of drugs, thus improving therapeutic efficacy. This study involved the synthesis of polymeric nanoparticles, based on amphiphilic diblock copolymers, comprising a biocompatible, poly (oligo (ethylene oxide) methyl ether methacrylate (POEOMA) as hydrophilic segment and a pH-sensitive block, the poly (2-diisopropylamino)ethyl methacrylate) (PDPA). The objective of this work was the development of polymeric pH-responsive nanoparticles to encapsulate and carry small RNAs as a model to further develop non-coding RNAs delivery systems with therapeutic value. The responsiveness of PDPA to pH allows the electrostatic interaction of these copolymers with nucleic acids at acidic pH, as a result of the protonation of the tertiary amine groups of this polymer at pH values below its pKa (around 6.2). Initially, the molecular weight parameters and chemical structure of the block copolymers were determined by size exclusion chromatography (SEC) and nuclear magnetic resonance (1H-NMR) spectroscopy, respectively. Then, the complexation with small RNAs was verified, generating polyplexes with sizes ranging from 300 to 600 nm and with encapsulation efficiencies around 80%, depending on the molecular weight of the polymers, their composition, and concentration used. The effect of pH on the morphology of nanoparticles was evaluated by scanning electron microscopy (SEM) being verified that at higher pH values, particles tend to lose their spherical shape. Since this work aims to develop systems for the delivery of non-coding RNAs, studies on RNA protection (contact with RNase, FBS, and Trypsin) and cell viability were also carried out. It was found that they induce some protection against constituents of the cellular environment and have no cellular toxicity. In summary, this research work contributes to the development of pH-sensitive polymers, capable of protecting and encapsulating RNA, in a relatively simple and efficient manner, to further be applied on drug delivery to specific sites where pH may have a critical role, as it can occur in several cancer environments.

Keywords: drug delivery systems, pH-responsive polymers, POEOMA-b-PDPA, small RNAs

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Authors: Sobia Shafqat, Saeed Ahmad Qaisrani

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MicroRNAs play an essential role in the regulation and development of all processes in most eukaryotes because of their prospective part as mediators controlling cell growth and differentiation towards the exact position of RNAs response in plants under biotic and abiotic factors or stressors. In a few cases, Zn is oblivious poisonous for plants due to its heavy metal status. Some other metals are extremely toxic, like Cd, Hg, and Pb, but these elements require in rice for the programming of genes under abiotic stress resembling Zn stress when micro RNAs268 was importantly introduced in rice. The micro RNAs overexpressed in transgenic plants with an accumulation of a large amount of melanin dialdehyde, hydrogen peroxide, and an excessive quantity of Zn in the seedlings stage. Let out results for rice pliability under Zn stress micro RNAs act as negative controllers. But the role of micro RNA268 act as a modulator in different ecological condition. It has been explained clearly with a long understanding of the role of micro RNA268 under stress conditions; pliability and practically showed outcome to increase plant sufferance under Zn stress because micro RNAs is an intervention technique for gene regulation in gene expression. The proposed study was experimented with by using genetic factors of Zn stress and toxicity effect on rice plants done at District Vehari, Pakistan. The trial was performed randomly with three replications in a complete block design (RCBD). These blocks were controlled with different concentrations of genetic factors. By overexpression of micro RNA268 rice, seedling growth was not stopped under Zn deficiency due to the accumulation of a large amount of melanin dialdehyde, hydrogen peroxide, and an excessive quantity of Zn in their seedlings. Results showed that micro RNA268 act as a negative controller under Zn stress. In the end, under stress conditions, micro RNA268 showed the necessary function in the tolerance of rice plants. The directorial work sketch gave out high agronomic applications and yield outcomes in rice with a specific amount of Zn application.

Keywords: micro RNA268, zinc, rice, agronomic approach

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Authors: Md. M. Hossain, Regan Roat, Jenica Christopherson, Colette Free, Zhiguang Guo

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Long noncoding RNA (lncRNA) mediated post-transcriptional gene regulation, and their epigenetic landscapes have been shown to be involved in many human diseases. However, their regulation in diabetes through governing islet’s β-cell function and survival needs to be elucidated. Due to the technical and ethical constraints, it is difficult to study their role in β-cell function and survival in human under in vivo condition. In this study, humanized mice have been developed through transplanting human pancreatic islet under the kidney capsule of NOD.SCID mice and induced β-cell death leading to diabetes condition to study lncRNA mediated regulation. For this, human islets from 3 donors (3000 IEQ, purity > 80%) were transplanted under the kidney capsule of STZ induced diabetic NOD.scid mice. After at least 2 weeks of normoglycecemia, lymphocytes from diabetic NOD mice were adoptively transferred and islet grafts were collected once blood glucose reached > 200 mg/dl. RNA from human donor islets, islet grafts from humanized mice with either adoptive lymphocyte transfer (ALT) or PBS control (CTL) were ribodepleted; barcoded fragment libraries were constructed and sequenced on the Ion Proton sequencer. lncRNA expression in isolated human islets, islet grafts from humanized mice with and without induced β-cell death and their regulation in human islets function in vitro under glucose challenge, cytokine mediated inflammation and induced apoptotic condition were investigated. Out of 3155 detected lncRNAs, 299 that highly expressed in islets were found to be significantly downregulated and 224 upregulated in ALT compared to CTL. Most of these are found to be collocated within 5 kb upstream and 1 kb downstream of 788 up- and 624 down-regulated mRNAs. Genomic Regions Enrichment of Annotations Analysis revealed deregulated and collocated genes are related to pancreas endocrine development; insulin synthesis, processing, and secretion; pancreatitis and diabetes. Many of them, that found to be located within enhancer domains for islet specific gene activity, are associated to the deregulation of known islet/βcell specific transcription factors and genes that are important for β-cell differentiation, identity, and function. RNA sequencing analysis revealed aberrant lncRNA expression which is associated to the deregulated mRNAs in β-cell function as well as in molecular pathways related to diabetes. A distinct set of candidate lncRNA isoforms were identified as highly enriched and specific to human islets, which are deregulated in human islets from donors with different BMIs and with type 2 diabetes. These RNAs show an interesting regulation in cultured human islets under glucose stimulation and with induced β-cell death by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the media of islets cultured with cytokines. Results of this study suggest that the islet specific lncRNAs are deregulated in human islet with β-cell death, hence important in diabetes. These lncRNAs might be important for human β-cell function and survival thus could be used as biomarkers and novel therapeutic targets for diabetes.

Keywords: β-cell, humanized mouse, pancreatic islet, LncRNAs

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Authors: S. S. Singh, A. Bhattacharya, S. Bhattacharya

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The eukaryotic exosome complex plays a pivotal role in RNA biogenesis, maturation, surveillance and differential expression of various RNAs in response to varying environmental signals. The exosome is composed of evolutionary conserved nine core subunits and the associated exonucleases Rrp6 and Rrp44. Rrp6p is crucial for the processing of rRNAs, other non-coding RNAs, regulation of polyA tail length and termination of transcription. Rrp6p, a 3’-5’ exonuclease is required for degradation of 5’-external transcribed spacer (ETS) released from the rRNA precursors during the early steps of pre-rRNA processing. In the parasitic protist Entamoeba histolytica in response to growth stress, there occurs the accumulation of unprocessed pre-rRNA and 5’ ETS sub fragment. To understand the processes leading to this accumulation, we looked for Rrp6 and the exosome subunits in E. histolytica, by in silico approaches. Of the nine core exosomal subunits, seven had high percentage of sequence similarity with the yeast and human. The EhRrp6 homolog contained exoribonuclease and HRDC domains like yeast but its N- terminus lacked the PMC2NT domain. EhRrp6 complemented the temperature sensitive phenotype of yeast rrp6Δ cells suggesting conservation of biological activity. We showed 3’-5’ exoribonuclease activity of EhRrp6p with in vitro-synthesized appropriate RNAs substrates. Like the yeast enzyme, EhRrp6p degraded unstructured RNA, but could degrade the stem-loops slowly. Furthermore, immunolocalization revealed that EhRrp6 was nuclear-localized in normal cells but was diminished from nucleus during serum starvation, which could explain the accumulation of 5’ETS during stress. Our study shows functional conservation of EhRrp6p in E.histolytica, an early-branching eukaryote, and will help to understand the evolution of exosomal components and their regulatory function.

Keywords: entamoeba histolytica, exosome complex, rRNA processing, Rrp6

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Authors: Mahdi Rahaie

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MicroRNAs are a novel class of small RNAs which regulate gene expression by translational repression or degradation of messenger RNAs. To produce sensitive, simple and cost-effective assays for microRNAs, detection is in urgent demand due to important role of these biomolecules in progression of human disease such as Alzheimer’s, Multiple sclerosis, and some other neurodegenerative diseases. Herein, we report several novel, sensitive and specific microRNA nanobiosensors which were designed based on colorimetric and fluorescence detection of nanoparticles and hybridization chain reaction amplification as an enzyme-free amplification. These new strategies eliminate the need for enzymatic reactions, chemical changes, separation processes and sophisticated equipment whereas less limit of detection with most specify are acceptable. The important features of these methods are high sensitivity and specificity to differentiate between perfectly matched, mismatched and non-complementary target microRNAs and also decent response in the real sample analysis with blood plasma. These nanobiosensors can clinically be used not only for the early detection of neuro diseases but also for every sickness related to miRNAs by direct detection of the plasma microRNAs in real clinical samples, without a need for sample preparation, RNA extraction and/or amplification.

Keywords: hybridization chain reaction, microRNA, nanobiosensor, neurodegenerative diseases

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Authors: Barsha Saha, Shouvik Chakravarty, Sukanta Ray, Kshaunish Das, Nidhan K. Biswas, Srikanta Goswami

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Pancreatic Ductal Adenocarcinoma is a well-recognised cause of cancer death with a five-year survival rate of about 9%, and its incidence in India has been found to be increased manifold in recent years. Due to delayed detection, this highly metastatic disease has a poor prognosis. Several molecular alterations happen during the progression of the disease from pre-cancerous conditions, and many such alterations could be investigated for their biomarker potential. MicroRNAs have been shown to be prognostic for PDAC patients in a variety of studies. We hereby used NGS technologies to evaluate the role of small RNA changes during pancreatic cancer development from chronic pancreatitis. Plasma samples were collected from pancreatic cancer patients (n=16), chronic pancreatitis patients (n=8), and also from normal individuals (n=16). Pancreatic tumour tissue (n=5) and adjacent normal tissue samples (n=5) were also collected. Sequencing of small RNAs was carried out after small RNAs were isolated from plasma samples and tissue samples. We find that certain microRNAs are highly deregulated in pancreatic cancer patients in comparison to normal samples. A combinatorial analysis of plasma and tissue microRNAs and subsequent exploration of their targets and altered molecular pathways could not only identify potential biomarkers for disease diagnosis but also help to understand the underlying mechanism.

Keywords: small RNA sequencing, pancreatic cancer, biomarkers, tissue sample

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Authors: Laura Gantley, Vanessa M. Conn, Stuart Webb, Kirsty Kirk, Marta Gabryelska, Duncan Holds, Brett W. Stringer, Simon J. Conn

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Trinucleotide repeat disorders comprise ~20 severe, inherited human neuromuscular and neurodegenerative disorders, which are a result of an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington’s disease, results from the expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Non-coding RNAs have been implicated in the initiation and progression of many diseases; thus, we focus on one circular RNA (circRNA) molecule arising from non-canonical splicing (back splicing) of HTT pre-mRNA. This circRNA and its mouse orthologue were transgenically overexpressed in human cells (SHSY-5Y and HEK293T) and mouse cells (Mb1), respectively. High-content imaging and flow cytometry demonstrated the overexpression of this circRNA reduces cell proliferation, reduces nuclear size independent of cellular size, and alters cell cycle progression. Analysis of protein by western blot and immunofluorescence demonstrated no change to HTT protein levels but altered nuclear-cytoplasmic distribution without impacting the expansion of the HTT repeat region. As these phenotypic and genotypic changes are found in Huntington’s disease patients, these results may suggest that this circRNA may play a functional role in the progression of Huntington’s disease.

Keywords: cell biology, circular RNAs, Huntington’s disease, molecular biology, neurodegenerative disorders

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Authors: Rinat Arbel-Goren, Joel Stavans

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Cell-to-cell variations in transcript or protein abundance, called noise, may give rise to phenotypic variability between isogenic cells, enhancing the probability of survival under stress conditions. These variations may be introduced by post-transcriptional regulatory processes such as non-coding, small RNAs stoichiometric degradation of target transcripts in bacteria. We study the iron homeostasis network in Escherichia coli, in which the RyhB small RNA regulates the expression of various targets as a model system. Using fluorescence reporter genes to detect protein levels and single-molecule fluorescence in situ hybridization to monitor transcripts levels in individual cells, allows us to compare noise at both transcript and protein levels. The experimental results and computer simulations show that extrinsic noise buffers through a feed-forward loop configuration the increase in variability introduced at the transcript level by iron deprivation, illuminating the important role that extrinsic noise plays during stress. Surprisingly, extrinsic noise also decouples of fluctuations of two different targets, in spite of RyhB being a common upstream factor degrading both. Thus, phenotypic variability increases under stress conditions by the decoupling of target fluctuations in the same cell rather than by increasing the noise of each. We also present preliminary results on the adaptation of cells to prolonged iron deprivation in order to shed light on the evolutionary role of post-transcriptional downregulation by small RNAs.

Keywords: cell-to-cell variability, Escherichia coli, noise, single-molecule fluorescence in situ hybridization (smFISH), transcript

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Authors: H. Haseena Banu, D. Karthick, R. Stalin, E. Nandha Kumar, T. P. Sachidanandam, P. Shanthi

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Background of the study: Type 2 diabetes is emerging as the predominant metabolic disorder in the world among adults characterized mainly by the resistance of the insulin sensitive tissues towards insulin followed by the decrease in the insulin secretion. The treatment for this disease usually involves treatment with oral synthetic drugs which are known to cause several side effects. Therefore, identification of new biomarkers as therapeutic target is the need of the hour. miRNAs are small, non–protein-coding RNAs that negatively regulate gene expression by promoting degradation and/or inhibit the translation of target mRNAs and have emerged as biomarkers in predicting diabetes mellitus. Objective of the study: To elucidate the therapeutic role of gallic acid in modulating the alterations in glucose metabolism induced by miRNAs 194 and 135a in Type 2 diabetic rats. Materials and Methods: T2D was induced in rats by feeding them with a high fat diet for 2 weeks followed by intraperitoneal injection of 35 mg/kg/body weight (b.wt.) of streptozotocin. Microarrays were used to assess the expression of miRNAs in control, diabetic and gallic acid treated rats. Gene expression studies were carried out by RT PCR analysis. Results: Forty one miRNAs were differentially expressed in Type 2 diabetic rats. Among these, the expression of miRNA 194 was significantly decreased whereas miRNA 135a was significantly increased in Type 2 diabetic rats. The glucose metabolism was also altered significantly in skeletal muscle of Type 2 diabetic rats. Conclusion: T2D is associated with alterations in the expression of miRNAs in skeletal muscle. Both these miRNAs 194 and 135a play an important role in glucose metabolism in skeletal muscle of diabetic rats. Gallic acid effectively ameliorated the alterations in glucose metabolism. Hence, both these miRNAs can serve as biomarkers and therapeutic targets in diabetes mellitus. The study also establishes the role of gallic acid as therapeutic agent. Acknowledgment: The financial assistance provided in the form of ICMR women scientist by ICMR DHR INDIA is gratefully acknowledged here.

Keywords: gallic acid, high fat diet, type 2 diabetes mellitus, miRNAs

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Authors: Iman Farasat, Howard M. Salis

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The CRISPR/Cas9 system has revolutionized genome engineering by enabling site-directed and high-throughput genome editing, genome insertion, and gene knockdowns in several species, including bacteria, yeast, flies, worms, and human cell lines. This technology has the potential to enable human gene therapy to treat genetic diseases and cancer at the molecular level; however, the current CRISPR/Cas9 system suffers from seemingly sporadic off-target genome mutagenesis that prevents its use in gene therapy. A comprehensive mechanistic model that explains how the CRISPR/Cas9 functions would enable the rational design of the guide-RNAs responsible for target site selection while minimizing unexpected genome mutagenesis. Here, we present the first quantitative model of the CRISPR/Cas9 genome mutagenesis system that predicts how guide-RNA sequences (crRNAs) control target site selection and cleavage activity. We used statistical thermodynamics and law of mass action to develop a five-step biophysical model of cas9 cleavage, and examined it in vivo and in vitro. To predict a crRNA's binding specificities and cleavage rates, we then compiled a nearest neighbor (NN) energy model that accounts for all possible base pairings and mismatches between the crRNA and the possible genomic DNA sites. These calculations correctly predicted crRNA specificity across 5518 sites. Our analysis reveals that cas9 activity and specificity are anti-correlated, and, the trade-off between them is the determining factor in performing an RNA-mediated cleavage with minimal off-targets. To find an optimal solution, we first created a scheme of safe-design criteria for Cas9 target selection by systematic analysis of available high throughput measurements. We then used our biophysical model to determine the optimal Cas9 expression levels and timing that maximizes on-target cleavage and minimizes off-target activity. We successfully applied this approach in bacterial and mammalian cell lines to reduce off-target activity to near background mutagenesis level while maintaining high on-target cleavage rate.

Keywords: biophysical model, CRISPR, Cas9, genome editing

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Authors: Ricardo Alberto Chiong Zevallos, Eduardo Moraes Rego Reis

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Pancreatic adenocarcinoma (PDAC) patients have a less than 10% 5-year survival rate. PDAC has no defined diagnostic and prognostic biomarkers. Gemcitabine is the first-line drug in PDAC and several other cancers. Long non-coding RNAs (lncRNAs) contribute to the tumorigenesis and are potential biomarkers for PDAC. Although lncRNAs aren’t translated into proteins, they have important functions. LncRNAs can decoy or recruit proteins from the epigenetic machinery, act as microRNA sponges, participate in protein translocation through different cellular compartments, and even promote chemoresistance. The chromatin remodeling enzyme EZH2 is a histone methyltransferase that catalyzes the methylation of histone 3 at lysine 27, silencing local expression. EZH2 is ambivalent, it can also activate gene expression independently of its histone methyltransferase activity. EZH2 is overexpressed in several cancers and interacts with lncRNAs, being recruited to a specific locus. EZH2 can be recruited to activate an oncogene or silence a tumor suppressor. The lncRNAs misregulation in cancer can result in the differential recruitment of EZH2 and in a distinct epigenetic landscape, promoting chemoresistance. The relevance of the EZH2-lncRNAs interaction to chemoresistant PDAC was assessed by Real Time quantitative PCR (RT-qPCR) and RNA Immunoprecipitation (RIP) experiments with naïve and gemcitabine-resistant PDAC cells. The expression of several lncRNAs and EZH2 gene targets was evaluated contrasting naïve and resistant cells. Selection of candidate genes was made by bioinformatic analysis and literature curation. Indeed, the resistant cell line showed higher expression of chemoresistant-associated lncRNAs and protein coding genes. RIP detected lncRNAs interacting with EZH2 with varying intensity levels in the cell lines. During RIP, the nuclear fraction of the cells was incubated with an antibody for EZH2 and with magnetic beads. The RNA precipitated with the beads-antibody-EZH2 complex was isolated and reverse transcribed. The presence of candidate lncRNAs was detected by RT-qPCR, and the enrichment was calculated relative to INPUT (total lysate control sample collected before RIP). The enrichment levels varied across the several lncRNAs and cell lines. The EZH2-lncRNA interaction might be responsible for the regulation of chemoresistance-associated genes in multiple cancers. The relevance of the lncRNA-EZH2 interaction to PDAC was assessed by siRNA knockdown of a lncRNA, followed by the analysis of the EZH2 target expression by RT-qPCR. The chromatin immunoprecipitation (ChIP) of EZH2 and H3K27me3 followed by RT-qPCR with primers for EZH2 targets also assess the specificity of the EZH2 recruitment by the lncRNA. This is the first report of the interaction of EZH2 and lncRNAs HOTTIP and PVT1 in chemoresistant PDAC. HOTTIP and PVT1 were described as promoting chemoresistance in several cancers, but the role of EZH2 is not clarified. For the first time, the lncRNA LINC01133 was detected in a chemoresistant cancer. The interaction of EZH2 with LINC02577, LINC00920, LINC00941, and LINC01559 have never been reported in any context. The novel lncRNAs-EZH2 interactions regulate chemoresistant-associated genes in PDAC and might be relevant to other cancers. Therapies targeting EZH2 alone weren’t successful, and a combinatorial approach also targeting the lncRNAs interacting with it might be key to overcome chemoresistance in several cancers.

Keywords: epigenetics, chemoresistance, long non-coding RNAs, pancreatic cancer, histone modification

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Authors: Giorgio Bertolazzi, Panayiotis Benos, Michele Tumminello, Claudia Coronnello

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MicroRNAs are small non-coding RNAs that post-transcriptionally regulate the expression levels of messenger RNAs. MicroRNA regulation activity depends on the recognition of binding sites located on mRNA molecules. ComiR (Combinatorial miRNA targeting) is a user friendly web tool realized to predict the targets of a set of microRNAs, starting from their expression profile. ComiR incorporates miRNA expression in a thermodynamic binding model, and it associates each gene with the probability of being a target of a set of miRNAs. ComiR algorithms were trained with the information regarding binding sites in the 3’UTR region, by using a reliable dataset containing the targets of endogenously expressed microRNA in D. melanogaster S2 cells. This dataset was obtained by comparing the results from two different experimental approaches, i.e., inhibition, and immunoprecipitation of the AGO1 protein; this protein is a component of the microRNA induced silencing complex. In this work, we tested whether including coding region binding sites in the ComiR algorithm improves the performance of the tool in predicting microRNA targets. We focused the analysis on the D. melanogaster species and updated the ComiR underlying database with the currently available releases of mRNA and microRNA sequences. As a result, we find that the ComiR algorithm trained with the information related to the coding regions is more efficient in predicting the microRNA targets, with respect to the algorithm trained with 3’utr information. On the other hand, we show that 3’utr based predictions can be seen as complementary to the coding region based predictions, which suggests that both predictions, from 3'UTR and coding regions, should be considered in a comprehensive analysis. Furthermore, we observed that the lists of targets obtained by analyzing data from one experimental approach only, that is, inhibition or immunoprecipitation of AGO1, are not reliable enough to test the performance of our microRNA target prediction algorithm. Further analysis will be conducted to investigate the effectiveness of the tool with data from other species, provided that validated datasets, as obtained from the comparison of RISC proteins inhibition and immunoprecipitation experiments, will be available for the same samples. Finally, we propose to upgrade the existing ComiR web-tool by including the coding region based trained model, available together with the 3’UTR based one.

Keywords: AGO1, coding region, Drosophila melanogaster, microRNA target prediction

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Authors: Tianqi Yu, Yingnan Ding, Yina Zhang, Yulan Liu, Yahui Li, Jing Lei, Jiyong Zhou, Suquan Song, Boli Hu

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Circular RNAs (circRNAs) play critical roles in various diseases. However, whether and how circular RNA regulates influenza A virus (IAV) infection is unknown. Here, we studied the role of circular RNA GATA Zinc Finger Domain Containing 2A (circ-GATAD2A) in the replication of IAV H1N1 in A549 cells. Circ-GATAD2A was formed upon H1N1 infection. Knockdown of circ-GATAD2A in A549 cells enhanced autophagy and inhibited H1N1 replication. By contrast, overexpression of circ-GATAD2A impaired autophagy and promoted H1N1 replication. Similarly, knockout of vacuolar protein sorting 34 (VPS34) blocked autophagy and increased H1N1 replication. However, the expression of circ-GATAD2A could not further enhance H1N1 replication in VPS34 knockout cells. Collectively, these data indicated that circ-GATAD2A promotes the replication of H1N1 by inhibiting autophagy.

Keywords: autophagy, circ-GATAD2A, H1N1, replication

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Authors: Narin Salehiyan, Ramin Ghasemi Shayan

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In order to investigate coronavirus RNA replication, transcription, recombination, protein processing and transport, virion assembly, the identification of coronavirus-specific cell receptors, and polymerase processing, the manipulation of coronavirus clones and complementary DNAs (cDNAs) of defective-interfering (DI) RNAs is the subject of this chapter. The idea of the Covid genome is nonsegmented, single-abandoned, and positive-sense RNA. When compared to other RNA viruses, its size is significantly greater, ranging from 27 to 32 kb. The quality encoding the enormous surface glycoprotein depends on 4.4 kb, encoding a forcing trimeric, profoundly glycosylated protein. This takes off exactly 20 nm over the virion envelope, giving the infection the appearance-with a little creative mind of a crown or coronet. Covid research has added to the comprehension of numerous parts of atomic science as a general rule, like the component of RNA union, translational control, and protein transport and handling. It stays a fortune equipped for creating startling experiences.

Keywords: covid-19, corona, virus, genome, genetic

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Authors: Mateen A. Khan, Elizabeth J. Theil, Dixie J. Goss

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Cellular iron homeostasis is accomplished by the coordinated regulated expression of iron uptake, storage, and export. Iron regulate the translation of ferritin and mitochondrial aconitase iron responsive element (IRE)-mRNA by interaction with an iron regulatory protein (IRPs). Iron increases protein biosynthesis encoded in iron responsive element. The noncoding structure IRE-mRNA, approximately 30-nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. Fluorescence anisotropy measurements showed the presence of one binding site on IRP1 for ferritin and mitochondrial aconitase IRE-mRNA. Scatchard analysis revealed the binding affinity (Kₐ) and average binding sites (n) for ferritin and mitochondrial aconitase IRE-mRNA were 68.7 x 10⁶ M⁻¹ and 9.2 x 10⁶ M⁻¹, respectively. In order to understand the relative importance of equilibrium and stability, we further report the contribution of electrostatic interactions in the overall binding of two IRE-mRNA with IRP1. The fluorescence quenching of IRP1 protein was measured at different ionic strengths. The binding affinity of IRE-mRNA to IRP1 decreases with increasing ionic strength, but the number of binding sites was independent of ionic strength. Such results indicate a differential contribution of electrostatics to the interaction of IRE-mRNA with IRP1, possibly related to helix bending or stem interactions and an overall conformational change. Selective destabilization of ferritin and mitochondrial aconitase RNA/protein complexes as reported here explain in part the quantitative differences in signal response to iron in vivo and indicate possible new regulatory interactions.

Keywords: IRE-mRNA, IRP1, binding, ionic strength

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Authors: Sachin Mulmi Shrestha, Xin Fang, Hui Ye, Lihua Ren, Qinghua Ji, Ruihua Shi

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Background: Esophageal squamous cell carcinoma (ESCC) is a tumor arising from esophageal epithelial cells and is one of the major disease subtype in Asian countries, including China. Esophageal cancer is the 7th highest incidence based on the 2020 data of GLOBOCAN. The pathogenesis of cancer is still not well understood as many molecular and genetic basis of esophageal carcinogenesis has yet to be clearly elucidated. Circular RNAs are RNA molecules that are formed by back-splicing covalently joined 3′- and 5′-endsrather than canonical splicing, and recent data suggest circular RNAs could sponge miRNAs and are enriched with functional miRNA binding sites. Hence, we studied the mechanism of circular RNA, its biological function, and the relationship between microRNA in the carcinogenesis of ESCC. Methods: 4 pairs of normal and esophageal cancer tissues were collected in Zhongda hospital, affiliated to Southeast University, and high-throughput RNA sequencing was done. The result revealed that circ_0032746 was upregulated, and thus we selected circ_0032746 for further study. The backsplice junction of circRNA was validated by sanger sequence, and stability was determined by RNASE R assay. The binding site of circRNA and microRNA was predicted by circinteractome,mirandaand RNAhybrid database. Furthermore, circRNA was silenced by siRNA and then by lentivirus. The regulatory axis of circ0032746/miR4270 was validated by shRNA, mimic, and inhibitor transfection. Then, in vitro experiments were performed to assess the role of circ0032746 on proliferation (CCK-8 assay and colon formation assay), migration and invasion (Transewell assay), and apoptosis of ESCC. Results: The upregulated circ0032746 was validated in 9 pairs of tissues and 5 types of cell lines by qPCR, which showed high expression and was statistically significant (P<0.005) ). Upregulated circ0032746 was silenced by shRNA, which showed significant knockdown in KYSE 30 and TE-1 cell lines expression compared to control. Nuclear and cytoplasmic mRNA fraction experiment displayed the cytoplasmic location of circ0032746. The sponging of miR4270 was validated by co-transfection of sh-circ0032746 and mimic or inhibitor. Transfection with mimic showed the decreased expression of circ_0032746, whereas inhibitor inhibited the result. In vitro experiments showed that silencing of circ_0032746 inhibited the proliferation, migration, and invasion compared to the negative control group. The apoptosis was seen higher in a knockdown group than in the control group. Furthermore, 11 common mircoRNA target mRNAs were predicted by Targetscan, MirTarbase, and miRanda database, which may further play role in the pathogenesis. Conclusion: Our results showed that novel circ_0032746 is upregulated in ESCC and plays role in itsoncogenicity. Silencing of circ_0032746 inhibits the proliferation and migration of ESCC whereas increases the apoptosis of cancer cells. Hence, circ0032746 acts as an oncogene in ESCC by sponging with miR4270 and could be a potential biomarker in the diagnosis of ESCC in the future.

Keywords: circRNA, esophageal squamous cell carcinoma, microRNA, upregulated

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Authors: Doaa Hashad, Amany Elbanna, Abeer Ibrahim, Gihan Khedr

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Background: H19 is one of the long non coding RNAs (LncRNA) that is related to the progression of many diseases including cancers. This work was carried out to study the level of the long non-coding RNA; H119, in plasma of patients with gastric cancer (GC) and to assess its significance in their clinical management. Methods: A total of sixty-two participants were enrolled in the present study. The first group included thirty-two GC patients, while the second group was formed of thirty age and sex matched healthy volunteers serving as a control group. Plasma samples were used to assess H19 gene expression using real time quantitative PCR technique. Results: H19 expression was up-regulated in GC patients with positive correlation to TNM cancer stages. Conclusions: Up-regulation of H19 is closely associated with gastric cancer and correlates well with tumor staging. Convenient, efficient quantification of H19 in plasma using real time PCR technique implements its role as a potential noninvasive prognostic biomarker in gastric cancer, that predicts patient’s outcome and most importantly as a novel target in gastric cancer treatment with better performance achieved on using both CEA and H19 simultaneously.

Keywords: biomarker, gastric, cancer, LncRNA

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Authors: Sheng-Hui Lan, Shan-Ying Wu, Shu-Ching Lin, Wei-Chen Wang, Hsiao-Sheng Liu

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Autophagy is an essential mechanism to maintain cellular homeostasis through its degradation function, and the autophagy deficiency is related various diseases including tumorigenesis in several cancers. MicroRNAs (miRNAs) are small none coding RNAs, which regulate gene expression through degradation of mRNA or inhibition of translation. However, the relationship between autophagy deficiency and dysregulated miRNAs is still unclear. We revealed a mechanism that autophagy up-regulates miR-449a expression at the transcriptional level through activation of forkhead transcription factor family member FoxO1 and then suppresses tumorigenesis in CRC. Our data showed that the autophagic activity and miR-449a expression were lower in colorectal cancer (CRC) and has a positive correlation. We further reveal that autophagy degrades p300 expression and then suppresses acetylation of FoxO1. Under autophagic induction conditions, FoxO1 is transported from the cytoplasm to the nucleus and binds to the miR-449a promoter and then promotes miR-449a expression. In addition, either miR-449a overexpression or amiodarone-induced autophagy inhibits cell cycle progression, proliferation, colony formation migration, invasion, and tumor formation of SW480 cells. Our findings indicate that autophagy inducers may have the potential to be used for prevention and treatment of CRC through upregulation of miR-449a expression.

Keywords: autophagy, MiR-449a, FoxO1, colorectal cancer

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Authors: Vanja Misic, Mohamed El-Mogy, Yousef Haj-Ahmad

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Endonuclease G (EndoG) is a well conserved mitochondrio-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from non-viral DNA vectors in gene therapy efforts.

Keywords: EndoG, silencing, exogenous DNA stability, HeLa cells

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Authors: Ghada Badr, Arwa Alturki

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The biological function of an RNA molecule depends on its structure. The objective of the alignment is finding the homology between two or more RNA secondary structures. Knowing the common functionalities between two RNA structures allows a better understanding and a discovery of other relationships between them. Besides, identifying non-coding RNAs -that is not translated into a protein- is a popular application in which RNA structural alignment is the first step A few methods for RNA structure-to-structure alignment have been developed. Most of these methods are partial structure-to-structure, sequence-to-structure, or structure-to-sequence alignment. Less attention is given in the literature to the use of efficient RNA structure representation and the structure-to-structure alignment methods are lacking. In this paper, we introduce an O(N2) Component-based Pairwise RNA Structure Alignment (CompPSA) algorithm, where structures are given as a component-based representation and where N is the maximum number of components in the two structures. The proposed algorithm compares the two RNA secondary structures based on their weighted component features rather than on their base-pair details. Extensive experiments are conducted illustrating the efficiency of the CompPSA algorithm when compared to other approaches and on different real and simulated datasets. The CompPSA algorithm shows an accurate similarity measure between components. The algorithm gives the flexibility for the user to align the two RNA structures based on their weighted features (position, full length, and/or stem length). Moreover, the algorithm proves scalability and efficiency in time and memory performance.

Keywords: alignment, RNA secondary structure, pairwise, component-based, data mining

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Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

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Authors: M. Gogniashvili, I. Maisaia, A. Kotorashvili, N. Kotaria, T. Beridze

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Three types of plasmon (A, B and G) is typical for genus Triticum. In polyploid species - Triticum turgidum L. and Triticum aestivum L. plasmon B is detected. In the forthcoming paper, complete nucleotide sequence of chloroplast DNA of 11 representatives of Georgian wheat polyploid species, carrying plasmon B was determined. Sequencing of chloroplast DNA was performed on an Illumina MiSeq platform. Chloroplast DNA molecules were assembled using the SOAPdenovo computer program. All contigs were aligned to the reference chloroplast genome sequence using BLASTN. For detection of SNPs and Indels and phylogeny tree construction computer programs Mafft and Blast were used. Using Triticum aestivum L. subsp. macha (Dekapr. & Menabde) Mackey var. paleocolchicum Dekapr. et Menabde as a reference, 5 SNPs can be identified in chloroplast DNA of Georgian endemic polyploid wheat. The number of noncoding substitutions is 2, coding substitutions - 3. In comparison with reference DNA two - 38 bp and 56 bp inversions were observed in paleocolchicum subspecies. There were six 1 bp indels detected in Georgian polyploid wheats, all of them at microsatellite stretches. The phylogeny tree shows that subspecies macha, carthlicum and paleocolchicum occupy different positions. According to the simplified scheme based on SNP and indel data, the ancestral, female parent of the all studied polyploid wheat is unknown X predecesor, from which four lines were formed. 1 SNP and two inversions (38 bp and 56 bp) caused the formation of subsp. paleocolchicum. Three other lines are macha, durum and carthlicum lines. Macha line is further divided into two sublines (M_1 and M_4). Carthlicum line includes subsp.carthlicum and T.aestivum - C_1 - C_2 - A_1. One of the central question of wheat domestication is which people(s) participated in wheat domestication? It is proposed that the predecessors of Georgian peoples (Proto-Kartvelians) must be placed, on the evidence of archaic lexical and toponymic data, in the mountainous regions of the western and central part of the Little Caucasus (the Transcaucasian foothills) at least 4,000 years ago. One of the possibility to explain the ‘wheat puzzle’ is that Kartvelian speakers brought domesticated wheat species and subspecis from Fertile Crescent further north to South Caucasus.

Keywords: chloroplast DNA, sequencing, SNP, triticum

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Authors: Rozhgar A. Khailany, Mehri Igci, Emine Bayraktar, Sakip Erturhan, Metin Karakok, Ahmet Arslan

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Kidney cancer is the most lethal urological cancer accounting for 3% of adult malignancies. VHL, a tumor-suppressor gene, is best known to be associated with renal cell carcinoma (RCC). The VHL functions as negative regulator of hypoxia inducible factors. Recent sequencing efforts have identified several novel frequent mutations of histone modifying and chromatin remodeling genes in ccRCC (clear cell RCC) including PBRM1 and SETD2. The PBRM1 gene encodes the BAF180 protein, which involved in transcriptional activation and repression of selected genes. SETD2 encodes a histone methyltransferase, which may play a role in suppressing tumor development. In this study, RNAs of 30 paired tumor and normal samples that were grouped according to the types of kidney cancer and clinical characteristics of patients, including gender and average age were examined by RT-PCR, SSCP and sequencing techniques. VHL, PBRM1 and SETD2 expressions were relatively down-regulated. However, statistically no significance was found (Wilcoxon signed rank test, p > 0.05). Interestingly, no mutation was observed on the contrary of previous studies. Understanding the molecular mechanisms involved in the pathogenesis of RCC has aided the development of molecular-targeted drugs for kidney cancer. Further analysis is required to identify the responsible genes rather than VHL, PBRM1 and SETD2 in kidney cancer.

Keywords: kidney cancer, molecular biomarker, expression analysis, mutation screening

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Authors: Zainab S. Ahmed, Mohammed S. Ali, Nadia A. Elshiekh, Sami Adam Ibrahim, Ghada M. El-Tayeb, Ahmed H. Elsadig, Rihab A. Omer, Sofia B. Mohamed

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Maple syrup urine (MSUD) is an autosomal recessive disease that causes a deficiency in the enzyme branched-chain alpha-keto acid (BCKA) dehydrogenase. The development of disease has been associated with SNPs in the DBT gene. Despite that, the computational analysis of SNPs in coding and noncoding and their functional impacts on protein level still remains unknown. Hence, in this study, we carried out a comprehensive in silico analysis of missense that was predicted to have a harmful influence on DBT structure and function. In this study, eight different in silico prediction algorithms; SIFT, PROVEAN, MutPred, SNP&GO, PhD-SNP, PANTHER, I-Mutant 2.0 and MUpo were used for screening nsSNPs in DBT including. Additionally, to understand the effect of mutations in the strength of the interactions that bind protein together the ELASPIC servers were used. Finally, the 3D structure of DBT was formed using Mutation3D and Chimera servers respectively. Our result showed that a total of 15 nsSNPs confirmed by 4 software (R301C, R376H, W84R, S268F, W84C, F276C, H452R, R178H, I355T, V191G, M444T, T174A, I200T, R113H, and R178C) were found damaging and can lead to a shift in DBT gene structure. Moreover, we found 7 nsSNPs located on the 2-oxoacid_dh catalytic domain, 5 nsSNPs on the E_3 binding domain and 3 nsSNPs on the Biotin Domain. So these nsSNPs may alter the putative structure of DBT’s domain. Furthermore, we detected all these nsSNPs are on the core residues of the protein and have the ability to change the stability of the protein. Additionally, we found W84R, S268F, and M444T have high significance, and they affected Leucine, Isoleucine, and Valine, which reduces or disrupt the function of BCKD complex, E2-subunit which the DBT gene encodes. In conclusion, based on our extensive in-silico analysis, we report 15 nsSNPs that have possible association with protein deteriorating and disease-causing abilities. These candidate SNPs can aid in future studies on Maple Syrup Urine Disease type II base in the genetic level.

Keywords: DBT gene, ELASPIC, in silico analysis, UCSF chimer

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Authors: Piotr J. Balwierz, Mikhail Pachkov, Phil Arnold, Andreas J. Gruber, Mihaela Zavolan, Erik van Nimwegen

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Understanding the key players and interactions in the regulatory networks that control gene expression and chromatin state across different cell types and tissues in metazoans remains one of the central challenges in systems biology. Our laboratory has pioneered a number of methods for automatically inferring core gene regulatory networks directly from high-throughput data by modeling gene expression (RNA-seq) and chromatin state (ChIP-seq) measurements in terms of genome-wide computational predictions of regulatory sites for hundreds of transcription factors and micro-RNAs. These methods have now been completely automated in an integrated webserver called ISMARA that allows researchers to analyze their own data by simply uploading RNA-seq or ChIP-seq data sets and provides results in an integrated web interface as well as in downloadable flat form. For any data set, ISMARA infers the key regulators in the system, their activities across the input samples, the genes and pathways they target, and the core interactions between the regulators. We believe that by empowering experimental researchers to apply cutting-edge computational systems biology tools to their data in a completely automated manner, ISMARA can play an important role in developing our understanding of regulatory networks across metazoans.

Keywords: gene expression analysis, high-throughput sequencing analysis, transcription factor activity, transcription regulation

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