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Dr. Scott Martin, Team Leader for RNAi Screening at NIH’s Chemical Genomics Center, to Present “Swimming in the Deep End – Sources Leading to a False Sense of Security in RNAi Screen Data” at Functional Genomics Screening Strategies Conference
There has been a growing skepticism surrounding RNAi data and the validity of hits arising from largescale RNAi screens. Much of this comes from a lack of agreement between screens conducted in similar biological systems and difficulty in validating published screen hits. In light of these realities, we must rethink some widely held beliefs about screening and validation strategies. These issues and relevant data will be discussed.
Functional Genomics Screening Strategies: Part Two
Exploring Novel Screening Platforms and Cellular
Models for Next-Generation Screens
Boston, MA | September 25-26, 2013
The second half of Functional Genomics Screening Strategies will explore the use of chemical genomics screens, microRNA (miRNA) and long non-coding RNA (lncRNA) screens and the transition into advanced cellular models such as, 3D cell cultures, co-cultures and stem cells that will launch the next generation of functional screens. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of such diverse screening platforms and models for a wide range of applications.
John Rinn – Genomic Garbage Man (Photo credit: ChimpLearnGood)
DNA: One man’s trash is another man’s treasure, but there is no JUNK after all
Author: Demet Sag, PhD
One man’s trash is another man’s treasure, but there is no JUNK after all:
The JUNK has a meaning
Long non-coding RNAs recognized after transcriptome sequencing and studied more closely recently thanks to genomic tiling arrays, cDNA sequencing and RNA-Seq, which they have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. How many are there in the genome? What are their mechanisms? How can we use them in molecular diagnostics and targeted therapies? How do they effect the function in a disease? Is it possible to modulate gene expression at the level of stem cell to redirect the cell differentiation? These are the main questions that we are looking for.
In early 90s actually first lincRNA was described, Xist. The main function was dosage compensation. Then in 2000s FANTOM consortium project changed the perspective on these long transcripts. Then they are called natural antisense transcripts (NATs), because very large number of these transcripts is overlapping with, and is transcribed in the antisense direction, to protein-coding genes. As a result of this study 11000 lincRNA discovered from full length cDNAs in mice. Later, yet another shift occur since these transcribed units are solely located in the introns or within “junk” DNA of protein-coding genes. Another independent study quantified that about 40% of protein-coding genes express NATs. Proven that there is nothing junk about DNA. Then, it was found that there are 8000 lincRNAs and among these 4000 are determined since they provide cell identity with multi-exogenic, polyadenylated, capped, ether in the cytoplasm or in the nucleus. However, even more recent studies show that there are about 20,000 lincRNAs. Furthermore, lincRNAs are classified under three distinct class: 1. Long-non-coding RNAs away from protein-coding genes, 2 NATs transcribed from the opposite strand of protein-coding genes, 3. Intronic lincRNAs expressed from within the introns of protein coding genes.
The human genome, categorized by function of each gene product, given both as number of genes and as percentage of all genes. (Photo credit: Wikipedia)
Their function is under study. However, keep in mind that they are redundant, so deleting or creating null mutations may or may not answer specific development questions. On the other hand, epigenetics, gene imprinting, and pathologies can be the best resource to identify their specific roles in biological functions and interactions. Distinct gene regulation either as a cis or trans element, gene imprinting, modulating alternative splicing, nuclear organization, determining a chromatin structure are under study. This will allow us to relate genome structure and function in health and disease better. Identification of their function during biological responses require a long way to be completed due to complexity since lincRNAs also regulate microRNAs. Regardless of many obstacles there is a progress. Disregulation of these lincRNA mainly observed in several cancer types, prostate, breast, hepatocellular carcinoma, colorectal, glioma and melanoma, possibly more. Most of the studies are done in vitro. However, there are many great model organism work as well, such as mice, zebra fish, and worm.
It was also not surprising that their regulation possibly under control of hormones based on circadian clock of our body. So better to sleep eight hour a day is not a cliché.
Next topic will include understanding of lincRNA mechanisms and epigenetics followed by lincRNAs during disease and cellular genesis.
… therapies in a variety of animal models and contributed to regulatory CMC and IND-enabling safety and toxicology studies for inclusion in … sequences sort a-cardiac and b-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J. Cell. Biol., …
… and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in … articles What about Circular RNAs? (pharmaceuticalintelligence.com) How Genes Function …
… due to their broad scope and non-specificity in the human genome. “I am extremely pro-patent, but I simply believe that people … believe that individuals have an innate right to their own genome, or to allow their doctor to look at that genome, just like the lungs or …
… Intelligence http://pharmaceuticalintelligence.com/2013/03/02/genome-sequenc…of-the-healthy/ Key Issues in Genome Sequencing of Healthy Individuals Eric Topol, MD, Genomic Medicine I … touching on important controversies in the use of whole genome …
… of recombination is highly uneven across the human genome, as in all studied organisms. Substantial recombination active regions … this variation would require comparison of recombination genome-wide among many single genomes. Whole-genome amplification (WGA) of …
… Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD Putting Genome Interpretation to the Test 01/30/2013 Ashley Yeager How well do methods for interpreting genome variation work? Ashley Yeager takes a look at a community experiment that is trying to assess just how useful genome interpretation tools in real-world situations. At the American …
… genomes — through the end of this year, National Human Genome Research Institute estimates indicate. And in his book, The Creative … the interpretation of an apparently healthy person’s genome and that of an individual who is already affected by a disease, whether …
… scary findings: the tale of John Lauerman’s whole genome sequencing FEBRUARY 15, 2012 Joe Thakuria draws John Lauerman’s blood for whole genome sequencing. By Madeleine Price Ball, licensed under … scary findings: the tale of John Lauerman’s whole genome sequencing » Joe Thakuria draws John …
… 2: LEADERS in the Competitive Space of Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in … Treatment http://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer- …
Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single … of DNA replication and the ability to amplify a whole genome. The amplicons are then sequenced either by whole-genome sequencing methods using Sanger-sequencing to verify any single …
… Aviva Lev-Ari, RN Genome Biol. 2012 Dec 13;13(12):R115. [Epub ahead of print] Whole-genome reconstruction and mutational signatures in gastric cancer. Nagarajan … read and DNA-PET sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal …
… by interpreting the mathematical patterns in the cancer genome. Researchers at the University of Oslo, Norway (UiO) have developed a … Hospital and UiO. Finds the changed patterns in the genome There is much talk about finding the special cancer gene. In reality, …
“The SILENCE of the Lambs” Introducing The Power of Uncoded RNA
Curator: Demet Sag, PhD
Word Cloud By Danielle Smolyar
An illustration of the central dogma of molecular biology annotated with the processes ncRNAs are involved in. (Photo credit: Wikipedia)
X-ray structure of the tRNA Phe from yeast. Data was obtained by PDB 1ehz and rendered with PyMOL. violet: acceptor stem wine red: D-loop blue: anticodon loop orange: variable loop green: TPsiC-loop yellow: CCA-3′ of the acceptor stem grey: anticodon (Photo credit: Wikipedia)
Our genome must be packed tightly to fit into the nucleus. Genome is the blue print of a living organism whether made up off a single or multiple cell. Recently, the genome seen as a functional network of physical contacts within (cis) and between (trans) chromosomes. It became necessary to map these physical DNA contacts at high-resolution with technologies such as the “chromosome conformation capture” (3C) and other 3C-related methods including 3C-Carbon Copy (5C) and Hi-C. Yet, we all know that in vivo conformation, gene to gene interactions from a long distance, histones and 3D have an impact on gene regulation and expression. The game is not just a sequence but functional genomics with a correct translation of sequence for development so that proper molecular diagnostics can be applied not only for prevention but also for monitoring the efficacy of the intervention. Thus, we can provide a targeted therapy for personalized medicine.
On the other hand, we still know very little about genome organization at the molecular level, although spatial genome organization can critically affect gene expression. It is important to recognize who is there to be present and who is there to create the functional impact for regulation in a specific tissue and time. In addition, mediation of these chromatin contacts based on a specific tissue is quite essential. For example, during long-range control mechanism specific enhancers and distal promoters needed to be invited to a close physical proximity to each other by transcription factors that has been found at other loci. Furthermore, chromatin-binding proteins such as the CCCTC-binding factor (CTCF) and cohesin seem to have critical roles in genome organization and gene expression. Let’s not forget about epigenetics, since there are so many methods to regulate chromatin interactions like cytosine methylation, maternal gene, gradient level, post-translational modifications and non-coding RNAs.
The non-coding RNAs (ncRNAs) are silent but they have the 99% power because ncRNAs are a broad class of transcripts consisting of structural (rRNAs, tRNAs, snRNAs, snoRNAs, etc.), regulatory (miRNAs, piRNAs, etc.), and of sense/antisense transcripts. Among these an interesting class is the latter group. This class includes transcriptional “features” (eRNAs, tiRNAs), and a very large number of long non-coding RNAs (lncRNAs), length from 200 nt to 100 kb. The magnificent future of lncRNAs comes from their production, as they can be transcribed nearby known protein-coding genes or from their introns. As a result, because of their intergenical production they are also called as “lincRNAs (long intergenical non-coding RNAs). They are abundant and specific as microRNAs. Hence, their inclusion into the biomarker list and assuming their roles during targeted therapy don’t require us to be a wizard but a functional genomicist knowing evolution, development and molecular genetics and plus signaling.
lincRNA can both activate and repress the gene either cis or trans acting to effect gene regulation will be discussed next.
As a result, one gene expression regulation needs from twenty to several hundred genes. As they say raising a child needs a village.
Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J. Science. 2009;326:289–293.
Folkersen L, Kyriakou T, Goel A, Peden J, Mälarstig A, Paulsson-Berne G, Hamsten A, Hugh Watkins, Franco-Cereceda A, Gabrielsen A, Eriksson P, PROCARDIS consortia
Heintzman ND, Hon GC, Hawkins RD, Kheradpour P, Stark A, Harp LF, Ye Z, Lee LK, Stuart RK, Ching CW, Ching KA, Antosiewicz-Bourget JE, Liu H, Zhang X, Green RD, Lobanenkov VV, Stewart R, Thomson JA, Crawford GE, Kellis M, Ren B. Nature. 2009 May 7; 459(7243):108-12.
O’Hara, Steven P.; Tabibian, James H.; Splinter, Patrick L.; LaRusso, Nicholas F. Journal of Hepatology vol. 58 issue 3 March, 2013. p. 575-582 DOI: 10.1016/j.jhep.2012.10.011. ISSN: 0168-8278
ABBREVIATIONS
3C = Chromosome conformation capture
rRNAs = Ribosomal RNAs
tRNAs = Transfer RNAs
snRNAs = Small nuclear RNAs
snoRNAs = Small nucleolar RNAs
miRNAs = MicroRNAs
piRNAs = Piwi-interacting RNAs
eRNAs = Enhancer RNAs
tiRNAs = Transcription initiation RNAs
spliRNAs = Splice-site RNAs
lincRNAs = Long intergenic non-coding RNAs
lncRNPs = Long non-coding ribonucleoprotein complexes
… noncoding RNA network regulates PTEN transcription Scientists Find Surprising New Influence On Cancer Genes A pseudogene long noncoding RNA networkregulates PTEN transcription
… Feb 17, 2013 Long segments of noncoding RNA are key to physically manipulating DNA in order to activate certain genes. These noncoding RNA-activators (ncRNA-a) have a crucial role in turning genes on and …
… The ENCODE project reveals that many genes encode for an RNA, not protein product, so called regulatory RNAs. However some of the … are capable of generating new insertions through
In the screening world there is definitely no one-size fits- all and no dearth of options to choose from in terms of assay platforms, protocols, cells or reagents. So how do you decide which screening strategy will work best for you? Can different screening techniques be utilized in tandem or be staggered to better validate results and overcome inherent shortcomings? Which type of screen will provide information that is most accurate and physiologically relevant to your biological query? Cambridge Healthtech Institute’s tenth annual conference on Functional Genomics Screening Strategies will cover the latest in the use of RNA interference (RNAi) screens, combination (RNAi and small molecule) screens, chemical genomics and phenotypic screens, for identifying and validating new drug targets and exploring unknown cellular pathways. The first half of the conference will focus on the design and use of RNAi screens, while the second half will explore the use of chemical genomics and long non-coding RNA (LncRNA) screens and the transition into advanced cellular models such as, 3D cell cultures and stem cells that will launch the next-generation of functional screens. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of such diverse screening platforms and models for a wide range of applications
UCSF researchers have uncovered a role in brain development and in neurological
disease for little appreciated molecules called long noncoding RNA. In this image,
fluorescent dyes track the presence of the RNA molecules and the genes they
affect in the developing mouse brain. Image courtesy of Alexander Ramos
Specific DNA once dismissed as junk plays an important role in brain development and might be involved in several devastating neurological diseases, UC San Francisco scientists have found.
Their discovery in mice is likely to further fuel a recent scramble by researchers to identify roles for long-neglected bits of DNA within the genomes of mice and humans alike.
While researchers have been busy exploring the roles of proteins encoded by the genes identified in various genome projects, most DNA is not in genes. This so-called junk DNA has largely been pushed aside and neglected in the wake of genomic gene discoveries, the UCSF scientists said.
In their own research, the UCSF team studies molecules called long noncoding RNA (lncRNA, often pronounced as “link” RNA), which are made from DNA templates in the same way as RNA from genes.
“The function of these mysterious RNA molecules in the brain is only beginning to be discovered,” said Daniel Lim, MD, PhD, assistant professor of neurological surgery, a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, and the senior author of the study, published online April 11 in the journal Cell Stem Cell.
Daniel Lim, MD, PhD
Alexander Ramos, a student enrolled in the MD/PhD program at UCSF and first author of the study, conducted extensive computational analysis to establish guilt by association, linking lncRNAs within cells to the activation of genes.
Ramos looked specifically at patterns associated with particular developmental pathways or with the progression of certain diseases. He found an association between a set of 88 long noncoding RNAs and Huntington’s disease, a deadly neurodegenerative disorder. He also found weaker associations between specific groups of long noncoding RNAs and Alzheimer’s disease, convulsive seizures, major depressive disorder and various cancers.
“Alex was the team member who developed this new research direction, did most of the experiments, and connected results to the lab’s ongoing work,” Lim said. The study was mostly funded through Lim’s grant – a National Institutes of Health (NIH) Director’s New Innovator Award, a competitive award for innovative projects that have the potential for unusually high impact.
LncRNA versus Messenger RNA
Unlike messenger RNA, which is transcribed from the DNA in genes and guides the production of proteins, lncRNA molecules do not carry the blueprints for proteins. Because of this fact, they were long thought to not influence a cell’s fate or actions.
Alexander Ramos
Nonetheless, lncRNAs also are transcribed from DNA in the same way as messenger RNA, and they, too, consist of unique sequences of nucleic acid building blocks.
Evidence indicates that lncRNAs can tether structural proteins to the DNA-containing chromosomes, and in so doing indirectly affect gene activation and cellular physiology without altering the genetic code. In other words, within the cell, lncRNA molecules act “epigenetically” — beyond genes — not through changes in DNA.
The brain cells that the scientists focused on the most give rise to various cell types of the central nervous system. They are found in a region of the brain called the subventricular zone, which directly overlies the striatum. This is the part of the brain where neurons are destroyed in Huntington’s disease, a condition triggered by a single genetic defect.
Ramos combined several advanced techniques for sequencing and analyzing DNA and RNA to identify where certain chemical changes happen to the chromosomes, and to identify lncRNAs on specific cell types found within the central nervous system. The research revealed roughly 2,000 such molecules that had not previously been described, out of about 9,000 thought to exist in mammals ranging from mice to humans.
In fact, the researchers generated far too much data to explore on their own. The UCSF scientists created a website through which their data can be used by others who want to study the role of lncRNAs in development and disease.
“There’s enough here for several labs to work on,” said Ramos, who has training grants from the California Institute for Regenerative Medicine (CIRM) and the NIH.
“It should be of interest to scientists who study long noncoding RNA, the generation of new nerve cells in the adult brain, neural stem cells and brain development, and embryonic stem cells,” he said.
Other co-authors who worked on the study include UCSF postdoctoral fellows Aaron Diaz, PhD, Abhinav Nellore, PhD, Michael Oldham, PhD, Jun Song, PhD, Ki-Youb Park, PhD, andGabriel Gonzales-Roybal, PhD; and MD/PhD student Ryan Delgado. Additional funders of the study included the Sontag Foundation and the Sandler Foundation.
Long Noncoding RNA Network regulates PTEN Transcription
Author: Larry H Bernstein, MD, FCAP
Scientists Find Surprising New Influence On Cancer Genes
A pseudogenelong noncoding RNA networkregulates PTEN transcription and translation in human cells
Per Johnsson, A Ackley, L Vidarsdottir, Weng-Onn Lui, M Corcoran, D Grandér, and KV Morris
Mol Cancer. 2011; 10: 38. Published online 2011 April 13. doi: 10.1186/1476-4598-10-38 PMCID: PMC3098824
New Type of Gene That Regulates Tumour Suppressor PTEN Identified
Feb. 24, 2013 — Researchers at Karolinska Institutet in Sweden have identified a new so-called pseudogene that regulates the tumour-suppressing PTEN gene.
They hope that this pseudogene will be able to control PTEN to
reverse the tumour process,
make the cancer tumour more sensitive to chemotherapy and
The development of tumours coincides with the activation of several cancer genes as well as the inactivation of other tumour-suppressing genes owing to
damage to the DNA and
to the fact that
the cancer cells manage to switch offthe transcription of tumour-suppressor genes.
To identify what might be regulating this silencing, the researchers studied PTEN,
one of the most commonly inactivated tumour-suppressor genes.
It has long been believed that the switching-off process is irreversible, but the team has now shown that
silenced PTEN genes in tumour cells can be ‘rescued’ and
re-activated by a ‘pseudogene’,
a type of gene that, unlike normal genes,
does not encode an entire protein.
“We identified a new non-protein encoding pseudogene, which
determines whether the expression of PTEN
is to be switched on or off,”
says research team member Per Johnsson, at Karolinska Institutet’s Department of Oncology-Pathology. “What makes this case spectacular is that the gene
only produces RNA,
the protein’s template.
It is this RNA that, through a sequence of mechanisms,
regulates PTEN.
Pseudogenes have been known about for many years, but
it was thought that they were only junk material.”
No less than 98 per cent of human DNA consists of non-protein encoding genes (i.e. pseudogenes), and by studying these formerly neglected genes the researchers
have begun to understand that they are very important and
can have an effect without encoding proteins.
Using model systems, the team has shown that the new pseudogene can
control the expression of PTEN and
make tumours more responsive to conventional chemotherapy.
Pre Johnssom suggests “we might one day be able to re-programme cancer cells
to proliferate less,
become more normal, and that
resistance to chemotherapy can hopefully be avoided.
“We also believe that our findings can be very important for the future development of cancer drugs. The human genome conceals no less than 15,000 or so pseudogenes, and it’s not unreasonable to think
that many of them are relevant to diseases such as cancer.”
The study was conducted in collaboration with scientists at The Scripps Research Institute, USA, and the University of New South Wales, Australia, and was made possible with
grants from the Swedish Childhood Cancer Foundation, the Swedish Cancer Society, the Cancer Research Funds of Radiumhemmet, Karolinska Institutet’s KID programme for doctoral studies, the Swedish Research Council, the Erik and Edith Fernström Foundation for Medical Research, the National Institute of Allergy and Infectious Diseases, the National Cancer Institute and the National Institutes of Health.
The functional role of long non-coding RNA in human carcinomas EA Gibb, CJ Brown, and WL Lam Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently
aberrantly expressed in a variety of human cancers,
however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into
the functional roles they may play in tumorigenesis.
In this review, we highlight the emerging functional role of lncRNAs in human cancer.
One of modern biology’s great surprises was the discovery that the human genome encodes only ~20,000 protein-coding genes, representing <2% of the total genome sequence [1,2]. However, with the advent of
tiling resolution genomic microarrays and
whole genome and transcriptome sequencing technologies
it was determined that at least 90% of the genome is actively transcribed [3,4].
The human transcriptome was found to be more complex than
a collection of protein-coding genes and their splice variants; showing
extensive antisense,
overlapping and non-coding RNA (ncRNA) expression [5-10].
Although initially argued to be spurious transcriptional noise, recent evidence suggests that the proverbial “dark matter” of the genome
may play a major biological role in cellular development and metabolism [11-17].
One such player, the newly discovered long non-coding RNA (lncRNA) genes, demonstrate
developmental and tissue specific expression patterns, and
aberrant regulation in a variety of diseases, including cancer [18-27].
NcRNAs are loosely grouped into two major classes based on transcript size; small ncRNAs and lncRNAs [28-30].
Small ncRNAs are represented by a broad range of known and newly discovered RNA species, with many being associated
with 5′ or 3′ regions of genes [4,31,32].
This class includes the well-documented miRNAs, RNAs ~22 nucleotides (nt) long involved in the specific regulation of both
protein-coding, and
putatively non-coding genes,
by post-transcriptional silencing or infrequently
by activation [33-35].
miRNAs serve as major
regulators of gene expression and as
intricate components of the cellular gene expression network [33-38].
Another newly described subclass are the transcription initiation RNAs (tiRNAs), which are
the smallest functional RNAs at only 18 nt in length [39,40].
small ncRNAs classes, including miRNAs, have established roles in tumorigenesis, an intriguing association between
the aberrant expression of ncRNA satellite repeats and cancer has been recently demonstrated [41-46].
Types of human non-coding RNAs
In contrast to miRNAs, lncRNAs, the focus of this article, are
mRNA-like transcripts ranging in length from 200 nt to ~100 kilobases (kb) lacking significant open reading frames.
Many identified lncRNAs are transcribed by RNA polymerase II (RNA pol II) and are polyadenylated, but this is not a fast rule [47,48].
There are examples of lncRNAs, such as the
antisense asOct4-pg5 or the
brain-associated BC200,
which are functional, but not polyadenylated [49-51].
lncRNA expression levels appear to be lower than protein-coding genes [52-55], and some
lncRNAs are preferentially expressed in specific tissues [21].
Novel lncRNAs may contribute a significant portion of the aforementioned ‘dark matter’ of the human transcriptome [56,57]. In an exciting report
by Kapranov et.al., it was revealed the bulk of the relative mass of RNA in a human cell, exclusive of the ribosomal and mitochondrial RNA,
is represented by non-coding transcripts with no known function [57].
Like miRNAs and protein-coding genes, some
transcriptionally active lncRNA genes display
histone H3K4 trimethylation at their 5′-end and
histone H3K36 trimethylation in the body of the gene [8,58,59].
The small number of characterized human lncRNAs have been associated with a spectrum of biological processes, for example,
epigenetics,
alternative splicing,
nuclear import,
as structural components,
as precursors to small RNAs and
even as regulators of mRNA decay [4,60-70].
Furthermore, accumulating reports of misregulated lncRNA expression across numerous cancer types suggest that
aberrant lncRNA expression may be a major contributor to tumorigenesis [71].
This surge in publications reflects the increasing attention to this subject and a number of useful lncRNA databases have been created .
In this review we highlight the emerging
functional role of aberrant lncRNA expression, including
transcribed ultraconserved regions (T-UCRs), within human carcinomas.
Publications describing cancer-associated ncRNAs. Entries are based on a National Library of Medicine Pubmed search using the terms
“ncRNA” or “non-coding RNA” or “noncoding RNA” or non-protein-coding RNA” with cancer and annual (Jan.1-Dec.31) date limitations. …
Publically available long non-coding RNA online databases
The definition ‘non-coding RNA’ is typically used to describe transcripts where
sequence analysis has failed to identify an open reading frame.
There are cases where ‘non-coding’ transcripts were found to encode short, functional peptides [72]. Currently, a
universal classification scheme to define lncRNAs does not exist. Terms such as
large non-coding RNA,
mRNA-like long RNA, and
intergenic RNA
all define cellular RNAs, exclusive of rRNAs,
greater than 200 nt in length and having no obvious protein-coding capacity [62].
This has led to confusion in the literature as to exactly which transcripts should constitute a lncRNA. One subclass of lncRNAs is called
large or long intergenic ncRNAs (lincRNAs). These lncRNAs are
exclusively intergenic and are
marked by a chromatin signature indicative of transcription [8,58].
RNA species that are bifunctional preclude categorization into either group of
protein-coding or
ncRNAs as
their transcripts function both at the RNA and protein levels [73].
The term ‘lncRNA‘ is used only to describe transcripts with no protein-coding capacity. In the meantime, and for the purposes of this review,
we will consider lncRNAs as a blanket term to encompass
mRNA-like ncRNAs,
lincRNAs, as well as
antisense and intron-encoded transcripts,
T-UCRs and
transcribed pseudogenes.
Discovery of LncRNAs
The earliest reports describing lncRNA predated the discovery of miRNAs, although the term ‘lncRNA‘ had not been coined at the time .
One of the first lncRNA genes reported was the imprinted H19 gene, which was quickly followed by the discovery of the
silencing X-inactive-specific transcript (XIST) lncRNA gene, which
plays a critical function in X-chromosome inactivation [74,75].
The discovery of the first miRNA lin-14 dramatically redirected the focus ofncRNA research from long ncRNAs to miRNAs [76], and
the discovery of miRNAs revealed RNA could
regulate gene expression and
entire gene networks could be affected by ncRNA expression and
Within the last decade miRNAs were discovered to be associated with cancer. At the time of this writing there are approximately
1049 human miRNAs described in miRBase V16 [80,81] with the potential of
affecting the expression of approximately 60% of protein -coding genes [82,83].
Conversely, the variety and dynamics of lncRNA expression was not to be fully appreciated until the introduction of whole transcriptome sequencing.
With the advent of the FANTOM and ENCODE transcript mapping projects, it was revealed that the mammalian genome is extensively transcribed,
although a large portion of this represented non-coding sequences [3,84]. Coupled with the novel functional annotation of a few lncRNAs, this discovery
promoted research focusing on lncRNAdiscovery and characterization. Recent reports have described new lncRNA classes such as lincRNAs and T-UCRs [8,58,85].
Current estimates of the lncRNA gene content in the human genome ranges from ~7000 – 23,000 unique lncRNAs, implying this class of ncRNA will
represent an enormous, yet undiscovered, component of normal cellular networks that may be disrupted in cancer biology [62].
Emerging Role of Long Non-Coding RNA in Tumorigenesis
A role for differential lncRNA expression in cancer had been suspected for many years, however, lacked strong supporting evidence [86]. With advancements
in cancer transcriptome profiling and accumulating evidence supporting lncRNA function, a number of differentially expressed lncRNAs have been associated
with cancer. LncRNAs have been implicated to
regulate a range of biological functions and
the disruption of some of these functions, such as
genomic imprinting and transcriptional regulation,
plays a critical role in cancer development.
Here we describe some of the better characterized lncRNAs that have been associated with cancer biology.
Human cancer-associated lncRNAs
Imprinted lncRNA genes
Imprinting is a process whereby the copy of a gene inherited from one parent is epigenetically silenced [87,88]. Intriguingly, imprinted regions often
include multiple maternal and paternally expressed genes with a high frequency of ncRNA genes. The imprinted ncRNA genes are implicated in the
imprinting of the region by a variety of mechanisms including
enhancer competition and chromatin remodeling [89].
A key feature of cancer is the loss of this imprinting resulting in altered gene expression [90,91]. Two of the best known imprinted genes
are in fact lncRNAs.
H19
The H19 gene encodes a 2.3 kb lncRNA that is expressed exclusively from the maternal allele. H19 and its reciprocally imprinted protein-coding neighbor
the Insulin-Like Growth Factor 2 or IGF2 gene at 11p15.5 were among the first genes, non-coding or otherwise, found to demonstrate genomic imprinting [74,92].
The expression of H19 is high during vertebrate embryo development, but is
downregulated in most tissues shortly after birth with the exception of skeletal tissue and cartilage [20,93,94].
Loss of imprinting and subsequent strong gene expression has been well-documented in human cancers. Likewise,
loss of imprinting at the H19 locus resulted in high H19 expression in cancers of the esophagus, colon, liver, bladder and with hepatic metastases [95-97].
H19 has been implicated as having both oncogenic and tumor suppression properties in cancer. H19 is upregulated in a number of human cancers, including
hepatocellular, bladder and breast carcinomas, suggesting an oncogenic function for this lncRNA [97-99]. In colon cancer H19 was shown to be directly activated
by the oncogenic transcription factorc-Myc, suggesting
H19 may be an intermediate functionary between c-Myc and downstream gene expression [98].
Conversely, the tumor suppressor gene and transcriptional activatorp53 has been shown to
down-regulate H19 expression [100,101].
H19 transcripts also serve as a precursor for miR-675, a miRNA involved in the regulation of developmental genes [102]. miR-675 is processed from the first exon of H19 and functionally
downregulates the tumor suppressor gene retinoblastoma (RB1) in human colorectal cancer, further implying an oncogenic role for H19 [103].
There is evidence suggesting H19 may also play a role in tumor suppression [104,105]. Using a mouse model for colorectal cancer, it was shown that mice lacking H19 manifested an increased polyp count compared to wild-type [106]. Secondly, a mouse teratocarcinoma model demonstrated larger
tumor growth when the embryo lacked H19, and finally in a hepatocarcinoma model, mice developed cancer much earlier when H19 was absent [107].
The discrepancy as to whether H19 has oncogenic or tumor suppressive potential may be due in part to the bifunctional nature of the lncRNA or may
be context dependent. In either case, the precise functional and biological role of H19 remains to be determined.
XIST – X-inactive-specific transcript
The 17 kb lncRNA XIST is arguably an archetype for the study of functionallncRNAs in mammalian cells, having been studied for nearly two decades.
In female cells, the XIST transcript plays a critical role in X-chromosomeinactivation by
physically coating one of the two X-chromosomes, and is necessary for the
cis-inactivation of the over one thousand X-linked genes [75,108-110].
Like the lncRNAs HOTAIR and ANRIL, XIST associates with polycomb-repressor proteins, suggesting
a common pathway of inducing silencing utilized by diverse lncRNAs.