Long non coding RNA lncRNAs

The length of lncRNAs
o Almost 98% of the human genome does not encode proteins.
o The non-coding transcripts less than 200 bases are called small non-
coding RNA and comprise of tRNA, rRNA, miRNA, snoRNA, piwi-
interacting RNA (pi-RNA).
o RNA molecules that are of more than 200 bases in length are known
as long non-coding RNA (lncRNA).
o lncRNAs are more than 200 nucleotides in length and also can be
more than 2 Kb.
o Such long noncoding RNAs usually have limited coding potential due
to the absence of open reading frames, 3`-UTR and termination
region; while their coding potential is less than 100 amino acids.
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Location of lncRNAs in the cells
• In the cell lncRNAs found in nucleus that form paraspeckle and
speckle domains.
• In the cytoplasm found free or inside exosomes that carry them out
the cell.
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Classification of
lncRNA
Broadly, the lncRNAs can be divided into 5 categories
according to their location in the genome
• Intergenic lncRNAs
• Intronic lncRNAs
• Sense lncRNAs
• Antisense lncRNAs
• Bidirectional lncRNAs
• Enhancer lncRNAs
• Promoter lncRNAs
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• Long intergenic non-coding RNAs (lincRNAs); lincRNAs
are synthesized by RNAP II from intergenic regions in
both sense and antisense orientations between two
protein-coding genes.
Intergenic lncRNAs
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• Intronic long non-coding RNAs; RNAP III, or mitochondrial
spRNAP IV synthesize various forms of long intronic non-coding
RNAs from intronic regions (broken black lines) of protein-coding
genes, in either sense or antisense directions, sometimes
including exonic sequences.
Intronic lncRNAs
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• Exonic, or sense long non-coding RNAs, are synthesized
by RNAP II from exonic sequences (pink boxes) of
protein-coding genes and processed via alternative
splicing mechanisms.
Exonic, or sense lncRNA
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• Natural antisense transcripts (NATs) are synthesized
by RNAP III from the antisense strand of protein-
coding genes, and give rise to three different forms.
Natural antisense lncRNA
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• Bi-directional long non-coding RNAs are synthesized
from sequences in close proximity (<1000 bases) to
the transcription start sites of protein coding genes
but proceed in the opposite direction
Bi-directional lncRNA
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• Enhancer long non-coding RNA (eRNAs) are
synthesized by RNAP II from the upstream enhancer
region (red box) of protein-coding genes to form
eRNAs with a 30-poly(A) tail, or by RNAP III to form
eRNA without a 30-poly(A) tail modification.
Enhancer lncRNA
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• Promoter long non-coding RNAs are synthesized from
the promoter region of protein-coding genes (purple box)
by RNAP II and regulate the expression of the associated
protein-coding gene.
Promoter lncRNA
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Similarities Between mRNA and lncRNA
o They are transcribed by RNA polymerase II (Pol II) with some exceptions.
o They have to some how chromatin states similarities
o lncRNA are often 5ʹ-capped, Some of them spliced and polyadenylated
o Both exhibit tissue specific expression
o They have conserved promoters in humans and mice
o Both are important markers for disease or developmental state, such as diverse
human cancers, T cell differentiation and development
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Differences between mRNA and lncRNA
o lncRNAs don’t have Open Reading Frame (ORF).
o lncRNAs tend to be shorter than mRNAs, have fewer but longer exons, be
expressed at relatively low levels and exhibit poorer primary sequence con-
servation.
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Chromatin effects on lncRNA expression
o At the level of the chromatin state, lncRNAs and mRNAs exhibit similar properties,
such as an enrichment of H3K4me3 at promoters; however, lncRNA genes have a
higher enrichment of H3K27ac and are more strongly repressed by certain
chromatin remodeling complexes, such as Swr1, Isw2, Rsc and Ino80.
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Divergent Transcription
o Divergent transcription is repressed by chromatin assembly factor complex CAF-1 and is
enhanced by H3K56ac and the chromatin remodeller SWI/SNF.
o The Pol II carboxy-terminal domain was enriched for a specific phosphorylation mark (Tyr1P)
at both upstream antisense RNAs and enhancers compared with Pol II at protein-coding genes.
o High expression of mRNAs also results in higher levels of the corresponding uaRNAs.
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lncRNAs and mRNAs Transcriptional Elongation
o DICER1 is an important factor in both the biogenesis of small ncRNAs and the
downstream activation of hundreds of lncRNAs.
o Transcriptional elongation is more strongly regulated by DICER1 enzyme and
MYC transcription factor for lncRNAs than for mRNAs
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Cont..
o Polyadenylation signals (PASs) are enriched in the nearby antisense
direction (lncRNAs), whereas the U1 snRNP splicing signal is enriched in the
nearby sense direction (mRNAs)
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lnRNA and mRNA localization
o Whereas mRNAs localize very specifically to ribosomes in the cytoplasm,
lncRNA localization is much more varied, as certain lncRNAs can occupy the
chromatin, subnuclear domains, the nucleoplasm or the cytoplasm.
lnRNA
mRNA
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lncRNA degradation
The short-lived non-coding RNAs, often referred to as cryptic unstable transcripts
(CUTs) in yeast are degraded through digestion by
• The nuclear exosome
• The cytoplasmic decapping complex (Dcp1–Dcp2) and subsequent 5ʹ-to-3ʹ exonuclear
degradation by Xrn1
• The nonsense-mediated decay (still unclear because lncRNAs have not ORF).
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Post-transcriptional processing events
in special lncRNA classes that not
found in mRNA
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MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and NEAT1 (nuclear
enriched abundant transcript 1) lncRNAs are processed at their 3ʹ ends by RNase P
• Generates tRNA-like small RNA products and the mature lncRNA, which possesses a
stabilizing 3ʹ-terminal RNA triplex structure.
• MALAT1 is localized to nuclear speckles and NEAT1 is localized to nuclear
paraspeckles.
• the tRNA-like structures cleaved from MALAT1 (mascRNAs) are stable and
cytoplasmic, whereas those from NEAT1 are unstable.
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Back splicing
o Canonical splicing of mRNAs produces linear transcripts but
back-splicing produces stable circular RNAs (circRNAs)
consisting of non-sequential exon–exon junctions.
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Intronic lariats
oIntronic lariats are typically unstable
after splicing, but some escape
debranching and degradation and
persist as non-coding circular intronic
long non-coding RNAs (ciRNAs).
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sno-lncRNA
• SnoRNAs are most often encoded within the
introns of some protein-coding genes, from
which they are trimmed.
• However, when tandem snoRNAs are
encoded within a single intron, trimming can
result in a sno-lncRNA, which consists of an
intronic lncRNA flanked by two snoRNAs and
thereby lacks a 5ʹ-cap or poly(A)-tail.
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Microprocessor cleavage
o Microprocessor — a protein complex that
processes miRNAs — cleaves the nascent
transcript to terminate transcription in a
polyadenylation-independent manner,
thereby producing lnc-pri-miRNAs, which
are 3ʹ-capped by the Microprocessor
complex70.
o lnc-pri-miRNAs are further processed into
miRNAs and unstable, non-polyadenylated
lncRNAs
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• Binding to RNAPII.
• Binding to TFs.
• Binding to chromatin remodelers and
modifiers.
• Binding to promoters, mRNAs, miRNAs.
• Binding to cofactors, activators and
repressors
• Repression and Activation
• X inactivation
• Imprinting specific genes.
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oNatural antisense transcript (NAT), is a single stranded RNA that is
complementary to a protein coding messenger RNA (mRNA) with which it
hybridizes, and thereby blocks its translation into protein.
• The trans-NATs and their respective targets are physically located in different loci
on the genome.
• The cis-NATs and their targets are located on the same locus, but opposite strands
of the DNA.
oThe regulation can be occurred at the transcriptional level or at the post
transcriptional level.
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Cont..
lncRNAs make Regulation at transcriptional level
o Influencing enhancer and promoter activity.
o Blocking RNAII elongation.
o Allows VDJ recombination.
o Chromatin change
Regulation at post transcriptional level
o Regulation of mRNA processing (Splicing and Editing).
o Increase the stability of mRNAs in the cytoplasm.
o Express mRNAs under stress conditions.
o Inhibit translation.
o Squestering miRNAs from binding to the target gene through making miRNAs sponge or binding
to the miRNAs binding site on the mRNAs.
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Phosphorylate the carboxyl terminal
domain (CTD) of RNAPII and disturbing its
ability to elongate the mRNA or to bind to
DNA by:
o Binding to transcription elongation
factor.
o Directly binding to RNAPII
At transcriptional level
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lncRNAs that found among clusters
regulate a set of genes at specific locus
through promoting chromatin
remodeling which leads to inhibition or
activation
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Cis-regulatory mechanisms of lncRNA function
Some enhancer RNAs, such as LUNAR1
near the insulin-like growth factor 1
receptor (IGF1R) locus, mediate
chromosome looping between enhancers
and nearby target genes via Mediator or
MLL protein complexes that leads to gene
transcription
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olncRNAs regulate in cis the expression of
coding genes in specific locus.
lncRNAs at imprinted loci
Mammalian x inactivation
olncRNAs regulate in cis the expression of coding
genes in specific locus.
oXist is activated on the inactive X chromosome in cis
and in trans by the lncRNA Jpx and is silenced in cis by
the antisense lncRNA Tsix.
oXist silence the x chromosome in cis. Rana Alhakimi

o When protein-coding genes and antisense lncRNA genes overlap, processing
RNA polymerase II (Pol II) particles may collide and thus abort transcription,
effectively inhibiting the expression of both genes.
RNA polymerase II (Pol II) particles collision
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Autoregulation
FMR1 (fragile X mental retardation 1) binds and silences its own promoter via RNA–
DNA hybrids at CGG repeat expansions that are characteristic of disease
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lncRNAs as nucleic acid templates
• TERC is one such example, in that it serves as a template for RNA-dependent DNA
polymerase activity (reverse transcription) carried out by the enzyme TERT
(telomerase reverse transcriptase) to elongate telomeres, thus combatting the
shortening of chromosome ends that inevitably results from DNA replication.
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mRNA degradation
Regulation at post transcription level
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miRNA Sponges
MicroRNA (miRNA) sponges are RNA
transcripts (in this case lncRNAs) containing
multiple high-affinity binding sites that
associate with and sequester specific
miRNAs to prevent them from interacting
with their target messenger (m)RNAs.
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The production pf mRNA from the
gene of interest
miRNAs act as a regulator that
regulate the production of mRNA
through different mechanisms
which lead to mRNAs degradation
and reduction of mRNAs output
In sometimes cells need to regulate the mRNAs degradation through
reducing miRNAs regulatory effect on mRNAs using lncRNAs.
In some diseases increase production of lncRNAs can lead to increase the
production of mRNAs and prevent their degradation.
mRNAs output
Gene of interest
Promoter
miRNAs
mRNAs degradation
miRNAs
lncRNA
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Mechanisms of action
o Signals
o Decoy
o Scaffold
o Guide
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Guide
o The lncRNAs are required for proper localization of
specific proteins including ribonucleoprotein
complexes.
o Homeobox antisense intergenic RNA (HOTAIR) is an
example of guide lncRNA to localize polycomb
repressor complex2 (PRC2) in developmental and
cancer-related gene expression.
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Signals
o The production and presence of signal factors of
lncRNAs are an indicator of their transcriptional activity
(e.g. KCNQ1ot1 and Xist).
o Some lncRNA transcripts such as CCND1 activate or
deactivate the natural functions of target protein
targets (that are allostericallymodified) via intrinsic
catalytic activities
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Scaffold
o The lncRNAs can serve as adaptors to bind more than
2 protein partners, thus are involved in structural
roles.
o The telomerase RNA TERC (TERRA), an example of
RNA scaffold, is responsible for telomerase function.
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Decoy o Molecular decoys (viz. Gas5, PANDA etc) are
polynucleotides that negatively regulate an effector by
preventing access of regulatory proteins to DNA.
o Gas5 is a hairpin-structured lncRNA (resembles
glucocorticoid receptors of DNA) that act as a decoy during
growth factor starvation.
o It releases the receptors of DNA during starvation
condition and prevents the transcription of metabolic genes
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Methods used to study lncRNAs inside the
cell
Methods used to predict lncRNA structure
o Selective 2′‐hydroxyl analysed by primer
extension (SHAPE)
o Parallel analysis of RNA structure (PARS)
o Domain‐specific chromatin isolation by RNA
purification (dChIRP)
Methods used to localize lncRNAs inside the cell
o Single‐molecule RNA in situ hybridization
(sm‐RNA FISH)
o RNA Mimics of GFP and RNA visualization
o Clustered regularly interspaced short palindromic
repeats (CRISPR) RNA Tracking
Methods utilized for determination of lncRNA
function
Knockout techniques
o Clustered regularly interspaced short palindromic
repeats (CRISPR) Cas technology
Knock‐down techniques
o RNA interference (RNAi)
o Small molecules
o using CRISPR interference/activation
(CRISPRi/a).
o Antisense oligonucleotides (ASOs)
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Cont..
Methods used for mapping lncRNA interactions
Mapping lncRNA–DNA interactions
o ChIRP (chromatin isolation by RNA purification)
o CHART (capture hybridization analysis of RNA targets)
o RAP (RNA antisense purification)
Mapping lncRNA–protein interactions by protein pull‐down
o RNA immunoprecipitation (RIP)
o Cross‐linking and immunoprecipitation (CLIP)
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• Choroidal neovascularization (CNV) involves the growth of new blood
vessels that originate from the choroid through a break in the Bruch
membrane into the sub–retinal pigment epithelium (sub-RPE) or
subretinal space. CNV is a major cause of visual loss.
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Methodology
oIn vitro
1. The Bone marrow-derived macrophages (BMDM) cells were isolated from mice
and cultured.
2. the cells were infected with the NEAT 1 Smart Silencer and with miR- 148a-3p
inhibitor and theirs negative controls.
3. It were induced by IL-4 for their differentiation to M2-type macrophages, then
step one was repeated.
4. the cells again were Cultured, Harvested, Lysed and then were incubated with
a RIP buffer containing magnetic beads conjugated with an anti-Ago2 antibody
or IgG isotype control.
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cont..
5. The total RNAs were isolated and converted theirs to cDNA using
revers transcriptase then subjected to real time PCR to detect the
genes of Arginase-1, TNF-a, iNOS, PTEN, miRNA-148a-3p, lncRNANEAT1
(u6 houskeeping gene used as control in real time PCR).
5. The isolated protein are detected using Waster blot are used to
detect the proteins of Arginase-1, TNF-a, and iNOS,(GAPDH using as
control ).
6. microarray analysis to detect the expression of lncRNAs
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CONT..
oIn vivo
1. The mice were subjected to laser to induce choroidal
neovascularization (CNV), after one day the mice were injected
intravitreally with NEAT 1 Smart, miR- 148a-3p inhibitor and NEAT 1
Smart Silencer + miR- 148a-3p inhibitor and and theirs negative
controls.
2. The control mice (vehicle group) were injected with the same
volume of saline or Lipofectin Reagent at day 1 after laser
photocoagulation.
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CONT..
3. On days 3 and 7 mice were intraperitoneally injected with
fluorescein sodium and late-phase angiograms were obtained five
minutes after the injection to study the leakage on the mice eyes.
4. RPE-choroid-sclera complexes were surgically isolated, fixed,
incubated with blocking buffer and then The CNV lesions were imaged
with a confocal microscope.
5. The 5, 6, 7 steps from invtro also were repeated invivo .
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Confirmation that miRNA-148a-3p was sponged by
lncRNA NEAT1
oFive types of pmirGLO vector were designed
1. luciferase gene +lncRNA NEAT1 (NEAT1-WT) gene.
2. luciferase gene+ PTEN 3′UTR (PTEN-WT) gene.
3. luciferase gene + NEAT1-MUT gene.
4. luciferase gene +PTEN-MUT gene
oThen the HEK293 T cells were transfected with the vectors alone and
in combination with miRNA-148a-3p mimics or negative control.
oluciferase activities were measured using the Dual-Glo® Luciferase
Assay System
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Fig 1. lncRNA expression in CNV mice.
The level of lncRNA NEAT1
significantly increased at days 3
and 7 in RPE-choroid-sclera
complexes after laser induction
of CNV. Which indicate the
direct role of lncRNA NEAT 1in
CNV formation.
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LncRNA NEAT1 mediates CNV formation and M2 macrophage in vivo
The mice that were injected
with lncRNA NEAT1 Smart
Silencer have showed lower
leakage compared to those
that were not injected.
Fig 2. Effects of lncRNA NEAT1 Smart Silencer on CNV leakage.
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lncRNA NEAT1 affect CNV growth by regulating
M2 polarization
Fig 3. RT-PCR analysis of gene expression of Arg-1 (A), Ym-1 (B), TNF-α (C), iNOS (D), miR-148a-3p (E), and
PTEN (F) in RPE-choroid-sclera complexes in normal tissues and at days 3, 7 after laser photocoagulation.
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Cont..
o The results showed that Arg-1 and YM-1 were significantly increased at days 3 and 7, iNOS and TNF-α
genes were not significantly decreased at day 3,while significantly decreased at day 7.
o It is considered that lncRNA NEAT1 could promote pro-angiogenesis Arg-1+Ym-1+M2 macrophage
polarization while inhibiting the pro-inflammation of iNOS + TNF-a + M1 type.
o M2 macrophage polarization was regulated by a lncRNA NEAT1/ miRNA-148a-3p/PTEN functional
axis. Our study also indicated that the expression of lncRNA NEAT1 might serve.
o The expression of miR-148a-3p (E) are significantly decreased while the expression of PTEN (F) are
significantly increased at day 3 and 7.
o It is considered that LncRNA NEAT1 sponged miR-148a-3p to regulate the expression of PTEN which
in turn stimulated CNV pathogenesis formation in mice model.
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Fig 4. RT-PCR analysis of the levels of Arg-1 (A), Ym-1 (B), TNF-α (C), iNOS (D), PTEN (E) and miRNA-148a-3p (F) in
RPE-choroid-sclera complexes at days 3 and 7 after lncRNA NEAT1 Smart Silencer intravitreally injected.
Cont..
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Cont..
oAfter inhibition of lncRNA NEAT1, Arg1 and Ym-1 decreased
significantly in the lncRNA NEAT1 Smart Silencer group at days 3 and 7
while the TNF-α and iNOS genes increased significantly in the lncRNA
NEAT1 Smart Silencer group at days 3 and 7.
oThese results confirm that lncRNA NEAT1 may affect CNV growth by
regulating M2 polarization.
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LncRNA NEAT1 sponged miR-148a-3p to regulate
the expression of PTEN which in turn mediate M2
macrophage
Figure 5. The relative luciferase activity in PTEN-WT
and PTEN-MUT after transfected with miR-148a-3p
mimics or NC.
• we found that miR-148a-3p mimics suppressed the
luciferase activity of the PTEN -WT reporter
plasmid, but no suppression was detected with the
mutant PTEN reporter plasmid (PTEN-MUT),
suggesting that PTEN might be a direct target of
miR-148a-3p.
• These results demonstrated that lncRNA NEAT1
could directly sponge miR-148a-3p to regulate PTEN
expression.
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Cont..
• A dual-luciferase reporter showed that the
luciferase activity was significantly inhibited by
the co-transfection of miR-148a-3p mimics and
the wild-type NEAT1 construct (NEAT1-WT).
• The results suggested that miR-148a-3p was a
direct target of lncRNA NEAT1. We further found
that luciferase activity was not altered by the
co-transfection of miR-148a-3p mimics and
mutated NEAT1 construct (NEAT1-MUT) (Fig.
10C), indicating that the binding site for miR-
148a-3p within lncRNA NEAT1 was functional.
Figure 6. The relative luciferase activity in NEAT 1-WT and
NEAT 1-MUT after transfected with miR-148a-3p mimics or NC.
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• Periodontal ligament stem cells PDLSCs (dental stem cells) had a distinct feature
to generate periodontal tissues that would be the ideal candidate for treatment
of periodontal bone loss.
• Recently, evidence has shown that exosomes are one type of extracellular vesicle
with promise for bone regeneration
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Methodology
o Periodontal ligament stem cells PDLSCs (dental stem cells) were isolated from
periodontal patients and healthy persons then were cultured.
o Exosome were extracted.
o PDLSCs differentiation to osteogenic cells were induced by growing in induction
medium and then exosomes were extracted by centrifugation (exosome were
extracted at 5 and 7 days of differentiation.
o RNAs were isolated and sequencing using RNA sequencing technique and the
expression was showed using RT-PCR.
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Results
Fig 1. The apparent variations and expression profiles of exosomal lncRNAs in EX0,
EX5 and EX7. (A) The distribution of identified exosomal lncRNAs in human
chromosomes. (B-D) Heatmap indicates differences in exosomal lncRNAs expression
profiling between EX0, EX5 and EX7.
A total of 118 lncRNAs and 43 lncRNAs
expressed differently in EX5 (70 up-regulated
and 48 down- regulated) and EX7 (24 up-
regulated and 19 down- regulated) comparing to
EX0 respectively, and there were 2 and 5
lncRNAs continuously up and down-regulated
through out of EX5 and EX7 compared to EX0
respectively.
The result suggest that lncRNAs play an
important role during osteogenic differentiation
of PDLSCs and promote bone formation
lncRNAs differential expression
during osteogenic differentiation of
PDLSCs
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Cont..
Fig 2. Exosomal lncRNAs expression profiling in EX0, EX5 and EX7 and validation by qRT-PCR. (A) Selected lncRNAs in the
heatmap. (B) Results of qRT-PCR, *p < 0.05, EX5 and EX7 compared with EX0. All qRT-PCRs were performed in triplicate.
3 of 7 lncRNAs that showing
consistent expression patterns
(up or down-regulated) were
selected for validation by qRT-
PCR.
The consistent regulation of
the 7 lncRNAs indicate their
significant role during
differentiation and more
studies are needed to open
the cover on their exact role.
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References
• Dhanoa, J. K., Sethi, R. S., Verma, R., Arora, J. S., & Mukhopadhyay, C. S. (2018). Long non-
coding RNA: its evolutionary relics and biological implications in mammals: a review. Journal of
animal science and technology, 60(1), 25.
• Kornienko, A. E., Guenzl, P. M., Barlow, D. P., & Pauler, F. M. (2013). Gene regulation by the act of
long non-coding RNA transcription. BMC biology, 11(1), 59.
• Quinn, J. J., & Chang, H. Y. (2016). Unique features of long non-coding RNA biogenesis and
function. Nature Reviews Genetics, 17(1), 47.
• Tsagakis, I., Douka, K., Birds, I., & Aspden, J. L. (2020). Long non‐coding RNAs in development
and disease: conservation to mechanisms. The Journal of pathology, 250(5), 480-495.
• Xie, L., Chen, J., Ren, X., Zhang, M., Thuaksuban, N., Nuntanaranont, T., & Guan, Z. (2020).
Alteration of circRNA and lncRNA expression profile in exosomes derived from periodontal ligament
stem cells undergoing osteogenic differentiation. Archives of Oral Biology, 104984.
• Zhang, P., Lu, B., Zhang, Q., Xu, F., Zhang, R., Wang, C., ... & Mei, L. (2020). LncRNA NEAT1
Sponges MiRNA-148a-3p to Suppress Choroidal Neovascularization and M2 macrophage
polarization. Molecular Immunology, 127, 212-222.
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