Si and Mi RNAs

1. IMPORTANCE OF SiRNA AND MICRORNA
Presented by Dr.SIBI P ITTIYAVIRAH,
PROFESSOR,
DIVISION OF PHARMACOLOGY
DEPARTMENT OF PHARMACEUTICAL
SCIENCES,CPAS,CHERUVANDOOR,KERALA,INDIA.

2. Small interfering RNA (siRNA), sometimes known as
short interfering RNA or silencing RNA, is a class of
double-stranded RNA non-coding RNA molecules,
typically 20-27 base pairs in length, similar to miRNA, and
operating within the RNA interference (RNAi) pathway.
It interferes with the expression of specific genes with
complementary nucleotide sequences by degrading
mRNA after transcription, preventing translation

3. RISC stands for RNA‐induced silencing complex. It is a multiprotein
complex responsible for recognizing double‐stranded RNA, target
mRNA, and for cleavage of the target resulting in its degradation.
MicroRNA RNA Interference in Mammalian Cells RNA‐Induced
Silencing Complex
A short hairpin RNA or small hairpin RNA ( shRNA) is an artificial RNA
molecule with a tight hairpin turn that can be used to silence target gene
expression via RNA interference (RNAi). Expression of shRNA in cells is
typically accomplished by delivery of plasmids or through viral or bacterial
vectors.

4. Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme
that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer
cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments
called small interfering RNA and microRNA, respectively.
Ago and DICER are two protein components, Argonaute (AGO) and Dicer
(DCL), are central to the RNAi machinery of eukaryotes. Plants encode for
several copies of AGO and DCL genes; in Arabidopsis thaliana, the AGO
protein family contains 10 members, and the DCL family contains four.

6. Si RNA
Naturally occurring siRNAs have a well-defined structure that is a short (usually 20 to
24-bp) double-stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3'
ends with two overhanging nucleotides.
siRNAs are an important tool for validating gene function and drug targeting in the
post-genomic era.
In 1998, Andrew Fire at Carnegie Institution for Science in Washington DC and Craig
Mello at University of Massachusetts in Worcester discovered the RNAi mechanism while
working on the gene expression in the nematode, Caenorhabditis elegans.
They won the Nobel prize for their research with RNAi in 2006.

8. Mechanism of Gene silencing
1. Long dsRNA (which can come from hairpin,
complementary RNAs, and RNA-dependent RNA
polymerases) is cleaved by an endo-ribonuclease called
Dicer.
2. Dicer cuts the long dsRNA to form short interfering RNA or
siRNA; this is what enables the molecules to form the
RNA-Induced Silencing Complex (RISC).

9. 1. Once siRNA enters the cell it gets incorporated into other proteins to form the
RISC.
2. Once the siRNA is part of the RISC complex, the siRNA is unwound to form
single stranded siRNA.
3. The strand that is thermodynamically less stable due to its base pairing at the
5´end is chosen to remain part of the RISC-complex
4. The single stranded siRNA which is part of the RISC complex now can
scan and find a complementary mRNA

10. 1. Once the single stranded siRNA (part of the RISC
complex) binds to its target mRNA, it induces mRNA
cleavage.
2. The mRNA is now cut and recognized as abnormal by the
cell.
3. This causes degradation of the mRNA and in turn no
translation of the mRNA into amino acids and then
proteins. Thus silencing the gene that encodes that
mRNA.

11. siRNA is also similar to miRNA, however, miRNAs are derived from shorter stemloop RNA
products, typically silence genes by repression of translation, and have broader specificity
of action, while siRNAs typically work by cleaving the mRNA before translation, and have
100% complementarity, thus very tight target specificity

12. Gene knockdown is an experimental technique by which the
expression of one or more of an organism's genes is reduced.
The reduction can occur either through genetic modification or by
treatment with a reagent such as a short DNA or RNA
oligonucleotide that has a sequence complementary to either
gene or an mRNA transcript.

13. Gene knockdown by transfection of exogenous siRNA is often
unsatisfactory because the effect is only transient, especially in
rapidly dividing cells. This may be overcome by creating an
expression vector for the siRNA.
The siRNA sequence is modified to introduce a short loop
between the two strands. The resulting transcript is a short hairpin
RNA (shRNA), which can be processed into a functional siRNA by
Dicer

15. RNA activation[
It has been found that dsRNA can also activate gene expression, a mechanism that
has been termed "small RNA-induced gene activation" or RNAa. It has been shown
that dsRNAs targeting gene promoters induce potent transcriptional activation of
associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs,
termed "small activating RNAs" (saRNAs)

16. Chemical modification
siRNAs have been chemically modified to enhance their
therapeutic properties, such as enhanced activity, increased
serum stability, fewer off-targets and decreased immunological
activation. Commonly, siRNA has been encapsulated in a
nanolipid particle to prevent degradation in the blood

17. Therapeutic applications and challenges[edit]
One of the biggest challenges to siRNA and RNAi based therapeutics is intracellular
delivery.[
siRNA also has weak stability and pharmacokinetic behavior.
Delivery of siRNA via nanoparticles has shown promise.[
siRNA oligos in vivo are
vulnerable to degradation by plasma and tissue endonucleases and exonucleases and
have shown only mild effectiveness in localized delivery sites, such as the human eye.
siRNA oligos allows delivery via nano-scale delivery vehicles called nanovectors.
A good nanovector for siRNA delivery should protect siRNA from degradation, enrich
siRNA in the target organ and facilitate the cellular uptake of siRNA.
The three main groups of siRNA nanovectors are: lipid based, non-lipid organic-based,
and inorganic
Lipid based nanovectors are excellent for delivering siRNA to solid tumors,but other
cancers may require different non-lipid based organic nanovectors such as cyclodextrin
based nanoparticles.

18. siRNAs delivered via lipid based nanoparticles have been shown to have
therapeutic potential for central nervous system (CNS) disorders.
Central nervous disorders are not uncommon, but the blood brain barrier (BBB)
often blocks access of potential therapeutics to the brain.
siRNAs that target and silence efflux proteins on the BBB surface have been
shown to create an increase in BBB permeability.
siRNA delivered via lipid based nanoparticles is able to cross the BBB
completely.

19. MicroRNAs
MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that function in regulation of gene expression.
MicroRNAs (miRNAs) are small noncoding RNAs that function as major
players of posttranscriptional gene regulation in diverse species.
In mammals, the biogenesis of miRNAs is executed by cooperation of
multiple biochemical reactions including processing of miRNA precursors
by two central endoribonucleases, Drosha and Dicer.
MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play
important regulatory roles in animals and plants by targeting mRNAs for cleavage or
translational repression. Although they escaped notice until relatively recently,
miRNAs comprise one of the more abundant classes of gene regulatory molecules in
multicellular organisms and likely influence the output of many protein-coding genes.

23. MicroRNAs play an essential role in posttranscriptional regulation of gene
expression. They are evolutionary conserved, small, noncoding,
19–22-nucleotide RNAs, whose abnormalities, such as up- or
downregulated expression, have been associated with several neoplasms,
including adrenocortical tumors
The miRNAs are involved in the regulation of several physiological processes
in almost all eukaryotes, including development, growth, differentiation, and
metabolism. Therefore, the involvement of miRNAs as an important player in
carcinogenesis was not surprising

24. MicroRNAs (or miRNAs) comprise a novel class of small,
non-coding endogenous RNAs that regulate gene expression by
directing their target mRNAs for degradation or translational
repression. Their discovery added a new dimension to the
understanding of complex gene regulatory networks in humans and
animals alike.

25. MicroRNA (miRNA) was initially discovered in Caenorhabditis elegans by
Victor Ambros' laboratory in 1993 while studying the gene lin-14.
At the same time, Gary Ravkun identified the first miRNA target gene.
Those two groundbreaking discoveries identified a novel mechanism of
posttranscriptional gene regulation.

26. The importance of microRNA
miRNAs represent small RNA molecules encoded in the genomes of plants and
animals. These highly conserved 22 nucleotides long RNA sequences regulate the
expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific
mRNAs. A growing body of evidence shows that miRNAs are one of the key players
in cell differentiation and growth, mobility and apoptosis (programmed cell death).

27. The most significant distinction between miRNAs and siRNAs is
whether they silence their own expression.
Almost all siRNAs (regardless of their viral or other origin) silence
the same locus from which they were derived.
On the other hand, most miRNAs do not silence their own loci, but
other genes instead.

28. The role of miRNAs in malignancies
Experimental approaches have shown that some miRNAs act as tumor suppressors, and other ones as
oncogenes; hence they have important roles in cancer development, progression of the disease and its
prognosis. Dysregulation of miRNAs is linked to the development of cancer.
The first example of a miRNA as an oncogene is miR-155, which is processed from the non-coding B-cell
integration cluster RNA. This was first identified as a common integration site in lymphoma induced by avian
leukosis virus (ALV). Later it was found that miR-155 expression is elevated in Hodgkin lymphoma samples
and cell lines, as well as in juvenile Burkitt's lymphoma.
The human ortholog of lin-4, miR-125b-1, has been implicated in the development of leukemia (especially at
an early step in leukemogenesis). This miRNA is also located in a fragile genomic region which is deleted in
patients with breast, cervical, ovary and lung cancers. miR-15a and miR-15-1 are involved in the
development of chronic lymphocytic leukemia.

29. Analysis of miRNA expression may provide valuable information, as
dysregulation of its function can lead to human diseases such as cancer,
cardiovascular and metabolic diseases, liver conditions and immune
dysfunction.
miRNAs and cardiovascular disease
The roles of miRNAs in cardiac hypertrophy and heart failure have been
demonstrated in several clinical studies. Specific miRNAs are disregulated in the
diseased heart; furthermore, up- and down-regulation of miRNAs are necessary
and often sufficient to explain different heart diseases.

30. A myriad of miRNAs are found to be regulated during cardiac hypertrophy and
two, miR-1 and miR-133, play a key role in inhibiting it.
Overexpression of miR-195 during cardiac hypertrophy results in pathological
cardiac growth and heart failure, while miR-199a is expressed in cardiomyocytes
where it maintains cell size and plays a role in the regulation of cardiac
hypertrophy.
miRNAs are also important regulators of cardiac fibrosis and are involved in
structural heart disease. Some specific miRNAs (such as miR-1) are
implicated in the development of arrhythmia, thus it may be a potential
antiarrhythmic target. Several miRNAs (miR-21, miR126, miR-221 and
miR-222) represent important modulators of vessel remodeling.

31. miRNA in other conditions
Recent research has shown that miR-33 controls cholesterol homeostasis
based on knockdown experiments using antisense technology. The
overexpression of this miRNA decreases cellular cholesterol efflux to
apolipoprotein A-I (ApoA-I), which is a key step in regulating reverse cholesterol
transport.
miR-375 is considered to be a regulatory inhibitor of insulin secretion and may
also constitute a novel drug target for the treatment of type 2 diabetes. Several
observations underscore the importance of miR-122 in liver disease. miRNAs
are also implicated in the proper functioning of human immune system.

32. As miRNA research has expanded into a huge number of disease areas,
it has become clear that expression levels of certain miRNAs are altered
in many diseases. As such, the potential of these small non-coding
RNAs as biomarkers has become obvious, and exploiting it has become
a focus for researchers around the globe.

33. MiRNAs in Alzhermer’s disease
One particularly active area of miRNA biomarker research is in Alzheimer's
disease. An effective method to screen for Alzheimer's early in disease
progression may aid in the development of much-needed therapeutic and
preventative drugs (Blennow et al., 2015; Framminella et al., 2015).
In a recent study, miRNAs present in the cerebrospinal fluid (CSF) were profiled
to detect expression differences between Alzheimer's disease patients and
controls. By taking forward a panel of three miRNAs, the researchers were able
to identify Alzheimer's disease in CSF with 95.5% accuracy (Denk et al., 2015).

39. References
● Blennow K, Dubois, B Fagan AM, Lewczuk P, de Leon MJ, Hampel H (2015). Clinical utility of cerebrospinal fluid biomarkers in the
diagnosis of early Alzheimer’s disease. Alzheimers Dement 11, 58–69.
● Bonci D, Coppola V, Patrizii M, Addario A, Cannistraci A, Francescangeli F, Pecci R, Muto G, Collura D, Bedini R, Zeuner A, Valtieri
M, Sentinelli S, Benassi MS, Gallucci M, Carlini P, Piccolo S, De Maria R (2015). A microRNA code for prostate cancer metastasis
[published online ahead of print 15 June, 2015]. Oncogene, doi:10.1038/onc.2015.176.
● Denk J, Boelmans K, Siegismund C, Lassner D, Arlt S, Jahn H (2015). MicroRNA profiling of CSF reveals potential biomarkers to
detect Alzheimer’s disease. PLoS ONE 10, e0126423.
● Femminella GD, Ferrara N, Rengo G (2015). The emerging role of microRNAs in Alzheimer’s disease. Front Physiol 6, 40.
● Mihelich BL, Maranville JC, Nolley R, Peehl DM, Nonn L (2015). Elevated serum microRNA levels associate with absence of
high-grade prostate cancer in a retrospective cohort. PLoS ONE 10, e0124245.
● Min PK, Chan SY (2015). The biology of circulating microRNAs in cardiovascular disease. Eur J Clin Invest 45, 860–74.
● Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A,
Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M (2008).
Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105, 10513–8.
● Montani F, Marzi MJ, Dezi F, Dama E, Carletti RM, Bonizzi G, Bertolotti R, Bellomi M, Rampinelli C, Maisonneuve P, Spaggiari L,
Veronesi G, Nicassio F, Di Fiore P, Bianchi F (2015). miR-Test: A blood test for lung cancer early detection. J Natl Cancer Inst 107.
● Rao P, Benito E, Fischer A (2013). MicroRNAs as biomarkers for CNS disease. Front Mol Neurosci 6, 39.
● Sozzi G, Boeri M, Rossi M, Verri C, Suatoni P, Bravi F, Roz L, Conte D, Grassi M, Sverzellati N, Marchiano A, Negri E, La Vecchia
C, Pastorino U (2014). Clinical utility of a plasma-based miRNA signature classifier within computed tomography lung cancer
screening: a correlative MILD trial study. J Clin Oncol 32, 768–773.
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