microRNA and Gene Regulation

1
microRNA and Gene Regulation

• Nicola M. Kouttab, Ph.D.
• Pathology
• University Pathologists/RWMC
• Providence, RI 02908

2
Characteristics of miRNAs

• Small non-coding double stranded RNAs
• Approximately 19-22 nt long
• Have been described in invertebrates and
  vertebrates flies, worms, fungi, plants, and
  mammals more recently in yeast.
• Many are conserved between vertebrates and
  invertebrates.

3
Historical Notes

• lin-4, first miRNA to be described in C.
  elegance important in development of the worm
  from larva to adult (Chalfie M et al, 1981).
  Lin-4 binds to 3UTR of lin-14 mRNA.
• let-7, second miRNA to be described also in C.
  elegans (Reinhart BJ et al, 2000). Also important
  in development of worm. Conserved in humans-
  created much interest.
• 1998-Fire and Mello, experiments in C. elegans,
  first to show that dsRNA is much more potent at
  inhibiting gene expression than antisense RNA.
  Set the stage for understanding the role of
  miRNAs in development and gene regulation. (
  Nobel Prize in Physiology and Medicine, 2007).
• 2001- First evidence that RNA interference
  (siRNA) occurs in mammals (Elbashir et al).
  Different mammalian cell lines were used
  including HEK-93, and HeLa cells.

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Localization of miRNA Genes

• miRNA genes can be localized in three areas
• Intronic miRNA in non-coding transcripts
• (miR-15a/16-1).
• Intronic miRNAs in protein-coding transcripts
• (miR-106b/93/25).
• Exonic miRNAs in non-coding transcripts
• (miR-21, miR-155)

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Transcription of miRNAs

• Primary-miRNA is transcribed in the nucleus, and
  is usually several kilobases long posses 5 cap
  and a poly-A tail.
• Cleaved in the nucleus by Drocha enzyme to 70nt
  hairpin transcript (pre-miRNA).
• Transported to the cytoplasm by Exportin 5
  through nuclear pores.
• Cleaved by Dicer enzyme (RNase III enzyme) into
  19-22nt ds-transcripts.

6
from Esquela-Kerscher, A., and Slack, F.J.
(2006). Nature Reviews 6, p. 262.
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Mechanism(s) of action

• In cytoplasm, miRNA is loaded into an RNA-induced
  silencing complex (RISC) and binds to Argonaute
  protein enzyme present in the complex.
• Within the RISC the sense strand is degraded.
• miRNA guides RISC to the target mRNA
  (complementary sequence).
• Target mRNA is degraded by the Argonaute enzyme.
• In mammalian cells, miRNA target sites are
  located in the 3-untranslated region (3 UTR) of
  target mRNA.
• Post-transcriptional silencing.

8
from Rana, T.M. (2007). Nature Reviews 8, p. 24.
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Mechanisms of Gene Silencing

• If homology between target mRNA and antisence
  strand in RISC is perfect- Induces cleavage of
  mRNA
• If homology is partial- Induces degradation of
  mRNA (no translation of mRNA into protein)
• Silencing of heterochromatin is another mechanism
  of gene silencing (not well understood)
• A complementary sequence of 6 to 7nt (seed
  sequense, base 2-7or 8 from 5 end) between mRNA
  and RISC strand is sufficient to induce mRNA
  degradation.

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Argonaute Enzymes

• In humans there is a family of four Argonaute
  (Ago) enzymes.
• Only Ago 2 is capable of target mRNA cleavage
  (endonucleolytic cleavage).

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Functions of miRNAs

• Coordinate the development and functions of cells
  and tissues Differentiation, proliferation
  apoptosis, cell cycle, drug sensitivity.
• Examples miR-181- development of T-cells in
  thymus, such as negative and positive selection.
• Multiple miRNAs required for stem cell
  development.
• Knockout of Dicer is lethal at day E7.5.
• Embryonic cells null for Dicer fail to
  proliferate- suggests role in stem cell
  maintenance.

12
from Stadler, B.M., and Ruohola-Baker, H.
(2008). Cell 132, p. 563.
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HUMAN STEM CELLS

• EMBRYONIC STEM CELLS- Derived from inner mass of
• blastocyt and can generate stem cells for all
  three
• germ layers (Rossant et al Cell, 2008).
• Express a unique repertoire of miRNAs (miR-302
  family in humans is orthologous to Zebrafish
  miR-430), which direct early embryonic
  development.
• Mutation of DICER causes embryonic lethality
• (Kloosterman Plaster, Dev Cell 2006).

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HUMAN STEM CELLS

• SOMATIC STEM CELLS- These cells undergo self
  renewal
• and multipotency.
• Deletion of DICER in limb mesoderm of
  mouse
• embryo induces defects in limb
  development
• (Kloosterman Plasterk, Dev Cell, 2006).
• A cardiac-enriched miRNA family has a
  critical role in
• progenitor cells development in the heart
  (Zhao et al,
• Nature, 2005).

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HUMAN STEM CELLS

• GERMLINE STEM CELLS-
• Genetic studies clearly show an imporant role
  for
• miRNA-silencing in gametogenesis- Impairment
  of function can
• cause loss of maintenance and sterility
  (Knight Bass, Science
• 2001 Yang et al, Development, 2007).

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Dysregulation of miRNA

• Ample evidence that dysregulation of miRNAs is
  implicated in loss of normal maturational
  ability, and development of diseases such as
  cancer.
• Examples Increase in
• miR-155 B-cell leukemias (mice)
• miR-17-92 B-cell lymphomas (mice)
• Inhibition of miR-21 inhibits growth of
  human
• mammary tumor cell line.
• In Contrast- miRNAs of the let-7 family, miR-15a,
  and
• miR-16-1, function as tumor suppressors.
  Downregulation
• of these miRNAs, increases the potential for
  cancer.

17
from Esquela-Kerscher, A., and Slack, F.J.
(2006). Nature Reviews 6, p. 263.
18
miRNA in Disease

• Deregulation of miRNA or RISC components has been
  implicated in various diseases.

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MiRNA-Target Interactions In Disease

• 1- A mutation may occur in a miRNA causing loss
  of
• function- Potential for disease.
• 2- Over-expression of miRNA may result in
  over-expression
• of an oncogene (He et al, 2005).
• 3- A target site may acquire a mutation and no
  longer
• binds miRNA- Releases gene from regulation.
• 4- A gene may acquire a new miRNA target that may
• result in aberrant silencing (Abelson et al,
  2005).

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miRNAs and Human Disease

• Reports have implicated miRNA in a variety of
  diseases
• Cancer- It is estimated that above 50 of human
  miRNA genes are located in cancer-associated
  genomic regions.
• Several miRNAs are involved in regulation of
  important cancer genes
• ras let-7
• PTEN miR-21
• BCL-2 miR-15/16

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miRNAs and Human Disease

• Viral Infections-- Hepatitis C virus (HCV), is
  able to use
• miR-122 in liver to promote viral replication.
• Metabolic Disorders-- miRNA is implicated in
  insulin secretion, blood lipids and cholesterol
  adipocyte differentiation.
• Heart Disease-- miR-1 and miR-133 modulate
  differentiation in muscle cells.
• In a murine model, level of miR-133 is
  inversely related to cardiac hypertrophy.

22
from Esquela-Kerscher, A., and Slack, F.J.
(2006). Nature Reviews 6, p. 264.
23
Therapeutic Opportunities Using siRNAs

• MiR-17-92 cluster is over-expressed in several
  different cancers- Inhibition of this cluster is
  potential therapy for these cancers.
• Let-7 is downregulated in lung cancer-
  Replenishing let-7 may be of therapeutic value.
• Inhibition of miR-375 increased insulin
  production in murine islet cells- Potential
  therapy for diabetes.
• In a murine model, inhibiton of miR122 reduced
  liver steatosis- Inhibition of miR-122 may be a
  target for metabolic disease.

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Therapeutic Strategies In Disease-Associated
miRNAs

• Down-regulation or over-expression of miRNAs has
  been associated with various disease states.
  Therapeutic strategies have been developed to
  correct these states.
• ANTISENSE OLIGONUCLEOTIDES (ASO)- ASOs can
  inhibit
• miRNA function at any level, from
  transcription, to processing, to
• maturation (Davis et al, Nucleic Acids Res,
  2006).
• MiRNA SPONGES This relies on expression of
  competitive transcripts
• with multiple binding sites for miRNA, which
  bind miRNAs, and prevent
• their binding to natural targets (Ebert et al,
  Nat Methods, 2007).
• TARGET MIMICRY- Addition of miRNA targets with
  mismatch base
• pairing. These bind to miRNA, and prevent them
  from regulation of
• natural targets (Franco-Zorrilla et al, Nat
  Genet, 2007).
• REPLACEMENT THERAPY- Over-expression of miRNAs
  can be
• counteracted, e.g by mimics.
• In down-regulation or loss-of-function mutation
  of miRNA, the miRNA can be restored by exogenous
  miRNA (Zeng et al, PNAS, 2003).

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siRNAs as Drugs

• MiRNA are considered as the next major
• breakthrough in therapeutic strategies.
• However
• What are some Problems to overcome before siRNAs
  can be effectively used as therapeutics, and
  become the next generation of drugs.

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siRNA as Drugs

• Phamacokinetics
• Naked or unmodified siRNA has very short
  half-life in blood (t1/2 is about 1hr).
• Form complex with other molecules, or,
• Chemical modification to avoid
• degradation by enzymes.

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siRNA as Drugs

• Off-Target effects
• Some non-targeted genes may be
• unintentially silenced due to some
  complementarity
• to siRNA tested.
• Triggering of immune and inflammatory effects.
• Activation of TLRs.

28
from Martin, S.E., and Caplen, N.J. (2007). Ann.
Rev. Genom. Hum. Gene. 8, p. 86.
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siRNA as Drugs

• Drug Resistance
• Especially important in cancer and viral
  infections where the mutation rate is high-
• Mutants are generated which can escape the
  effect of the drug.
• Solution- Use combinations of siRNAs.

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siRNA as Drugs

• Interference with endogenous miRNA
• Competing with limiting amounts of Dicer.
• Competing for adequate amounts of
• Exportin 5, for transport to the cytoplasm.

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Therapeutic Aims For siRNA Must Address

• To become the next class of drugs requires
• Delivery systems that allow siRNA to enter
  cytoplasm of cell.
• In humans, the most effective delivery
• has been through
• inhalation into respiratory tract
• topical application to the genital tract
• direct injection into tissue.

32
from Dykshoorn, D.M., and Lieberman, J. (2006).
Cell 126, p. 232.
33
Delivery

• For delivery to deeper targets conjugation of
  siRNA to other molecules shows promise
• Cholesterol-conjugated
• Bound to antibody fragment
• Liposomes-nanoparticles
• Viral vectors (Adenoviruses, HIV vectors)
• Electroporation

34
from Rana, T.M. (2007). Nature Reviews 8, p. 28.
35
from Akhtar, S., and Benter, I.F. (2007). JCI
12, p. 3627.
36
from Martin, S.E., and Caplen, N.J. (2007). Ann.
Rev. Genom. Hum. Gene. 8, p. 94.
37
MiRNA in Mouse Skin Epidermis and Hair Follicle

• Many miRNA are differentially expressed in
  epidermis and hair follicle.
• There are distinctive patterns of expression of
  miRNA.
• Most abundant miRNA is mmu-miR-16, which is also
  present in many other tissues of the body.
• Yi et al Nature Genetics, 2006

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Significance of miRNA in Skin Development

• Dicer knocked out in skin epithelial cells,
  therefore no maturation of miRNAs.
• No survival of mice beyond postnatal 4-6 days.
• Hair germs evagination into the epidermis,
  instead of invagination downward into the dermis.
• Continual proliferation of follicle cells destroy
  the integrity of the skin of mutant mice.
• Results miRNA is important in skin
  morphogenesis, and skin is important in life and
  death processes. (Yi et al 2006).

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Significance of MiRNAs in Skin (2)

• Similar study in Dicer deleted mice (Andl et al
  2006) showed similar results to study by Yi et al
  2006.
• Cyst-like structures replaced hair follicles.
• Elevation in numbers of basal and supra-basal
  cell layers in Dicer mutant mice.
• Reduction of Notch1- hyperproliferation and tumor
  development.

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Phases of Wound Healing

• Inflammatory - Hemostasis and inflammation.
• Proliferation - Epithelialization,
  angiogenesis,granulation tissue formation,
  collagen deposition.
• Maturation - Remodeling, collagen depositon.

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MiRNA and Angiogenesis

• C-myc has an important role in neo-vascularization
  in neoplasms (Dews et al, 2006).
• In tumor angiogenesis, miR-18 and miR-19
  (cleavage products of miRNA-17-92 cluster) are
  upregulated by c-myc.
• c-kit is involved in neo-vascularization. MiRNA
  221/222 decrease c-kit protein level,which
  inhibited the ability of endothelial cells to
  promote tube formation (Strumberg 2005 Roboz et
  al, 2006).

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siRNA in Wound Healing
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siRNA in Wound Healing
44
from Akhtar, S., and Benter, I.F. (2007). JCI
12, p. 3624.
45
Future Directions and Challenges

• We are just beginning to understand the
  importance of miRNAs and siRNAs in development
  and therapeutic potential.
• miRNAs and siRNAs show specificity and efficacy-
  Well suited as therapeutics.
• To continue to evaluate miRNAs and siRNAs in
  disease states-by inhibiting miRNAs and observing
  the consequences.
• To better target genes that are responsible for
  disease.
• Expand delivery systems and understand
  phamacokinetics.
• Modifications to increase longevity in vivo.
• Evaluate efficacy using combinations of siRNAs,
  or in combinations with other drugs.