microrna’s applications in medicine
DESCRIPTION
MicroRNA’s applications in medicine . microRNA Related Publications. Source: PubMed. lin-14; lin-4 relationship. 1. lin-4 encodes small RNAs with antisense complementarity to lin-14 3’ UTR .[Cell, 75, 843 (1993)] - PowerPoint PPT PresentationTRANSCRIPT
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MicroRNA’s applications in medicine
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microRNA Related Publications
Source: PubMed
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1.lin-4 encodes small RNAs with antisense complementarity to lin-14 3’ UTR.[Cell, 75, 843 (1993)]2. The LIN-14 protein concentration drops in late development, whereas the lin-4 RNA increases. 3. MECHANISM:lin-4 RNA increases in later development and inhibits translation of the lin-14 gene and thus specifies the pattern of late-stage development.
lin-14 mRNAlin-4 RNAtranslation
lin-14; lin-4 relationship
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1. lin-4 encodes two mRNAs: 61 and 22.2. 61 forms a stem-loop structure and is the
precursor of 22 nt3. The red nt sequence is the 22 nt
microRNA.
Cell, 106, 23 (2001)
lin-4 RNA Structure
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microRNA (miRNA)
Cell, 116, 281, 2004
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miRNA biogenesis pathway
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• following debranching of lariat-structures known as mirtrons, (iii)
• alternative folding of transfer RNAs (tRNAs) or small nucleolar RNAs (snoRNAs), or (iv)
• By tRNAse Z cleavage of pri-miRNAs containing tRNA-like structures linked to pre-miRNAstem-loops
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RNA Pol II—intron encoded miRNARNA Pol III—independently encoded miRNA minor group
Formation of MicroRNA cont.
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miRNA Gene• multiple isoforms
• 12 loci for let‑7‑family miRNAs• Approximately 50% of mammalian miRNA loci are found in
close proximity• a single polycistronic transcription unit
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miRNA Gene
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Evolutionarily conserved small noncoding RNAs in eukaryotes
phylogenetically conserved; ~55%
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• ~30% of mammalian genes are regulated by miRNAs• miRNAs are often expressed at more than 2,000 copies per cell in a
cell-type and tissue-specific manner
NATURE REVIEWS | MICROBIOLOGY-VOLUME 4 -2006 | 651
MIRNA feature
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miRNA function Variation • Repressing Translation
• with individual sites less than a half and often by less than a third• many more targets dramatically repressed
• modest mRNA destabilization (2-fold)• heterochromatin formation• Enhancing translation
• HCV
• Enhancing gene expression aRNAs (small activating RNAs)• Promoter targeting
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miRNA Function
Mode of Action of miRNAs in Plants and Animals
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nucleotides 2–8 prearranged in a geometry resembling an A-form helix
miRNA Target Sites
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Processing bodies are sites of storage and/or degradation of mRNA
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microRNAs and Pbodies
• Localization of RISC-bound mRNAs often to cytoplasmic processing bodies (Pbodies)
• which exclude the translational machinery• mRNA remodeling• decapping• de-adenylation, as well as exonucleases
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Regulation of miRNA • Alternative cleavage and polyadenylation
• In proliferating cells the shorter UTRs • only of conserved miRNA sites
• longer isoforms that dominate in nonproliferating cells• Blocking by the binding of Deadend RNA-binding protein in
the germline• Site masking by protein • blockage of Drosha processing• interference with Dicer processing• controlled at the nuclear export step• uridylation of pre-miRNA (14 u in 3’ end)
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Target-Prediction Tools
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Target-Prediction Tools
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microRNA; small is mighty!
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MicroRNAs in disease diagnostics• miRNAs displayed high stability in paraffin-embedded tissues
from clinical samples or in human plasma• miRNA profiles could distinguish a tumor’s developmental
origin• miRNA signatures were further used to define subtypes of
cancers• such as the distinction between basal and luminal breast cancers
• miRNA expression profiles to distinguish forms of heart disease
• muscular disorders• neurodegenerative diseases
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Rosetta Genomics diagnostic panels
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MicroRNAs have been reported to be:
• Differentially expressed in tissues• Critical in the development of organisms• Associated with oncogenes• Involved in viral infection processes• Neurodegenerative diseases• Cardiovascular diseases
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Differential expression of MicroRNAs
• MicroRNAs (miRNAs) are an abundant class of negative gene regulators that have been shown to control a wide range of biological functions such as cellular proliferation, differentiation and apoptosis.
• Different tissues express different types of miRNAsIt indicates that:• Specific miRNAs have specific roles in different
conditions
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MicroRNAs regulate cell cycle
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MicroRNAs regulate ES
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MicroRNAs regulate Hematopoisis
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MicroRNAs regulate Myogenesis
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MicroRNAs regulate Neurogenesis
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MicroRNAs regulate osteogenesis & Skin
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Distribution of US patents in miRNA’s fields
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MicroRNAs in Clinic
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Some MicroRNAs Pepline
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STEM CELLS technology research center projects
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MicroRNAs
Diagnosis
Biomarkers
Stem Cells
Differentiation
Diseases
Cancer
Prostate
Breast
Glioma
ALL
Liver & Metabolics
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miRNA expression on metastatic breast cancer
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• The most incident malignancy in women • The leading cause of cancer-related death in women in developed countries• Accounts for one-sixth of cancer deaths in the United States. • 1.15 million new breast cancer cases and over 500,000 deaths reported
around the world annuallyIn Iran:
• The incidence rate is 7000 case annually• The incidence age is 10 years lower in Iran(35-45y)• 60% of all patients treated for early-stage disease ultimately developed
recurrence, mostly metastatic (30% in the world)• 15.8% Triple-Negative Breast cancer(TNBC)• Survival time in 65-70% of cases is <=5 years
Breast cancer
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Oncotarget 2011.1: 563-57641
Selection of Wnt signaling pathway interfering in metastasis of breast cancer
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1. Signaling pathway study(selection of target genes)
2. miRNA predictionI. miRWalk, TargetScan 6.2 ، Miranda ، PicTar ,MicroCosmII. miRWalk , miR Path
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CTNNB1(β-catenin)RhoA
ROCK1
miR-340miR-381
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Aggregate Pct
Total Contex Score
Total Poorly conserved
Conserved miRNA Gene Name
<0.1 -0.03 1 7mer-m8 - miR-340CTNNB1<0.1 -0.04 1 - 7mer-1A miR-381
<0.1 -0.07 1 - 8mer miR-340RhoA<0.1 -0.03 1 7mer-1A - miR-381
<0.1 -0.05 3 8mer
7mer-1A
7mer-1A miR-340
ROCK1<0.1 -0.08 2 7mer-1A 7mer-1A miR-381
Gene Name miRNA mirSVR score PhastCons score Position in 3’UTR
CTNNB1
miR-340 -1.0515 0.7864 293
miR-381 -0.4484 0.7454 816
RhoA
miR-340 -1.2938 0.7720 315
miR-381 -0.5789 0.8181 598
ROCK1
miR-340 -1.0044 0.5830 1050
miR-381 -0.1714 0.6752 1725
Gene Name Pictar Score Score per species miRNA Probabilities Free Energies kcal/mol
CTNNB1 1.09 0.98 - -381hsa miR 0.63 -18.6RhoA 1.53 1.55 - -381hsa miR 0.79 -15.5
ROCK1 0.79 0.58 - -381hsa miR 0.45 -22.6
Target Scan
microRNA.org
Pictar
Example of Bioinformatics:
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Results of Luciferase assay
Genes expression in MCF-7 and MDA-MB-231 miRs expression in MCF-7 and MDA-MB-231
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Compare of Gene expression before and after miRNA transduction
Migration AssayInvasion assay
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Pathological interpretation
Liver: Diffused and patchy hepatic necrosis
Presenting of inflammatory cellsGeographical liver infarction
Lung: filling of alveolar space by diffuse hemorrhage and collapseAcute and severe inflammation
Brain: NegativeBone: Negative
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10 nude female mice5mice in each groupInjection of 5×105 cellsMDA-miR-340/ control vectorTo the tail vain28 days follow upScarify the miceSeparation of LungLiver, Bone and Brain
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11. Usage of bioinformatics for miRNA predictions2. Confirmation of prediction3. Stem-loop assumption for miRNA expression analysis4. New therapeutics approaches base on molecular techniques for
metastatic Breast cancer5. Tumor suppressor miRNAs for adjuvant therapy6. New and less harmful miRNA delivery systems7. Directly ushering miRNAs to the Cancerous cells
Suggestions for future
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microRNA-mediated suppression of Androgen receptor pathway in prostate cancer
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Prostate Cancer• Prostate cancer (PC) is one of the major
problems in public health around the world.• It is estimated that 1 in every 6 men may develop
PC and 1 in 30 will die of this metastatic disease.
• About 80% of all men in their eighties show pathologic evidence of prostate cancer when they died
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Cancer death rate among men 1930-2006
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Androgen Receptor Pathway• Central role in initiation and progression of
prostate cancer.
Taplin,2007
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Prostate cancer therapeutic strategies
Loriot 2012
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Target Genes• Receptors: AR, IGF1R, IL6R, • Growth factors: VEGFa, IGF1, EGF• AR co activators and TFs: HSP90, HDAC6,
NCOA1,NCOA2,NCOA3, NCOA4, PAG1, KAT2B, KLK3,KLK2, Tnk2, Carm1, ERG, HIF1a, RAC3, KAT5 ,EP300 ,TRAM1,CYP17A1
• 5α reductase:SRD5A1,SRD5A2• AR downstream genes: ERBB2, TMPRSS2,PSA
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Targetscan
Excell File for Each Gene
Pictar Microcosm
Miranda
Mirwalk
MiRNApath
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Viability of prostate cancer cell lines after miR transduction
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DU-145 flow cytometry after miR transduction
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NeuroGlioma and microRNAs
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miRNAs and Glioma
Glioblastoma multiforme (GBM) is the most common and aggressive type of human brain tumor.
Despite advances in surgery, radiation therapy, and chemotherapy, the prognosis of patients with gliomas has not significantly improved.
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Methods and Results• Rao and his partner investigated expression profile of miRNAs in glioma patient and
normal case and introduce various miRNAs associated with GBM pathogenesis such as miR-129-5p.
• U87, A172 and U373 cell lines were infected with lentiviruses containing mir-129-5p precursor sequence. The effects of ectopic expression of miR-129-5p on GBM phenotype were examined by cell cycle analysis and apoptosis assays. MiR-129-5p cytotoxicity effect was measured by the MTT assay.
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Cell cycle results
In addition to induction of apoptosis, miR-129-5p could
declined cell growth and inhibited cell cycle and amplification of
glioma cells
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Luciferase assay results
Control R-Virus NR-virus0
0.2
0.4
0.6
0.8
1
1.2
Rel
ativ
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cife
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act
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Control 72h 96h
According to Target Scan, miRwalk, MiRand and other predicted programs, CDK6 is a target of miR-129-5p. CDK6 have 6 recognition sites for miR-129-5p.
CDK6 was validated by luciferase assay as direct target of miR-129-5p.
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Acute Lymphoblastic Leukemia(ALL)
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Acute lymphoblastic leukemia (ALL) is a form of leukemia, or cancer of the white blood cells characterized by excess lymphoblasts.
ALL accounts for approximately 70% of all childhood (ages 0 to 19 years) leukemia cases, making it the most common type of childhood cancer
leukemia is the 12th(But in Iran 5th) most common class of neoplastic disease
And the 11th most common cause of cancer related death
Acute Lymphoblastic Leukemia
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The Notch signaling cascade
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Selection of hub genes
Signaling pathway study(selection of target genes)
Notch signaling pathway study Determination of associated genes in ALL (NCSTN-ADAM17-CMY-NOTCH1-PSEN1-APH1)
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miRNA predictionI. miRBase, TargetScan 5.1, Miranda, PicTar, MicroCosm
II. Diana, miR Path, mirZ
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QPCR for two cell lines
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NCSTN
APH1
PSEN1
c-MYC
ADAM17
NOTCH1Roc
k1Roc
k2
CTNNB10
102030405060708090
100
JurkatMolt-4
Gen
e E
xpre
ssio
n
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QPCR for two cell lines after miRNA transduction
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luciferase assay, Flow cytometry and Western bloting
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Flowcytometry
Western blotting
Expression-149
14192429
Plex-jred-turboGFPmiRNA-15miRNA-150
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Conclusion
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miRNAs are next important class of therapeutic molecules after siRNA
the theory of “miRNA replacement therapy”miR-15 and miR-150 new candidates for miRNA replacement
therapyNew delivery systems based on liposomemiRNA targeted therapy in patients
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Differentiation of CD133 + cells into T-lymphoid lineage
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miR-146a and miR-150 promote the differentiation of CD133 + cells into T-lymphoid lineage
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Surface marker expression
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Monocyte differentiation
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Bypassing the maturation arrest in myeloid cell line U937 by over-expression of microRNA-424
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Expression analysis of monocyte markers using real-time PCR and flowcytometry
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Specific microRNA expression in Chronic myelogenous leukemia (CML) as a diagnostic biomarker
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Chronic myelogenous leukemia (CML)
• CML is a clonal bone marrow stem cell stem disorder. • It is a type of myeloproliferative disease associated
with a characteristic chromosomal translocation called the Philadelphia chromosome
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The evaluation of Specific microRNA expression by Real time PCR in CML
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Effects of microRNAs on fatty liver
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What is fatty liver?Abnormal accumulation of TAG in liver, causing cirrhosis, liver failure, and hepatocellular carcinoma
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miR-122 correlation with fatty liver
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Bioinformatic data mining
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Supporting data
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What is the use?Lipin-1 and CTDNAEP-1 are two important protein in TAG biosynthesis in liver, targeted by miR-122 So it is logically possible to interfere with the normal biosynthesis of the pathway by introducing miR-122 in liver
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Future Activity MicroRNA
in Vivo
Small Animal
Large Animal (primates)
Safe Transfection
Safe Viral Vectors
Targeted Nanoparticles
Targeted Oligo RNA’s & Gene Constructs
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Thank You
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