MicroRNA’s applications in medicine

 

microRNA Related Publications

Source: PubMed

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

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

microRNA (miRNA)

Cell, 116, 281, 2004

miRNA biogenesis pathway

• 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.

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

miRNA Gene

Evolutionarily conserved small noncoding RNAs in eukaryotes

phylogenetically conserved; ~55%

• ~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

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MIRNA feature

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

miRNA Function

Mode of Action of miRNAs in Plants and Animals

nucleotides 2–8 prearranged in a geometry resembling an A-form helix

miRNA Target Sites

Processing bodies are sites of storage and/or degradation of mRNA

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

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)

Target-Prediction Tools

Target-Prediction Tools

microRNA; small is mighty!

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

Rosetta Genomics diagnostic panels

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

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

MicroRNAs regulate cell cycle

MicroRNAs regulate ES

MicroRNAs regulate Hematopoisis

MicroRNAs regulate Myogenesis

MicroRNAs regulate Neurogenesis

MicroRNAs regulate osteogenesis & Skin

Distribution of US patents in miRNA’s fields

MicroRNAs in Clinic

Some MicroRNAs Pepline

STEM CELLS technology research center projects

MicroRNAs

Diagnosis

Biomarkers

Stem Cells

Differentiation

Diseases

Cancer

Prostate

Breast

Glioma

ALL

Liver & Metabolics

miRNA expression on metastatic breast cancer

• 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

Oncotarget 2011.1: 563-57641

Selection of Wnt signaling pathway interfering in metastasis of breast cancer

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:

Results of Luciferase assay

Genes expression in MCF-7 and MDA-MB-231 miRs expression in MCF-7 and MDA-MB-231

Compare of Gene expression before and after miRNA transduction

Migration AssayInvasion assay

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

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

microRNA-mediated suppression of Androgen receptor pathway in prostate cancer

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

Cancer death rate among men 1930-2006

Androgen Receptor Pathway• Central role in initiation and progression of

prostate cancer.

Taplin,2007

Prostate cancer therapeutic strategies

Loriot 2012

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

Targetscan

Excell File for Each Gene

Pictar Microcosm

Miranda

Mirwalk

MiRNApath

Viability of prostate cancer cell lines after miR transduction

DU-145 flow cytometry after miR transduction

NeuroGlioma and microRNAs

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.

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.

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

Luciferase assay results

Control R-Virus NR-virus0

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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.

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

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

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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

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

Differentiation of CD133 + cells into T-lymphoid lineage

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

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

What is fatty liver?Abnormal accumulation of TAG in liver, causing cirrhosis, liver failure, and hepatocellular carcinoma

miR-122 correlation with fatty liver

Bioinformatic data mining

Supporting data

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

Future Activity MicroRNA

in Vivo

Small Animal

Large Animal (primates)

Safe Transfection

Safe Viral Vectors

Targeted Nanoparticles

Targeted Oligo RNA’s & Gene Constructs

Thank You

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