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Noncoding RNAs in cardiovascular disease – potential as biomarkers and more

Ali Bierly, PhDallison.bierly@qiagen.comApril 19, 2016

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Agenda

1. Introduction to cardiovascular system and diseases

2. Introduction to noncoding RNAs (microRNA and lncRNA)

3. Noncoding RNAs in cardiovascular diseases – what’s known, and new research

4. Tools for total RNA research (mRNA, miRNA and lncRNA)

5. Additional resources

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Cardiovascular diseases

Diseases of the heart and blood vessels

Coronary artery

disease

High blood pressure

Congestive heart failure

Cardiac arrestArrythmia

Peripheral artery

disease

Stroke

Top cause of death globally – includesdiseases of the heart and blood vessels

Behavioral risk factors: Tobacco use Diet/obesity Physical inactivity Excessive alcohol use

Early detection and management is key to successful control*

* Source: WHO Cardiovascular diseases (CVDs) fact sheet

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The cardiovascular system

Image CC BY-SA 2.1 JP http://creativecommons.org/licenses/by-sa/2.1/jp/

Primary responsibilities: Blood circulation, leading to:

Nutrient transport, such as amino acids Oxygen transport CO2 transport Hormone circulation Blood cell circulation

Primary organs/vessels involved: Heart (pumps blood to body and lungs) Lungs (pulmonary circulation, oxygenates blood) Arteries (deliver oxygenated blood) Veins (return blood to the heart) Capillaries (connect arteries and veins)

Blood component of the circulatory system, which also includes the lymphatic system

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Major cardiovascular diseases

Vascular diseases

Coronary artery disease (can lead to myocardial infarction)Peripheral arterial diseaseCerebrovascular disease (including stroke)Renal artery stenosis

Heart diseases

Cardiomyopathy – diminished ability of heart muscle to contractHypertrophic cardiomyopathy – the above, due to thickening of myocardium Hypertensive heart disease – complications of high blood pressure; left ventricular hypertrophy, atherosclerosis, congestive heart failure, atrial fibrillationHeart failure – heart can’t pump enough anymore, can result from a number of CVDsValvular heart disease

Question: Can noncoding RNA biomarkers help us detect CVD early?

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Noncoding RNAs – microRNA overview

microRNA (miRNA) Naturally occurring, ~22-nt noncoding RNAs Mediate post-transcriptional gene regulation Circulating miRNAs are detectable in serum and plasma – good potential biomarkers Detectable by qPCR, sequencing or microarray or a discovery-verification combination of

sequencing followed by qPCR Involved in many biological pathways, including apoptosis, cell differentiation and

development and immunity, as well as diseases from cancer to cardiovascular to diabetes

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microRNA biogenesis

Noncoding RNAs – microRNA overview

Transcribed by RNA polymerase II as long primary transcripts (pri-miRNAs), which may contain more than one miRNA

In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III-like enzyme Drosha

Pre-miRNAs are then exported to the cytosol by Exportin 5

In the cytosol, RNAse III-like Dicer processes pre-miRNAs into mature miRNAs

Mature miRNAs are incorporated into RISC

miRNAs with high homology to target mRNAs lead to mRNA cleavage

miRNAs with imperfect base pairing to target mRNAs lead to translational repression and/or mRNA degradation

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Long noncoding RNA (lncRNA) Noncoding transcripts >200 nt (some may code for “micropeptides”)

Not strongly conserved across species in general, but some strongly conserved elements

Involved in regulating gene transcription, post-transcriptional regulation and epigenetic regulation

Noncoding RNAs – lncRNA overview

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lncRNAs are a novel class of RNAs larger than 200 nucleotides

Noncoding RNAs – lncRNA overview

Martin, L., Chang, H.Y. (2012) Uncovering the role of genomic "dark matter" in human disease. J Clin. Invest. 122, 1589

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What’s currently known about ncRNA in CVD research?

Acute coronary syndromes Coronary artery disease Heart failure diagnosis

Upregulated

miR-1, miR-21, miR-30a, miR-30c, miR-34a, miR-122, miR-126, miR-133a/b, miR-134, miR-145, miR-146a, miR-155, miR-186, miR-195, miR-198, miR-199, miR-208, miR-208b, miR-223, miR-320a, miR-328, miR-370, miR-423-5p, miR-433, miR-485-3p, miR499

Upregulated

miR-21, miR-25, miR-92a, miR-106b, miR-122, miR-133a, miR-135a, miR-140-3p, miR-146a, miR-155, miR-182, miR-186, miR-208b, miR-370, miR-451, miR-490-3p

Upregulated

miR-18b*, miR-21, miR22, miR-29b, miR-30a, miR-92b, miR-122, miR-129-5p, miR-133a, miR-142-3p, miR-200b, miR-210, miR-320a, miR-423-5p, miR-499, miR-519e*, miR-520d-5p, miR-622, miR-675, miR-1254, HS_202.1

Downregulated

let-7b, miR-29a, miR-122, miR-125b, miR-126, miR-155, miR-223, miR-320b, miR-375, miR-663b, miR1291

Downregulated

miR-21, miR-29a, miR-31, miR-125b, miR-147, miR-181a, miR-214, miR-320b

Downregulated

miR-30b, miR-103, miR-107, miR-125b, miR-126, miR-139, miR-142-3p, miR-142-5p, miR-342-3p, miR-497

Information from Romaine, S.P.R. et al. (2015) microRNAs in cardiovascular disease: an introduction for clinicians. Heart doi:10.1136/heartjnl-2013-305402

microRNAs with differential expression in cardiovascular diseases

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* Information from Uchida, S. and Dimmeler, S. (2015) Long noncoding RNAs in cardiovascular diseases. Circ. Res. 116, 737.

** Information from Kataoka, M. and Wang, D.-Z. (2014) Non-coding RNAs I including miRNAs and lncRNAs in cardiovascular biology and disease. Cells 3, 883.

What’s currently known about ncRNA in CVD research?

lncRNAs that have been associated with cardiovascular diseases

Acute myocardial infarction: HIF-1 AS*, MALAT1*, KCNQ1OT1*, MIAT**, ANRIL NR_003529*

Cardiac hypertrophy: CHRF (induces hypertrophy by acting as a sponge formiR-489, de-repressing its target MyD88)*, Mhrt (downregulated after pressure overload, protective of heart function when overexpressed)*

Coronary artery disease susceptibility: ANRIL**

Many more are being investigated by RNA-seq and greater characterization is yet to come.

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Recent advances in noncoding RNA biomarkers for CVDs

microRNAs in circulating HDL help predict vulnerability of coronary artery disease patients

Niculescu, L.S. et al. (2015) miR-486 and miR-92a Identified in Circulating HDL Discriminate between Stable and Vulnerable Coronary Artery Disease Patients.

PLoS One 10, e0140958.

Aim: Determine if there’s any relationship between the microRNAs associated with circulating lipoproteins and stability or vulnerability in CAD patients

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What is coronary artery disease?

Cause: Buildup of plaque in the arteries (atherosclerosis), restricting flow of oxygen-enriched blood to the heart by narrowing the arteries. Plaque breaking up can result in blood clot formation on top of the ruptured plaque, blocking blood flow.

Possible consequences: Angina (chest pain)

Myocardial infarction (heart attack, death of heart muscle due to lack of blood flow)

Arrhythmias

Heart failure

What is a stable vs vulnerable plaque?

A vulnerable plaque is at risk for rupturing, causing blood clots and driving patients toward heart attack. A key goal in the CAD field is to find an early detection method for vulnerable plaques / vulnerable patients in order to help prevent coronary events.

Image: Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. Used under Creative Commons Attribution 3.0 Unported license.

Question: Can microRNA biomarkers help identify vulnerable CAD patients?

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Niculescu et al.’s approach: qPCR and statistical analysis

Four groups of subjects: stable angina (SA), unstable angina (UA), 1 month post-myocardial infarction (MI) and healthy controls

Isolated serum for biochemical and microRNA analysis, and generated 3 independent pools for each group of subjects.

Profiled serum parameters (cholesterol, triglycerides, fasting glucose, etc.) Isolated/characterized serum lipoproteins (IDL, LDL, HDL, HDL2, HDL3) by

ultracentrifugation

Isolated and analyzed miRNAs from sera and lipoprotein fractions

Used miRNeasy Serum/Plasma Kit for isolation Screened serum miRNAs in a pool of sera from 8 random individuals per group with

Human CVD miScript miRNA PCR Array Used individual miRNA TaqMan assays for 7 microRNAs

in all 111 (95 CAD, 16 control) subjects Used binary logistic regression model to determine predictive

value of the microRNAs for vulnerable CAD patients

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Findings and relevance – miRNA biomarkers in CAD

From the array, they found 48 upregulated miRNAs in serum of SA, 38 in UA and 38 in MI – MI group’s microRNAs were considered top-rank

The highest ranked were miR-486, miR-92a and miR-122, and the group analyzed them with individual qPCR assays as well as 3 others due to previous literature (miR-146a, miR-125a and miR-33a)

No significance in differences between SA and UA serum in the larger cohort for these microRNAs; small & significant increase for miR-122, miR-146a and miR-125a in MI vs SA

The 6 selected microRNAs were mostly associated with HDL, varying by subfraction

HDL from CAD patients showed highest levels of miR-486 and miR-92a (predominantly associated with HDL2 and HDL3, respectively)

miR-486 was much higher in HDL2 from UA and MI compared to SA miR-92a in HDL3 was higher in UA and MI compared to SA

miR-486 and miR-92a associated with HDL can distinguish between vulnerable and stable CAD patients – may warrant further study along with a combination of these miRNAs with apolipoprotein levels, PON1 activity and HDL/LDL ratio (data not discussed here)

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Recent advances in noncoding RNA biomarkers for CVDs

microRNA biomarkers identify diffuse myocardial fibrosis

Fang, L. et al. (2015) Circulating microRNAs as biomarkers for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy. J. Transl. Med. 13, PMID: 26404540

Aim: To find circulating biomarkers for myocardial fibrosis to alleviate the problems of traditional cardiac magnetic resonance (CMR) imaging techniques for diagnosis.

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Diffuse myocardial fibrosis and hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM): Thickening of the myocardium that damages cardiac muscle function and can lead to heart

failure or unexpected cardiac death.

Usually asymptomatic

Genetic component – can be inherited (often a mutation in the beta myosin heavy chain or the cardiac myosin binding protein C gene) or de novo mutation in beta myosin heavy chain gene

Diffuse myocardial fibrosis: Early feature of HCM

Associated with poor prognosis

Difficult to diagnose

Image: Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. Used under Creative Commons Attribution 3.0 Unported license.

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Fang et al.’s approach

Used cardiac magnetic resonance (CMR) imaging and postcontrast T1 mapping time to identify diffuse myocardial fibrosis in HCM patients

Collected blood samples from the same patients prior to CMR and isolated RNA from plasma using the miRNeasy Mini Kit.

microRNA was surveyed with the miScript Serum & Plasma miRNA PCR Array

Individual miRNA assays were used to follow up the array results on dysregulated miRNAs

Receiver operating characteristic (ROC) curve was used to determine how individual miRNAs could diagnose fibrosis, and a logistic regression model was used to determine probabilities.

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Findings and relevance: biomarkers for diffuse myocardial fibrosis

Identified 14 miRNAs that were increased in patients with diffuse myocardial fibrosis compared to healthy controls in the initial screen of 8 HCM patients and 4 controls

12 of these were confirmed by individual qPCR assays in a larger cohort of 55 HCM patients

AUC for all 14 miRNAs, the 12 validated miRNAs from the screen plus miR-29a-3p and miR-133-3p, was 0.87 for prediction of diffuse myocardial fibrosis

The logistic regression model with backward stepwise method narrowed this down to 8 miRNAs, still with an AUC of 0.87

miRNAs identified as potential biomarkers for myocardial fibrosis

miR-18a-5p miR-30d-5p miR-21-5p miR-193-5p

miR-10b-5p miR-296-5p miR-29a-3p miR-15a-5p

This microRNA signature might someday be developed into an alternative diagnostic option that is less expensive and more available than CMR, with no contraindications for renal dysfunction or implanted cardiac devices.

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Recent advances in noncoding RNA biomarkers for CVDs

High plasma expression of microRNAs in coronary artery disease

Zhou, J. et al. (2016) miRNA 206 and miRNA 574-5p are highly expression [sic] in coronary artery disease. Biosci. Rep. 36, e00295

Aim: Identify key microRNAs in CAD that could be turned into diagnostic biomarkers in the future.

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Zhou et al.’s approach:

Isolated blood from CAD patients and healthy controls (defined as individuals with no coronary stenosis or atherosclerotic vascular disease)

Initial screening of microRNAs was done via microarray

Individual microRNA assays (miScript Primer Assays) were used to follow up by qPCR

Used statistical analysis to determine the strength of the microRNAs’ predictive ability for CAD

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Findings and relevance: high miRNA expression in CAD

Identified 33 microRNAs differentially expressed in CAD patient plasma (3 samples)

2 microRNAs were selected for analysis in a larger sample group, miR-206 and miR-574p – they were upregulated very strongly compared to controls (8.74-fold and 29.53-fold) and their targets were possibly related to CAD development

miR-206 and miR-574p were confirmed as consistently upregulated in CAD via qPCR assay in 67 CAD patients and 67 healthy controls

ROC curve analysis showed a 0.607 AUC value for miR-206 and a 0.696 value for miR-574-5p

These 2 microRNAs are possibly promising for development of early biomarkers for CAD

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Technologies for total RNA discovery – Isolation

Total RNA isolation – miRNeasy Kits

FFPE KitMicro KitMini KitSerum/Plasma Kit

Purify total RNA 18 nt and up, including mRNA, miRNA and lncRNA

Can also purify separate miRNA-enriched fraction and total RNA >200 nt fractions (Mini/Micro kits)

Products are suitable for quantitative RT-PCR, Northern blot and microarray analysis

Automatable on QIAcube

Use with up to 200 µl serum or plasma

Internal normalization controlavailable

Visit webpage for more info

Animal cells/tissues, including difficult-to-lyse tissues

Visit webpage for more info

Works with small amounts of cultured cells (1 x 106 cells)

Works with small amounts of animal/human tissue (≤5 mg)

Visit webpage for more info

Lysis buffer efficiently releases

RNA while avoiding degradation

Subsequent incubation at 80°Creverses formalin crosslinking

Visit webpage for more info

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Technologies for total RNA discovery – mRNA expression

RT2 Profiler PCR Arrays and Assays Available for 13 species

Lab-verified assays

Multiple arrays for CVD-related pathways, with gene lists selected by our experts:

Endothelial cell biology Atherosclerosis Lipoprotein signaling & cholesterol metabolism Angiogenesis VEGF signaling Cardiotoxicity Hypertension View the full list

Preamplification Optional for small samples – RT2 PreAMP cDNA Synthesis

Kit and Primer Mixes

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Profile 84 different genes using one array

Appropriate controls for

data normalization sample quality reaction performance

Lab-verified assays for guaranteed performance

Gene expression profiling – RT2 Profiler PCR Arrays & Assays

Technologies for total RNA discovery – mRNA expression

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Technologies for total RNA discovery – microRNA expression

microRNA expression profiling and functional analysis

qPCR arrays and assays

miScript microRNA PCR Arrays and Primer Assays

CVD-related arrays: Cardiovascular Disease

Apoptosis

Cell Differentiation & Development

Serum and Plasma

miFinder

Full miRNome arrays: Through miRBase V21 for human and

mouse

Leading coverage for dog, rat, rhesus macaque and cow

Functional analysis miScript miRNA Mimics

miScript miRNA Inhibitors

miScript miRNA Target Protectors

qPCR reagents and preamplification miScript PreAMP PCR Kit and Primer

Mixes (optional step for small samples)

miScript II RT Kit

miScript SYBR Green PCR Kit

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Technologies for total RNA discovery – microRNA expression

Profile 84 different miRNAs using one array

Appropriate controls for

data normalization sample quality reaction performance

Lab-verified assays for guaranteed performance

miScript miRNA PCR Arrays

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microRNA list for the Human Cardiovascular Disease miScript array

Technologies for total RNA discovery – microRNA expression

Myocardial Infarction:Up-Regulated: let-7e-5p, miR-122-5p, miR-126-5p, miR-133b, miR-145-5p, miR-146a-5p, miR-15b-5p, miR-208a, miR-208b, miR-223-3p, miR-320a, miR-499a-5p.Down-Regulated: miR-1, miR-107, miR-130a-3p miR-133a, miR-143-3p, miR-155-5p, miR-16-5p, miR-195-5p, miR-21-5p, miR-214-3p, miR-22-3p, miR-24-3p, miR-26a-5p, miR-26b-5p, miR-494.

Cardiac Hypertrophy:Up-Regulated: let-7b-5p, let-7c, miR-103a-3p miR-125b-5p, miR-140-5p, miR-142-3p, miR-146a-5p, miR-18b-5p, miR-195-5p, miR-199a-5p, miR-208a, miR-208b, miR-21-5p, miR-214-3p, miR-221-3p, miR-222-3p, miR-224-5p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-25-3p, miR-27a-3p, miR-27b-3p, miR-31-5p, miR-424-5p.Down-Regulated: miR-1, miR-126-5p, miR-133a, miR-133b, miR-149-5p, miR-150-5p, miR-181b-5p, miR-185-5p, miR-29a-3p, miR-29b-3p, miR-29c-3p, miR-30e-5p, miR-451a, miR-486-5p, miR-93-5p.Regulated: miR-182-5p.

Cardiomyopathy:Up-Regulated: let-7c, miR-100-5p, miR-103a-3p, miR-10b-5p, miR-125b-5p, miR-140-5p, miR-145-5p, miR-146a-5p, miR-181b-5p, miR-195-5p, miR-208a, miR-208b, miR-21-5p, miR-210, miR-214-3p, miR-221-3p, miR-222-3p, miR-23a-3p, miR-328, miR-342-3p, miR-423-3p, miR-499a-5p.Down-Regulated: miR-1, miR-125a-5p, miR-126-5p, miR-133a, miR-133b, miR-143-3p, miR-29b-3p, miR-365a-3p, miR-378a-3p, miR-7-5p, miR-92a-3p.

Differentiation/Development:Up-Regulated: let-7a-5p, let-7b-5p, let-7c, let-7d-5p, let-7f-5p, miR-1, miR-133a, miR-143-3p, miR-144-3p, miR-145-5p, miR-17-5p, miR-181a-5p, miR-206, miR-208a, miR-21-5p, miR-24-3p, miR-26a-5p, miR-27a-3p, miR-27b-3p, miR-30a-5p, miR-30c-5p, miR-30d-5p, miR-378a-3p, miR-93-5p, miR-98-5p, miR-99a-5p.Down-Regulated: miR-124-3p, miR-125b-5p, miR-183-5p, miR-302a-3p, miR-302b-3p.

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Technologies for total RNA discovery – lncRNA expression

qPCR arrays and assays

RT2 lncRNA PCR Arrays (link):Four pathways, available in human and mouse lncFinder Cancer PathwayFinder

Cell Development & Differentiation

Inflammatory Response & Autoimmunity

Custom lncRNA PCR arrays (link): >25,000 human lncRNA assays available

>10,000 mouse lncRNA assays

Can combine lncRNA with mRNA assays

Individual lncRNA qPCR assays (link)

Designed against combined NCBI RefSeq and Ensembl GENCODE database

Compatible with RT2 First Strand Kit and RT2 SYBR Green Mastermix

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Cardiovascular Diseases Research Portal

Visit the research portal here

Links to 24 ebiology stores on CVD-related topics, such as: Angiogenesis

Apoptosis

ECM and adhesion molecules

Hypertension

And many more

Includes tools for: mRNA profiling

microRNA profiling

lncRNA profiling

NGS

Reporter assays

Methylation arrays

RNAi

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Cardiovascular system pathway maps at GeneGlobe

500+ downloadable, editable pathway maps

Multiple maps related to cardiovascular system and diseases:

Cardiomyocyte differentiation Factors promoting cardiogenesis Angiopoietin-TIE2 signaling C. pneumoniae infection in

atherosclerosis Embryonic cell differentiation into

cardiac lineage NFAT in cardiac hypertrophy VEGF pathway Many more

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Questions?

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Ali Bierly, Ph.D.

allison.bierly@qiagen.com