miR-142-3p Directly Regulates Autophagy Dependent Gene ATG16L1

by

Frances Dang

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology

University of Toronto

© Copyright by Frances Dang 2014

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miR-142-3p DIRECTLY REGULATES AUTOPHAGY-DEPENDENT GENE ATG16L1

Frances Dang

Master of Science

Department of Physiology University of Toronto

2014

Numerous genome-wide association studies demonstrate that a variant in the autophagy-

dependent gene ATG16L1 is associated with Crohn’s Disease (CD). miRNA regulation

has been shown to contribute to the pathogenesis of many inflammatory diseases

including inflammatory bowel disease. miRNA bind to the complementary 3’

untranslated region (UTR) of the target mRNA to repress their translation and promote

their degradation. Here we investigated the functional effect of miR-142-3p on ATG16L1

and autophagy. HeLa and HCT116 cells were transfected with either a miR-142-3p

mimic or antagonist and qPCR for ATG16L1 and western blotting was performed. In

comparison to control cells, a decrease in ATG16L1 transcripts was detected in cells

transfected with the miR-142-3p mimic. A reduction in ATG16L1 and LC3-II protein

level was also seen in immunoblots. Taken together, these results indicate that miR-142-

3p can directly regulate ATG16L1 and repress autophagy, implicating miRNA regulation

in the pathogenesis of CD.

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Acknowledgements

First and foremost, I would like to express my sincerest and deepest gratitude to

my supervisor, Dr. Nicola Jones, who I am privileged to have had as a mentor over the

past two years. Thank you for being so supportive and understanding to the challenges in

the lab and beyond. You’ve inspired and helped me to grow both as a scientist and a

person. Thank you for helping me in every way you can to succeed with my thesis and

career goals. Many thanks to my supervisory committee, Dr. Patricia Brubaker, Dr.

Stephen Girardin and Dr. Mark Silverberg for their helpful guidance with my project.

They have provided me with great support and motivation to complete this thesis.

To the Jones Lab: thank you for making this research experience so enjoyable.

Thank you Esther Galindo-Mata for your positive outlook to everything and for teaching

me all the lab skills and techniques to help get me started. To Laura Greenfield, thank

you for your support both as a mentor and friend. To Majd Albanna, thank you for being

a good lab mate by working and studying with me at odd hours!

I would like to thank my family, especially my mother and sister for their

encouragement, support and keeping things in perspective. To my amazing girlfriends:

Thank you Lynn for being my rock throughout the past two years. It’s been a tumultuous

ride and I thank you from the bottom of my heart for sticking it out with me through the

highs and lows (we made it!). Thank you to Selina and May for their enthusiasm with the

project and for encouraging me when things don’t work out. Finally, thank you to my

dear Steve, I’m glad that things have worked themselves out and that you were there to

support me whenever I needed you.

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TABLE OF CONTENTS !

Abstract ii Acknowledgements iii Table of Contents iv List of Figures vi List of Tables vii List of Abbreviations viii Chapter 1 Introduction 1 1.1. Inflammatory Bowel Disease 1 1.2. Genetics of IBD 4 1.3. Autophagy 6 1.4. Dysregulation of ATG16L1 in Crohn’s Disease 10

1.5. Additional Genetic Evidence Implicating Autophagy as a Critical Pathway in Crohn’s disease 14

1.6 Beyond Genetics: The Role of the Environment in IBD 16 1.7. Epigenetic Regulation 18

1.8. miRNA Regulation and Biogenesis 20 1.9. miRNA Implicated in Crohn’s Disease 21 1.10. Summary and Hypothesis 22 Chapter 2 Methods 2.1. Cell Culture Conditions 24 2.2. Transfection 24 2.3. Chemically Enhanced Autophagy 25 2.4. Dual Luciferase Assay 25

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2.5. RNA Extraction 27 2.6. cDNA Synthesis 27 2.7. Quantitative Real Time PCR 27 2.8. Immunoblotting 27 2.9. Densitometric Analysis 28 2.10. Immunofluorescence of LC3-GFP tagged cells 29 2.11. LC3 Immunostaining

2.12. Statistical Analysis 30

Chapter 3: Results 3.1. miR-142-3p Inhibits ATG16L1 mRNA Expression 31 3.2. miR-142-3p Targets ATG16L1 33

3.3 miR-142-3p Reduces Endogenous Autophagy 36 3.4. miR-142-3p Does Not Reduce Chemically Induced Autophagy 43

Chapter 4: Discussion and Future directions 50 References 61 !!!!!!!!!!!!!!!!!

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LIST OF FIGURES !

Title&&

Figure 1 Model of factors involved in the pathogenesis of IBD 3 Figure 2 Overview of the autophagy pathway in mammalian cells 9 Figure 3 Consequences of ATG16L1 Dysfunction 13 Figure 4 Vector backbone of miRNA 3’UTR target clones 26 Figure 5 miR-142-3p reduces ATG16L1 mRNA and protein expression in HeLa

and HCT116 cells 32 Figure 6 miR-142-3p directly targets the 3’UTR of ATG16L1 35 Figure 7 miR-142-3p reduces endogenous autophagy: Western Blot 39 Figure 8 miR-142-3p reduces endogenous autophagy: LC3-GFP

Immunofluorescence 40 Figure 9 miR-142-3p reduces endogenous autophagy: LC3 Immunostaining 42 Figure 10 Induction of autophagy with rapamycin treatment 44 Figure 11 miR-142-3p does not reduce chemically induced autophagy: Western Blot

Analysis 46 Figure 12 miR-142-3p does not reduce chemically induced autophagy: LC3

Immunostaining 48 Figure 13 How the environment and epigenetics could alter expression of ATG16L1 55 Figure 14 Proposed model of miRNA regulation of autophagy in IBD 57

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LIST OF TABLES

Table 1 miRNAs predicted to target ATG16L1 23 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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LIST OF ABBREVIATIONS

Ago Argonaute

AIEC Adherent Invasive E. coli

ATG Autophagy Dependent Gene

CD Crohn’s Disease

DMEM Dulbecco’s Modification of Eagle’s Medium

DSS Dextran Sodium Sulphate

E. coli Escherichia coli

FBS Fetal Bovine Serum

GI Gastrointestinal

GWAS Genome-wide Association Studies

H. pylori Helicobacter pylori

HeLa Human Cervical Carcinoma Epithelial Cells

IBD Inflammatory Bowel Disease

IFN-� Interferon gamma

IL-6 Interleukin-6

IL-1� Interleukin-1�

IRGM Immunity Related Guanosine Triphosphatase M

LC3 Microtubule-Associated Protein Light Chain 3

LRRK2 Leucine-Rich Repeat Kinase 2

miRNA microRNA

mRNA Messenger RNA

mTOR Mammalian target of rapamycin

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NF�B Nuclear Factor Kappa-Light-Chain-Enhancer of

Activated B Cells

Nod2 Nucleotide Binding Oligomerization Domain Containing

Protein 2

Nod2fs NOD2 Frameshift Mutation

PAMP Pathogen Associated Molecular Patterns

PBS Phosphate-Buffered Saline

PI3K Phosphatidylinositol 3-kinase

PPAR Peroxisome Proliferator-Activator

RISC RNA-induced Silencing Complex

qPCR Quantitative Real Time PCR

S. typhimurium Salmonella enterica serovar Typhimurium

siRNA Small Interfering RNA

SNP Single Nucleotide Polymorphism

T300A rs2241880 SNP Nonsynonymous Mutation Encodes for a

Threonine to Alanine substitution at the 300 Position

TBST Tris-buffered Saline with Tween

TNF Tumor necrosis factor

UC Ulcerative Colitis

UTR Untranslated region

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CHAPTER 1: INTRODUCTION

1.1 Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a group of diseases that causes chronic

inflammation of the gastrointestinal tract and is associated with significant morbidity

(Knights, 2013). IBD is a multifactorial disease with genetic, immunological, bacterial,

and environmental factors all contributing to its development (Figure 1). The exact

causative factors in disease pathology are not fully understood. However, the current

hypothesis is that IBD results from the interaction between genetic and environmental

factors that influence the normal intestinal flora and microbiome to elicit an inappropriate

immune response. These contributing factors lead to either excessive up regulation or

impaired resolution of inflammatory events, which ultimately results in the development

of chronic inflammation (Knights, 2013).

IBD is thought to be a disease of industrialization, with the prevalence being

highest in North America, northern Europe and the United Kingdom. The average

number of cases of IBD ranges from 100-200 for every 100 000 persons (Loftus, 2004).

Crohn’s disease (CD) and ulcerative colitis (UC) are the two main subtypes of IBD. CD

is hallmarked by segmental transmural inflammation with sparing intermediary areas,

which can involve any region along the GI tract. UC is characterized by inflammation

that is confined to the colon and involves only the superficial mucosal and submucosal

layers of the intestinal wall. CD most commonly involves the ileum and colon but can

affect any region of the gastrointestinal tract whereas UC always involves the rectum and

inflammation may extend as far as the caecum in a continuous pattern (Cho, 2008). With

time, patients with CD can progress to develop more complications such as strictures,

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fistulas and abscesses (Solberg et al., 2007). Both diseases have considerable morbidity

and shorter life expectancy (Cosnes et al., 2002). There is currently no cure for either CD

or UC and treatment focuses on alleviating symptoms and minimizing recurrence.

Therefore understanding disease mechanisms is of paramount importance in order to

develop novel therapeutic agents.

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!

!!!!Figure!1:!Interactions!of!different!factors!involved!in!the!pathogenesis!of!IBD.!

The!current!model!for!the!etiology!of!IBD!is!interactions!of!various!factors!such!as!

the!environment!and!the!microbiota!in!genetically!susceptible!individuals!trigger!

inappropriate!immune!responses!that!targets!the!GI!tract.!(This!information!was!

adapted!from!Nature!Clinical!Practice,!Gastroenterology!and!Hepatology,!!April!

2006,!Volume!3:!390M407).!

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1.2. Genetics of IBD:

Population based studies demonstrate that genetics contribute to the pathogenesis

of IBD. For example, there is an 8-10 fold greater risk of IBD among relatives of UC and

CD probands and a concordance rate of approximately 35% amongst twins (Lowe et al.,

2009). The advent of genome wide association studies (GWAS) has revolutionized our

understanding of the genetics of IBD. GWAS involve the genotyping of hundred

thousands of variants throughout the genome in very large cohorts to see if they are

associated with any particular trait. Some markers will have significantly different allele

frequencies between disease cases and controls, suggesting that the functional allele can

modify disease susceptibility. The non-biased approach of using GWAS allows for the

identification of novel candidate genes not identified through traditional methodological

approaches and does not require knowledge of the biological pathway of the trait

involved (Hardy and Singleton, 2009). GWAS have identified replicated variations in 99

gene loci associated with IBD. Although there are roughly 28 gene loci associated with

the development of both UC and CD, a large number are attributed specifically to either

one or the other (Lees et al. 2011).

NOD2 polymorphisms were the first definitive risk factors associated with CD

(Cho, 2008). Subsequently, numerous GWAS consistently confirmed the association with

CD. NOD2 is a pattern recognition receptor that senses intracellular bacterial

peptidoglycan (Hisamatsu, 2003). NOD2 is expressed in intestinal epithelial cells and

immune cells such as antigen-presenting cells, macrophages and lymphocytes. Activation

of NOD2 by microbial ligands activates the transcription factor nuclear factor kappa B

(NF-kB) and mitogen activated protein kinase signaling (Abraham, 2006). The three

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polymorphisms (Arg702Trp, Gly908Arg and Leu1007fsinsC) of NOD2 associated with

CD lie within or near the C-terminal, leucine-rich repeat domain that is required for

microbial sensing (Hugot et al., 2001; Ogura et al., 2001). These polymorphisms result in

reduced activation of NFkB, which functions as a positive regulator of immune defense.

Loss of function of NOD2 alone is not sufficient to cause CD. In individuals of

European descent, heterozygous carriers of one of the risk alleles have approximately a

2.4 fold increased risk of CD while homozygous or compound heterozygous carriers have

a 17.1 fold increased risk (Economu et al., 2004). The exact mechanisms responsible for

the increased risk of CD conferred by NOD2 remain unclear. However, NOD2-deficient

mice express lower levels of antimicrobial defensins, increased colonization by

commensal bacteria and impaired clearance of pathogens (Cho and Brant, 2011). Recent

work has shown that NOD2 interacts directly with an autophagy dependent gene

ATG16L1 and recruits ATG16L1 to the site of bacterial entry to promote bacterial

clearance through autophagy. Autophagy is a cellular degradation pathway that is also

important for clearance of intracellular pathogens. The protective NOD2 variant is

localized to the plasma membrane. In contrast, the Leu1007fsinsC polymorphism is

present in the cytosol and displays impaired recruitment of ATG16L1 and subsequently

alters autophagy (Travassos et al., 2010).

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1. 3. Autophagy

Autophagy is an evolutionarily conserved catabolic cellular process by which

cytoplasmic content is delivered for degradation in double membrane-enclosed vesicles

called autophagosomes (Figure 2) (Fritz et al., 2011). One of the fundamental roles of

autophagy is maintaining cellular homeostasis by getting rid of injured, unnecessary or

excessive proteins and organelles. Autophagy also plays a vital role in the innate immune

response by encapsulating and clearing intracellular pathogens such as Helicobacter

pylori, Salmonella typhimurium or Listeria monocytogenes (Scharl and Rogler, 2012).

Therefore, defects in autophagy may underlie the pathogenesis of many different diseases

and inflammatory responses.

The process of autophagy can be divided into several distinct steps including

induction, nucleation and elongation of the phagophore, completion of the

autophagosome, vesicle maturation/fusion with the lysosome and finally cargo

degradation (He, 2009). During autophagy, cytoplasmic content is engulfed by a double

membrane structure called the autophagosome. The autophagosomes fuse with lysosomes

where acidic hydrolases degrade large macromolecules into their basic constituents (Fritz

et al., 2011).

Autophagy is triggered by a variety of signals such as nutrients, growth factors,

hormones, hypoxia and accumulation of misfolded or damaged proteins (Wirawan et al.,

2012). Many of these signals converge at the level of mammalian target of rapamycin

complex (mTORC). mTORC regulates a number of cellular processes including cell

growth, proliferation, protein synthesis and autophagy. The initiation of autophagy

requires inhibition of mTORC, which is triggered by conditions such as starvation,

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growth factor depletion and infection (Misushima et al., 2011). Inhibition of mTORC

promotes downstream activation of ULK1/2 that phosphorylates ATG13 and RB1CC1.

Subsequent ATG proteins stabilize ATG13 and form the mTOR substrate termed the

ULK complex. The ULK complex translocates from the cytosol to the endoplasmic

reticulum (ER). The association of the ULK complex with the ER activates an ER-

localized autophagy-specific class III phosphatidylinositol 3-kinase (PI3K) complex,

which is composed of PIK3C3, PIK3R4, BECN1 and ATG14. BECN1 is an important

regulator in the induction of autophagy and is regulated by BCL2 in a nutrient-dependent

manner. Autophagy is inhibited under nutrient-rich conditions because BCL2 binds to

BECN1, whereas starvation induces autophagy through the dissociation of BCL2 from

BECN1 (He et al., 2009).

Following autophagy induction, two ubiquitin-like conjugation systems are

necessary for autophagosome biogenesis (Wirawan et al., 2012). These systems are

responsible for membrane expansion, shaping and sealing. A complex consisting of

ATG5, ATG12 and ATG16L1 is conjugated to the outer membrane and is required for

autophagosome maturation. This complex is responsible for shaping the membrane and

acting as an E3 ligase, to allow the second conjugation reaction to be completed. The

second ubiquitin-like conjugation process involves microtubule-associated light chain-3

(LC3) lipidation. Through a series of reactions, LC3 is conjugated to

phosphatidylethanolamine in the autophagosome membrane to produce LC3-II. LC3-II

remains associated with the autophagosomal membrane and only becomes degraded after

fusion with the lysosome. Because LC3-II associates with the membrane of the

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autophagosome whereas LC3-I is cytosolic, LC3-II is used as an ideal marker for

autophagy (Yasuko et al., 2006).

The maturation of the autophagosome involves fusion to acidic lysosomes to form

autolysosomes. The autolysosomes contain lysosomal proteases to degrade sequestered

cargo (He et al., 2009). Together these steps encompass the autophagic flux, a continuous

intracellular flow from autophagosome formation to sequestration and degradation of

cargo and final release of breakdown molecules back into the cytosol.

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!!

Figure!2:!Overview!of!the!autophagy!pathway!in!mammalian!cells.!During!

autophagy,!cytoplasmic!material!is!engulfed!by!a!double!membrane!structure!called!

the!autophagosome.!The!autophagosome!then!fuses!with!the!lysosome!where!

sequestered!materials!get!degraded.!The!formation!of!autophagosomes!requires!

two!ubiquitin!like!systems.!Complexes!of!ATG!proteins!5,12!and!16L1!are!

conjugated!together!to!form!the!outer!membrane!of!the!autophagosome.!Through!a!

series!of!reactions,!LC3!is!conjugated!to!produce!LC3MII.!LC3MII!remains!associated!

with!the!autophagosomal!membrane!and!only!becomes!degraded!after!fusion!with!

the!lysosome.!(This!figure!was!originally!published!in!Trends!in!Microbiology!

November!2013,!Volume!21;!602M612.!Copyright!permission!obtained!from!journal.)!!

Initiation: formation ofsequestration crescent

Isolation membrane

Elongation: expansion and cargo recognition

Completion

Maturation: dockingand fusion of lysosome

Degradation

Lysosome

Lysosomalhydrolases

Autophagy

LC3 II

Atg5-Atg12-Atg16 complex

Autophagosome

Autolysosome

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1.4. Dysregulation of ATG16L1 in Crohn’s Disease

As noted above, ATG16L1 is a key protein in the autophagy pathway. The

ATG16L1 protein is comprised of an N-terminal APG16 domain with coil-coiled

domains and eight C-terminal beta-transducin (WD) repeat regions (Mizushima et al.,

2003). The rs2241880 single nucleotide polymorphism (SNP) encodes for a threonine to

alanine substitution (T300A) at the C-terminal of the WD-repeat region, which is thought

to mediate protein-protein interactions. The T300A risk allele frequency is 50% in

healthy individuals and 60% in CD patients of Caucasian descent resulting in an increase

odds ratio for CD of between 1.4-1.9 (Zhang et al., 2009). The exact functional relevance

of the ATG161 CD variant remains unknown. ATG16L1 is broadly expressed in

intestinal epithelial cells and immune cells such as CD4+, CD8+ and CD19+ primary

human T cells (Hampe et al., 2007; Rioux et al., 2007).

Autophagy plays an important role in maintaining intestinal homeostasis and

dysfunction in this pathway poses a risk factor for chronic intestinal inflammation. The

dysregulation of ATG16L1 leads to numerous consequences pertaining to bacterial

clearance, defective antigen presentation, upregulation of inflammatory signaling and

abnormal Paneth cell function (Figure 3).

The Crohn’s disease ATG16L1 variant results in prolonged survival and increased

numbers and replication of intracellular Salmonella, Escherichia coli and Shigella

flexneri (Scharl and Rogler, 2012). A study by Raju et al., show that the CD ATG16L1

variant increases susceptibility to H. pylori infection and peripheral blood monocytes

from individuals with the variant have impaired autophagic responses to VacA exposure

(Raju et al., 2012). In addition, cells with the ATG16L1 CD variant are deficient in

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limiting the replication of adherent invasive E. coli (AIEC), a pathogen associated with

the presence of ileal CD (Lapaquette et al., 2010). Additional studies demonstrate that

ATG16L1 mutant cells infected with AIEC had reduced bacterial clearance and increased

secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF- α) and

interleukin-6 (IL-6) (Lapaquette et al., 2012).

In addition to its role in bacterial clearance, ATG16L1 is also critical in Paneth cell

morphology and function in the crypts of Lieberkuhn in the small intestine. Paneth cells

are epithelial cells that secrete antimicrobial factors such as defensins and lysozymes into

the lumen. In a study by Cadwell et al., mice that were hypomorphic for ATG16L1 had

severe Paneth cell morphology and dysfunction in their granule exocytosis pathway

(Cadwell et al., 2008; Cadwell et al., 2010). Paneth cells from these hypomorphic mice

had increased expression of molecules involved in pro-inflammatory responses such as

peroxisome proliferator-activator receptor (PPAR), adipocytokines, leptin and

adiponectin. Paneth cells from patients homozygous for the ATG16L1 CD risk allele

show similar Paneth cell abnormalities as compared to the ATG16L1 hypomorphic mice,

further suggesting an important role for dysfunction of ATG16L1 in the pathogenesis of

CD (Cadwell et al., 2008; Cadwell et al., 2010).

ATG16L1 is also associated with the inflammasome, a multi-protein complex

activated by various stress factors and is required for maturation of pro-inflammatory

cytokines interleukin-β (IL-1β) and interleukin-18 (IL-18). Saitoh et al., demonstrate that

autophagy is induced following inflammasome activation and serves to limit its activity

by regulating IL-1β activity. In addition, mice deficient for ATG16L1 in hematopoietic

cells have increased susceptibility to dextran sodium sulfate (DSS) induced colonic

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inflammation, which is alleviated by injection with anti-IL-1β and anti-IL-18 antibodies.

These results demonstrate the importance of ATG16L1 in controlling inflammatory

immune responses and suppressing intestinal inflammation (Saitoh et al., 2008).

A recent study by Murthy et al. showed that the ATG16L1 CD variant results in

impaired autophagy due to increased sensitivity of caspase-3-mediated processing of

ATG16L1. The amino acids 296-299 make up a caspase cleavage motif in ATG16L1.

Death receptor activation or starvation-induced stress in both human and murine

macrophages resulted in increased degradation of the T300A or T316A variants of

ATG16L1, leading to decreased autophagy. In addition, knock-in mice that contained the

T316A variant had defective handling and clearance of the ileal pathogen Yersinia

enterocolitica and increased inflammatory cytokine production. However, when the

caspase-3-encoding gene (Casp3) was deleted or the caspase cleavage site was eliminated

by site-directed mutagenesis, starvation-induced autophagy and pathogen clearance was

rescued (Murthy et al., 2014). These findings suggest a possible mechanism of

degradation of ATG16L1 and ties cellular stress, apoptotic stimuli and impaired

autophagy in a unified pathway that leads to genetic predisposition to Crohn’s disease.

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!!

!

Figure!3:!Consequences!of!ATG16L1!dysregulation.!Autophagy helps maintain

intestinal homeostasis and dysfunction in this pathway poses a risk factor for chronic

intestinal inflammation. The dysregulation of ATG16L1 leads to numerous consequences

pertaining to decreased bacterial clearance, defective antigen presentation, upregulation

of inflammatory signaling and abnormal Paneth cell function.

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1.5. Additional genetic evidence implicating autophagy as a critical pathway in

Crohn’s disease

Following the identification of the CD-related polymorphism in ATG16L1,

additional polymorphisms in autophagy related genes were identified. Leucine-rich repeat

kinase 2 (LRRK2) is a protein that gets recruited to the endosomal-autophagic pathway

and polymorphisms in the gene results in impaired autophagic balance (Alegre-

Abarrategui et al., 2009). The rs376186 SNP found within its coding region results in

decreased stability of the protein product and lower expression levels (Barrett et al.,

2008). LRRK2 inhibits activation of NFAT1, a transcription factor that plays a role in

expression of pro-inflammatory cytokines. Multiple lines of evidence suggest a possible

role of LRRK2 in CD pathogenesis. LRRK2 deficient mice are more susceptible to DSS-

induced colitis (Tong et al., 2010) and knock down of LRRK2 in cells results in impaired

capabilities of killing intracellular bacteria. A study by Gardet et al. investigated the

involvement of the interferon- γ (IFN- γ) response of LRRK2, whose locus is located

downstream of the SNP associated with higher risk of CD. LRRK2 was shown to be a

gene target of IFN- γ and its expression is highly upregulated by bacteria-induced or CD

inflammation, suggesting that its transcription is tightly regulated by physiologic

conditions (Gardet et al., 2011). These observations implicate LRRK2 to be involved in

the regulation of mucosal IFN- γ immune responses that are relevant to host responses to

pathogens in CD (Gardet et al., 2011).

The Immunity related guanosine triphosphatase M (IRGM) protein plays an

important role in the innate immunity by regulating autophagy in response to intracellular

pathogens. Functional IRGM regulates the maturation of pathogen-containing vacuoles as

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well as adhesion and motility of macrophages (Scharl and Rogler, 2012). A non-

synonymous polymorphism in the coding region of IRGM is associated with CD. The

polymorphism exists in perfect linkage disequilibrium with a 20-kb deletion

polymorphism upstream of the IRGM transcriptional start site, affecting multiple

transcription-factor binding sites (McCarroll et al., 2008). This results in impaired IRGM

expression and reduced autophagy among specific cell types. Low IRGM levels are

detected in lymphocytes of CD patients with the CD-associated IRGM polymorphisms

(Prescott et al., 2010). In addition, McCarroll et al., show that the efficiency of the

autophagic machinery against S. typhimurium infection is compromised through siRNA

knockdown of IRGM but enhanced by overexpression of IRGM. Consequently, mice

deficient for IRGM have impaired capabilities of eliminating pathogens such as S.

typhimurium, Toxoplasma gondii and L. monocytogenes (Collazo et al., 2001). These

studies demonstrate that a threshold level of IRGM expression is necessary for proper

bacterial handling (Scharl and Rogler, 2012).

Taken together, the associations of SNPs in NOD2, IRGM, LRRK2 and

ATG16L1 specifically in CD demonstrate that dysregulation of bacterial processing

mechanisms is a central feature in the pathogenesis of CD and warrants further studies in

how the autophagy pathway may be implicated in disease.

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1.6. Beyond Genetics: The Role of the Environment in IBD

The incidence and prevalence of IBD is rising worldwide, particularly in the

pediatric population (Aujnaurin, 2013). However, genetic predisposition itself does not

explain fully the increases in incidence and prevalence of CD and UC over the past few

decades (Aujnarain et al., 2013). Although many genetic risk loci have been identified,

incomplete penetrance and overlapping genotypes among patients with different

phenotypes inadequately explain its etiology. IBD has been characterized primarily as a

disease of westernized nations, with increased prevalence in developed nations. Migrant

studies have shown that individuals immigrating from countries of low to countries of

high prevalence are at increased risk of developing IBD (Bernstein, 2008). Taken

together, these findings suggest that environmental factors contribute to the development

of IBD and the observed increased incidence in the last decades.

A number of theories have been proposed to answer the unknown environmental

exposures that may interact with the host immune system and elicit an abnormal

inflammatory response. The most predominant theory is the hygiene hypothesis, which

suggests that the increase of immunologic disorders is due to lack of childhood

sensitization to enteric antigens (Shanahan, 2011). In addition, a number of

environmental risk factors have been identified for IBD, including smoking, oral

contraceptives, diet, appendectomy, and non-steroidal anti-inflammatory drugs

(Molodecky et al., 2010).

A complete review of environmental factors associated with IBD is beyond the

scope of the current thesis. However, current evidence indicates a role for environmental

factors in regulating the host genetic and microbial interactions that are thought to

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contribute to susceptibility to disease. Understanding the potential mechanisms that

mediate these interactions should provide novel insights into disease pathogenesis.

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1.7. Epigenetic Regulation

Epigenetics studies heritable changes in gene expression that are not caused by

changes in the DNA sequence (Richards, 2006). Epigenetic mechanisms involve

methylation, acetylation and modifications of chromatin proteins that organize DNA

(Youngson et al., 2008). DNA methylation involves methylation of cytosine residues and

a large proportion of genes exhibit correlation between gene expression and degree of

methylation. Similarly, histone hyperacetylation results in high gene expression while a

low degree of gene expression is correlated with hypoacetylation. In addition to the

aforementioned mechanisms of epigenetic regulation, miRNAs have recently become of

great interest in gene regulation and are described in more detail below.

Epigenetic changes have been proposed as an explanation for inherited causes of risk

in complex genetic diseases that have not yet been identified in GWAS (Maher 2008).

Furthermore, differences in environmental exposures are associated with epigenetic

changes that can alter phenotype and predispose individuals to increased risk of disease.

For example, in mice, nutritional supplementation to the mother has been shown to

induce epigenetic changes resulting in altered state of a viable yellow allele of agouti

(Avy) in the germ line, which is retained through epigenetic resetting during

gametogenesis and embryogenesis (Cropley et al., 2006). Therefore, a mother’s diet

could have enduring influence on future generations, independent of later alterations in

diet. Similarly, adults who were prenatally exposed to famine had reduced DNA

methylation of the imprinted insulin-like growth factor 2 (IGF2) gene compared to

unexposed same sex siblings (Heijmans et al., 2008). In summary, environmentally

induced heritable epigenetic changes may be common and can influence disease risk.

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Although the genetic risk loci of ATG16L1 predisposes individuals to CD,

incomplete penetrance and overlapping genotypes among patients with different

phenotypes inadequately explain its etiology (Aujnarain et al., 2013). Therefore, it is

possible that environmental factors could alter epigenetic regulation of ATG16L1

thereby contributing to increased disease risk.

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1.8. miRNA Regulation and Biogenesis

A variety of studies indicate that the miRNA silencing pathway is altered in tissues

from patients with CD (Iborra et al. 2010). microRNAs (miRNAs) are single stranded,

non-coding RNAs that are approximately 19-25 nucleotides in length and function as

post-transcriptional repressors of their target genes by binding to specific sites in the

3’untranslated region (UTR) of the target mRNA. There are over approximately 1800

miRNAs that are either functionally confirmed or predicted (Griffiths-Jones, 2004, 2006;

Kozomara and Griffiths-Jones, 2011), which can have profound effects on gene

regulatory networks.

The primary miRNA transcript (pri-miRNA) is produced by RNA polymerase II. pri-

miRNA is cleaved by the microprocessor complex Drosha–DGCR8 (Pasha) in the

nucleus (Sushila et al., 2010). The resulting precursor hairpin, the pre-miRNA, is

exported into the cytoplasm where the RNase Dicer cleaves the pre-miRNA hairpin to its

mature length. The passenger strand is degraded while the functional strand of the mature

miRNA is loaded together with Argonaute (Ago) proteins (Kim, 2005). Ago2 binds to

GW182, another protein required for gene silencing and together, form the RNA-induced

silencing complex (RISC). The RISC goes on to silence target mRNAs through mRNA

cleavage and translational repression.

!21!

1.9. miRNA implicated in Crohn’s Disease

Recent studies have implicated altered miRNA expression in IBD. Studies have

shown that intestinal tissues from patients with IBD have altered miRNA expression

compared to healthy normal controls. For example, intestinal tissue obtained from

patients with ileal CD or colonic CD have altered expression of 5 and 4 miRNAs

respectively when compared to healthy controls (Wu et al. 2010). Of interest, a number

of miRNAs have recently been identified to directly regulate autophagic signaling in

Crohn’s disease. A SNP in IRGM gene associated with CD alters the miRNA-binding

site of miR-196 and leads to loss of regulation under inflammatory conditions (Brest et

al., 2011). In addition, miR-196 is overexpressed in inflamed intestinal epithelium in CD

patients and causes downregulation of the protective IRGM variant without affecting

levels of the CD associated variant. The decrease in the protective IRGM expression

leads to impaired autophagy and enhanced intracellular replication of AIEC (Brest et al.,

2011). In another study, miR-106b reduced ATG16L1-mediated autophagy and disrupted

autophagy dependent clearance of CD associated AIEC strain LF82 bacteria. In addition,

miR-106b was found to be overexpressed in intestinal epithelial of individuals with active

CD (Lu et al. 2013). Taken together, these studies emphasize miRNAs as important

regulators of gene expression and intestinal inflammation in CD.

!22!

1.10. Summary and Hypothesis:

Autophagy plays an important role in maintaining intestinal homeostasis and

dysfunction in this cellular pathway poses as a risk factor for chronic intestinal

inflammation. While many genetic loci have been identified to predispose individuals to

IBD, they are not enough to adequately explain the etiology of these diseases. Epigenetic

regulation of gene expression can provide an additional layer in the pathophysiology of

IBD. I propose that the miRNA silencing pathway is involved in regulating autophagy

and ATG16L1 to promote inflammation and disease.

Through the use of miRecords (http://mirecords.biolead.org/), which integrates

predicted miRNA from 11 established bioinformatic target prediction programs, a

previous graduate student determined the miRNA sequences predicted to regulate

ATG16L1. Using a cutoff of at least 4 programs predicting the target, 36 specific miRNA

were identified to target and regulate ATG16L1 (Table 1). Of those 36, the list was

narrowed down to 10 based on degree of homology between species, which is to be

expected of a regulator of such a conserved process as autophagy. From this list,

miR142-3p was chosen because its expression pattern of changes in several inflammatory

diseases including psoriasis (Joyce et al., 2011), systemic lupis erythematosus (Ding et

al., 2012) and scleroderma (Makino et al., 2012). In addition, in the IL-10-/- mouse

model of inflammatory bowel disease, upregulation of miR-142-3p corresponded with

the severity of inflammation (Schaefer et al., 2011).

Therefore, I hypothesize that miR-142-3p directly targets ATG16L1 to suppress

autophagosome formation and increase susceptibility to CD. The aim of this project is to

investigate the interaction between miR-142-3p, ATG16L1 and autophagy.

!23!

!!

!Table!1:!miRNAs!predicted!to!target!ATG16L1.!miRNAs!predicted!to!target!

ATG16L1!were!found!using!miRecords,!a!database!that!integrates!results!of!11!

leading!miRNA!target!prediction!algorithms.!From!this!list,!miRM142M3p!was!selected!

based!on!degree!of!homology!across!species!and!its!upregulated!expression!in!both!

an!ILM10M/M!deficient!and!DSSMinduced!mouse!model.!This!work!was!done!by!a!

previous!student!in!the!lab,!Michal!Sibony.!

!

!24!

CHAPTER 2: METHODS !

!

2.1.!Cell!Growth!Conditions!

Human!cervical carcinoma!(HeLa)!and!colorectal!carcinoma!(HCT116)!epithelial!cells!

were!grown!from!frozen!stocks!originally!obtained!from!the!American!Type!Culture!

Collection!(ATCC).!HeLa!and!HCT116!cells!were!grown!in!Dulbecco’s modification of

eagle’s medium!(DMEM)!and!McCoy!5A!medium!respectively.!Media!was!

supplemented!with!10%!Fetal!Bovine!Serum.!Cells!were!grown!at!37°!C!in!a!5%!CO2!

incubator.!Cells!were!regularly!split!and!used!up!to!passage!20.!HeLa!cells!were!

chosen!for!initial!experiments!as!they!are!the!most!commonly!used!human!cell!line!

in!biological!experiments!and!easily!transfected.!Experiments!were!subsequently!

repeated!in!HCT116!cells!because!they!are!more!disease!relevant.!

!!

2.2.!Transfection:!!

Cells!were!grown!to!50%!confluence!in!appropriate!culture!plates.!Cells!were!

transfected!with!Lipofectamine!RNAimax!by!Invitrogen!in!OPTIMEM1!reducedM

serum!medium!for!a!total!of!48!hours.!Culture!media!was!replaced!6M8!hours!after!

transfection.!Depending!on!the!experiment,!cells!were!transfected!with!either!50!or!

100!nM!concentrations!of!the!miRM142M3p!mimic,!miRM142M3p!inhibitor!or!a!miRNA!

Negative!Control!(all!obtained!from!Life!Technologies).!!

!

!

!

!25!

2.3.!Chemically!Enhanced!Autophagy:!

Autophagy!was!chemically!induced!by!incubating!cells!in!the!presence!of!100!ng/mL!

of!Rapamycin!(Sigma!Aldrich)!in!appropriate!culture!medium!at!37°!C!for!4!hours.!!

!

2.4.!Dual!Luciferase!Assay:!

The!3’UTR/Control!target!clones!from!Genecopoeia!allows!for!miRNA!target!

identification!and!functional!validation!of!predicted!targets.!In!the!pmiRMGLO!vector!

from!Genecopoeia,!the!3’UTR!!for!ATG16L1!or!control!sequence!was!inserted!

downstream!of!a!firefly!luciferase!reporter!gene,!driven!by!an!SV40!promoter!for!

expression!in!mammalian!cells.!A!second!renilla!luciferase!reporter!driven!by!a!CMV!

promoter!is!also!cloned!into!the!same!vector!and!serves!as!an!internal!control!by!

measuring!transfection!efficiency!and!cell!viability!(Figure!4).!!Cells!were!coM

transfected!using!Lipofectamine!2000!(Invitrogen)!with!a!plasmid!(500!ng)!

containing!the!3’UTR!of!ATG16L1!(Genecopoeia)!alongside!the!negative!control!

miRNA,!miRM142M3p!mimic!or!miRM142M3p!antagonist!(50!nM).!In!a!similar!

experiment,!cells!were!coMtransfected!using!Lipofectamine!2000!with!a!similar!

plasmid!(500!ng)!that!contained!a!control!sequence!with!no!reported!miRNA!

binding!site!(Genecopoeia)!alongside!the!negative!control!miRNA,!miRM142M3p!

mimic!or!miRM142M3p!antagonist.!!Cells!were!then!lysed!in!passive!lysing!buffer!and!

analyzed!for!firefly!and!renilla!luciferase!activities!using!the!DualMLuciferase!

reporter!assay!system!(Promega)!on!a!luminometer.!Firefly!luciferase!activity!was!

normalized!to!renilla!luciferase!activity.!!

!

!26!

!

!

!

!

!

!

!

!

!

!

!

!

!

Figure!4:!pmiRLGLO!Vector!backbone!of!miRNA!3’UTR!target!clones!from!

Genecopoeia.!The!3’UTR!sequence!is!obtained!from!public!domain!gene!sequence!

databases!and!inserted!downstream!of!a!firefly!luciferase!reporter!gene,!driven!by!

an!SV40!promoter.!A!renilla!luciferase!reporter!driven!by!CMV!is!also!cloned!into!

the!same!vector!to!allow!transfection!normalization!and!accurate!comparison!

between!sample!groups.!!

!

!

!

!

!27!

2.5.!RNA!Extraction:!

Cells!were!washed!3!times!with!PBS!and!total!RNA!was!extracted!using!the!RNeasy!

mini!kit!(Qiagen).!Integrity,!quantity!and!purity!of!RNA!was!examined!using!a!NanoM

Drop!Spectrophotometer.!Commonly!expected!260/230!or!260/280!absorbance!

ratios!of!1.8M2.0!was!used!to!determine!whether!there!was!residual!chemical!

contamination.!!

!

2.6.!cDNA!Synthesis:!

cDNA!was!synthesized!from!total!RNA!using!the!iScript!cDNA!Synthesis!kit!(Biorad)!

on!a!Mastercycler!(Eppendorf).!1000!ng!of!total!RNA!was!converted!to!cDNA!

according!to!manufacturers!instructions!and!stored!at!4°C!until!use.!!

!

2.7.!Quantitative!RealLTime!PCR:!

Quantitative!PCR!amplifications!were!performed!on!a!Step!One!Plus!RealMTime!PCR!

System!(Applied!Biosystems).!The!thermal!profile!was!95°!C!for!2!minutes!followed!

by!40!cycles!of!denaturation!at!95°!C!for!5!seconds!and!annealing!at!60°!C!for!30!

seconds.!Validated!primer!sets!for!ATG16L1!and!BetaMActin!were!obtained!from!

Biorad.!!

!

2.8.!Immunoblotting:!

Following!48!hours!of!experimental!treatments,!cells!were!put!on!ice,!washed!3X!

with!PBS!and!scraped!with!100!μl!of!RIPA!buffer!containing!phosphatase!and!

protease!inhibitors!(all!from!SigmaMAldrich).!Cell!suspensions!were!centrifuged!at!

!28!

high!speed!for!10!minutes!and!supernatants!were!either!stored!at!M80°!C!or!

immediately!boiled!at!100!°!C!with!1x!Laemmeli!buffer!for!5!minutes.!Equal!amounts!

of!protein!were!run!on!a!12!%!sodium!dodecyl!sulfate!polyacrylamide!gel!

electrophoresis!(SDSMPAGE)!at!120!V!for!1.5!hours!at!room!temperature.!Proteins!

were!then!transferred!onto!PVDF!membrane!(Millipore)!at!30V!for!8!hours!at!4°!C.!

PVDF!membranes!were!blocked!with!5%!milk!in!TrisMbuffered!saline!with!tween!

(TBST)!and!probed!with!a!1:500!dilution!of!LC3!rabbit!polyclonal!antibody!(LC3MI!

band!is!seen!at!17!kDa!and!LC3MII!band!at!19!kDa)!(Novus!Biologicals),!a!1:500!

dilution!of!ATG16L1!(68!kDa)!rabbit!polyclonal!antibody!(Novus!Biologicals)!and!a!

1:3000!dilution!of!actin!(42!kDa)!rabbit!polyclonal!antibody!(Sigma!Aldrich).!

Immunoblots!were!developed!with!the!appropriate!HRPMconjugated!secondary!

antibody!(1:1000!dilution).!!

!

2.9.!Densitometric!Analysis!

Immunoblots!were!captured!and!quantitated!using!the!LiCor!2300!machine.!For!

densitometry,!signals!of!ATG16L1!and!LC3!were!measured!for!each!treatment!and!

expressed!as!a!ratio!over!βMactin!(loading!control)!from!the!same!samples.!The!

ATG16L1!primary!antibody!results!in!two!bands,!representing!the!two!main!

isoforms!of!ATG16L1!(α!and!β).!Signal!intensities!of!both!bands!were!measured!

together!for!densitometry.!The!LC3!antibody!results!in!two!bands!as!well,!LC3MI!and!

LC3MII.!Only!the!signal!intensity!of!the!LC3MII!band!was!measured!as!a!ratio!over!βM

actin!as!it!correlates!with!autophagosome!numbers.!!

!

!29!

2.10.!Immunofluorescence!LC3LGFP!Tagged!Cells!

Cells!were!seeded!on!12Mwell!cover!slips!and!transfected!using!Lipofectamine!2000!

(Invitrogen)!with!a!LC3MGFP!plasmid!alongside!either!a!miR!Negative!Control,!miRM

142M3p!mimic!or!miRM142M3p!antogonist!(50!nM)!(all!from!Life!Technologies).!

Transfection!media!was!changed!to!regular!media!after!4!hours.!48!hours!following!

transfection,!cells!were!washed!3!times!with!PhosphateMBuffered!Saline!(PBS),!fixed!

for!20!minutes!in!4%!paraformaldehyde!(SigmaMAldrich),!rinsed!in!3!times!in!PBS!

and!stained!with!4’M6MdiamidinoM2Mphenylindole!(DAPI)!(1:5000!dilution)!for!10!

minutes!and!mounted.!Representative!images!were!taken!on!the!Olympus!DSU!

spinning!confocal!with!a!60X!oil!lens.!Quantitation!of!cells!was!done!in!a!blinded!

fashion!under!an!epifluorescent!microscope.!100!cells!from!each!transfection!group!

was!graded!as!having!either!low!or!high!levels!of!puncta.!A!cell!was!considered!as!

having!high!levels!of!puncta!if!it!had!greater!than!8!visible!puncta!as!seen!under!an!

epifluorescent!microscope.!!

!

2.11.!LC3!Immunostaining!

Cells!were!seeded!on!12Mwell!cover!slips!and!transfected!using!RNAimax!

(Invitrogen)!with!a!miR!Negative!Control,!miRM142M3p!mimic!or!miRM142M3p!

inhibitor!(all!from!Life!Technologies).!Transfection!media!was!replaced!the!

following!day.!48!hours!postMtransfection,!cells!were!washed!3!times!with!PBS!

(Wisent),!fixed!for!20!minutes!in!4%!paraformaldehyde!(SigmaMAldrich,!Oakville,!

Ontario,!Canada),!rinsed!in!PBS,!permeabilized!by!incubation!in!methanol!for!15!

minutes,!rinsed!with!PBS!and!blocked!for!an!hour!in!1%!BSA!in!PBS!(weight/vol).!!

!30!

Cells!were!incubated!with!a!1:200!dilution!of!LC3!rabbit!polyclonal!antibody!(Cell!

Novus!Biologicals)!in!5%!BSA!in!PBS!(weight/vol)!overnight!at!4°!C.!Cells!were!

washed!3x!with!PBS!for!5!minutes!each!time!and!then!put!in!a!1:1000!dilution!of!

antiMrabbit!IgG!CY3!secondary!antibody!(Jackson!Immunoresearch)!for!one!hour.!

Finally,!cells!were!rinsed!3!times!with!PBS!and!stained!with!DAPI!(1:5000!dilution)!

for!10!minutes!and!mounted.!Cells!were!quantitated!in!a!blinded!fashion.!

Immunostaining!was!observed!under!the!Olympus!DSU!spinning!disk!confocal!

microscope!(Olympus!America)!with!a!60x!oil!lens.!25!cells!from!each!group!were!

quantitated!as!having!high!or!low!levels!of!puncta.!A!cell!was!graded!as!having!high!

levels!if!it!had!over!~15!puncta.!!

!

2.12.!Statistical!Analysis!

All!experiments!were!performed!at!least!3!times!(n≥3).!OneMway!ANOVA!and!

Tukey’s!postMhoc!tests!were!performed!to!compare!the!means!±!standard!error!(SE)!

for!treatment!groups!using!GraphPad!Prism!6!(GraphPad!Inc.).!A!pMvalue!of!less!than!

0.05!was!used!to!determine!statistical!significance.!!

!

!

!

!

!

!

!

!31!

CHAPTER 3: RESULTS

3.1. miR-142-3p Inhibits ATG16L1 mRNA Expression

I first investigated the effects of miR-142-3p on ATG16L1 expression by

transfecting HeLa cells with a miRNA negative control, miR-142-3p mimic or miR-142-

3p antagonist. Quantitative real time - PCR (qPCR) analysis showed that transfection of

cells with the miR-142-3p mimic significantly reduced ATG16L1 mRNA expression in

comparison with the miRNA negative control. In contrast, ATG16L1 mRNA levels were

comparable to the negative control when transfected with the miR-142-3p antagonist

(Figure 5a).

I next assessed the effect of miR-142-3p in intestinal cells. A reduction in

ATG16L1 mRNA was detected in HCT116 cells transfected with the miR-142-3p mimic.

In HCT116 cells transfected with the miR-142-3p antagonist, ATG16L1 mRNA levels

were comparable to the negative control (Figure 5b). Taken together, these data suggest

that miR-142-3p reduces ATG16L1 mRNA expression levels. Similar results can be seen

for ATG16L1 protein levels in subsequent experiments (Figure 7).

!

!

!

!32!

!

!!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

Figure!5:!miRL142L3p!reduces!ATG16L1!mRNA!and!protein!expression!in!HeLa!

and!HCT116!cells.!a)!HeLa!and!b)!HCT116!cells!were!transfected!with!individual!

miRM142M3p!mimic,!miRM142M3p!antagonist!or!a!miRNA!negative!control!(100!nM).!

48!hours!post!transfection,!total!RNA!was!extracted!and!cDNA!synthesized.!qPCR!of!

cDNA!was!performed!to!assay!for!ATG16L1!mRNA!levels.!(n!=!3!independent!

experiments,!*pMvalue!<0.05).!!

ATG

16L1

mR

NA

exp

ress

ion

(Rel

ativ

e to

Act

in)

Negat

ive C

ontrol

miR

-142

-3p m

imic

miR

-142

-3p in

hibito

r0.0

0.5

1.0

1.5

*

**

n = 3* p < 0.05** p < 0.01

ATG

16L1

mR

NA

exp

ress

ion

(Rel

ativ

e to

Act

in)

Negat

ive C

ontrol

miR

-142

-3p m

imic

miR

-142

-3p in

hibito

r0.0

0.5

1.0

1.5

*

n = 3* p < 0.05

a)!HeLa!! b)!HCT116!

!

2

!

!

!33!

3.2. miR-142-3p targets ATG16L1

My studies with the miR mimic and antogonist suggest that ATG16L1 is a target

for miR-142-3p. In order to determine if the 3’UTR of ATG16L was a direct target of

miR-142-3p, I transfected HeLa cells with a pmiR-GLO dual luciferase reporter vector

containing the ATG16L1 3’UTR vector or control 3’UTR without a miRNA binding site

(details about the vectors can be found in materials and methods and Figure 4). HeLa

cells were then co-transfected with a miRNA negative control, a miR-142-3p mimic or a

miR-142-3p antagonist. In cells co-transfected with the miR-142-3p mimic and the pmiR-

GLO vector containing the 3’ UTR of ATG16L1 a significant decrease in luciferase

activity was observed in comparison with cells transfected with the negative control.

Transfection with the miR-142-3p antagonist caused an increase in luciferase activity in

comparison with cells transfected with the miRNA negative control (Figure 6a). In

contrast, when cells were co-transfected with a control 3’UTR vector lacking miRNA

binding sites and the miRNA negative control, miR-142-3p mimic or miR-142-3p

antagonist, no significant difference in luciferase activity was detected between

transfection groups (Figure 6b). These findings suggest that miR-142-3p directly targets

the 3’UTR of ATG16L1.

!!

!

!

!

!

!

!34!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

b)!

!

a)!!

!

!

!35!

Figure!6:!miRL142L3p!directly!targets!the!3’UTR!of!ATG16L1.!a)!HeLa!cells!were!

coMtransfected!with!the!3’UTR!target!sequence!expression!plasmid!clone!for!

ATG16L1!and!either!the!miRM142M3p!mimic!or!antagonist.!b)!HeLa!cells!were!coM

transfected!with!the!3’UTR!of!a!control!sequence!without!a!miRNA!binding!site!and!

either!the!miRM142M3p!mimic!or!miRM142M3p!antagonist.!Cell!lysates!were!collected!

and!firefly!luciferase!activity!was!measured!and!normalized!to!renilla!luciferase!

activity.!(n!=!3!independent!experiments,!*!pMvalue!<!0.05).!!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!36!

3.3. miR-142-3p Reduces Endogenous Autophagy

I next determined if the observed miR-142-3p dependent changes in ATG16L1

expression had a functional effect on the autophagy pathway. To determine the effect of

miR-142-3p on endogenous autophagy, cells were transfected with the miR-142-3p

mimic or antagonist and autophagic activity was compared to cells transfected with the

miRNA negative control. A reduction in ATG16L1 and LC3-II protein levels was

detected in both HeLa (Figure 7a) and HCT116 cells (Figure 7b) transfected with the

miR-142-3p mimic as determined by western blotting. In contrast, in cells transfected

with the miR-142-3p antagonist an increase in ATG16L1 and LC3-II protein levels was

detected. The results suggest that miR-142-3p is capable of inhibiting autophagic activity

through regulation of ATG16L1 protein expression.

To confirm the functional effect of miR-142-3p on autophagy, HeLa cells were

co-transfected with a miR-142-3p mimic or antagonist and LC3-GFP plasmid to visualize

autophagosomes. In comparison with sham-transfected cells, a decrease in LC3-GFP

puncta was detected in cells transfected with the miR-142-3p mimic (Figure 8).

In order to ensure these findings were not an artifact of LC3-GFP transfection, I

assessed endogenous LC3-II labeled autophagic puncta using immunocytochemistry.

HeLa and HCT116 cells were transfected with a miR-142-3p mimic, miR-142-3p

antagonist or negative control miRNA and stained with an LC3 primary and an anti-

rabbit CY3 secondary antibody. Quantitation of autophagic puncta using confocal

microscopy showed that both HeLa and HCT116 cells transfected with the miR-142-3p

mimic had both a reduced number and smaller size of puncta as compared to cells

transfected with the negative control miRNA (Figure 9a and 9b). Taken together, these

!37!

results demonstrate that transfection with the miR-142-3p mimic decreases ATG16L1

protein levels in association with reduced endogenous autophagic activity.

!

!38!

a)!HeLa!Cells! !!!!!!!!!!!!!!!!!!!!!!!

!

!

!

!

!

!

!

!

!

!

!

ATG16L1

Actin Actin

LC3-II

! !

LC3-I

!39!

b)!HCT116!Cells!!!!!!!!!!

!

!

!

!

!

!

!

!

!

!

!

Figure!7:!miRL142L3p!reduces!endogenous!autophagy.!a)!HeLa!and!b)!HCT116!

cells!were!transfected!with!a!miRNA!negative!control,!miRM142M3p!mimic!or!miRM

142M3p!antagonist!(100!nM).!At!48!hours!post!transfection,!cell!lysates!were!

collected.!ATG16L1!expression!and!autophagy!was!monitored!by!assessing!LC3MII!

conversion!by!western!blot.!Graph!below!depicts!densitometry!of!ATG16L1!and!

LC3MII!relative!to!βMactin!protein!levels.!(n=!3!independent!experiments,!*pMvalue!

<0.05).!

!

!

!

!

ATG

16L1

/Act

in

Negat

ive C

ontrol

miR

-142

-3p m

imic

miR

-142

-3p in

hibito

r0.0

0.5

1.0

1.5

n = 3* p < 0.05

*

LC3I

I/Act

in

Negat

ive C

ontrol

miR

-142

-3p m

imic

miR

-142

-3p in

hibito

r0.0

0.5

1.0

1.5

2.0

n = 3

*

ATG16L1

Actin Actin

LC3-II

! !

LC3-I

!40!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

Figure!8:!miRL142L3p!reduces!endogenous!autophagy.!Representative!images!

from!HeLa!cells!coMtransfected!with!the!LC3MGFP!plasmid!and!either!miRM142M3p!

mimic!(Panel!B)!or!miRM142M3p!antagonist!(Panel!C)!(50!nM)!using!Lipofectamine!

2000.!Panel!A!shows!representative!image!of!a!miR!sham!transfected!control!cell.!

Graph!below!depicts!quantitation!of!cells!graded!as!having!high!levels!of!puncta!

from!each!transfection!group.!(Green!=!LC3!GFP,!Blue!=DAPI,!Images!are!

representative!of!n!=!3!experiments,!*pMvalue!<0.05)!

!

A B C

!41!

a)!HeLa!Cells!

!

!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!

!

!

!

!

!

0

10

20

30

40

50 *

n = 3* p < 0.05

B C A

!

!42!

b)!HCT116!Cells!

!

!!!!!!!!!!!!!!!!!!!!!!

!

!

Figure!9:!miRL142L3p!reduces!endogenous!autophagy.!a)!HeLa!and!b)!HCT116!

cells!were!transfected!with!a!miRNA!negative!control,!miRM142M3p!mimic!or!miRM

142M3p!antagonist!(100!nm).!Cells!were!fixed!and!stained!with!an!LC3!primary!and!a!

CY3!secondary!antibody.!Panel!A!is!a!representative!image!of!cells!transfected!with!

a!negative!control!miRNA,!Panel!B!is!of!cells!transfected!with!the!miRM142M3p!mimic!

and!Panel!C!is!of!cells!transfected!with!the!miRM142M3p!antagonist.!Graph!depicts!

quantitation!of!cells!from!each!transfection!group.!(Red!=!LC3,!Blue!=!DAPI,!images!

are!representative!of!n!=!3!experiments,!*pMvalue!<0.05).!

A B C

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3.4!miR-142-3p does not reduce chemically induced autophagy

Next we assessed the effect of miR-142-3p in cells when autophagy was induced

using rapamycin, a pharmacological inhibitor of mTOR. Preliminary data show that

rapamycin treatment results in increased conversion of LC3-I to LC3-II, indicative of

increased autophagy (Figure 10). In rapamycin-treated HeLa cells, transfection with the

miR-142-3p mimic did not result in a decrease in ATG16L1 protein levels. A

corresponding decrease in LC3-II protein levels was also not detected in HeLa cells

transfected with the miR-142-3p mimic (Figure 9a). The same experiment was repeated

in HCT116 cells and transfection with the miR-142-3p mimic and antagonist did not

result in changes in ATG16L1 and LC3-II protein levels (Figure 9b).

Quantitation of autophagic puncta using immunofluorescence showed comparable

levels of autophagosomes in cells transfected with the miR-142-3p mimic, antagonist or

negative control (Figure 11 and 12).

!!!!!!!!!!!!!!!!!!!!

!44!

!!!!!!!!!!!!!!!!!!!!!!!!!!Figure!10:!Induction!of!autophagy!with!rapamycin!treatment.!HeLa!cells!were!

treated!with!rapamycin!(100!ng/mL)!for!4!hours!and!cell!lysates!were!collected.!

Autophagy!was!monitored!using!LC3MII!by!western!blot!(n!=1!experiment).!!

!!!!!!!!!!!!!!

Actin

LC3-II

LC3-I

Control

Rap

amyc

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Negat

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miR

-142

-3p m

imic

miR

-142

-3p in

hibito

r0.0

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ATG

16L1

/Act

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miR

-142

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imic

miR

-142

-3p in

hibito

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a)!HeLa!!

ATG16L1

Actin

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Actin

LC3-I

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!

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!!!!!!!Figure!11:!miRL142L3p!does!not!reduce!chemically!induced!autophagy.!a)!HeLa!

and!b)!HCT116!cells!were!transfected!with!100!nM!of!a!miRNA!negative!control,!

miRM142M3p!mimic!and!miRM142M3p!antagonist.!At!40!hours!post!transfection,!

Rapamycin!was!added!to!each!group!for!4!hours!and!then!cell!lysates!were!collected.!

Autophagy!was!monitored!using!ATG16L1!and!LC3MII!by!western!blot.!Graphs!

depicts!corresponding!densitometric!analysis!of!immunoblots!from!n!=!3!

experiments.!!

!!!!!!!!

ATG16L1

Actin

LC3-II

Actin

b)!HCT116!!A

TG16

L1/A

ctin

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miR

-142

-3p m

imic

miR

-142

-3p in

hibito

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ative

Contro

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-142

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LC3-I

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a)!HeLa!Cells!!a)!HeLa!Cells!!!!!!!!!!!!!!!!!!!!!!!!!!

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b)!HCT116!Cells!!!

!

!

!!

!!!!!!!!!!!!!!!!!

!

!

!

Figure!12:!miRL142L3p!does!not!reduce!rapamycinLenhanced!autophagy.!!

a)!HeLa!and!b)!HCT116!cells!were!transfected!with!a!miRNA!negative!control,!miRM

142M3p!mimic!or!miRM142M3p!inhibitor!(100!nm)!and!treated!with!Rapamycin.!Cells!

were!fixed!and!stained!with!an!LC3!primary!and!a!CY3!secondary!antibody.!Panel!A!

is!a!representative!image!of!cells!transfected!with!a!negative!control!miRNA,!Panel!B!

is!of!cells!transfected!with!the!miRM142M3p!mimic!and!Panel!C!is!cells!transfected!

with!the!miRM142M3p!antagonist.!Graph!depicts!quantitation!of!cells!from!each!

A B C

Negati

ve C

ontrol

miR-14

2-3p m

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miR-14

2-3p in

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transfection!group.!(Red!=!LC3,!Blue!=!DAPI,!images!are!representative!of!n!=!3!

experiments,!*p<0.05)!

!50!

CHAPTER 4: DISCUSSION

The exact mechanism by which the genetic polymorphism in ATG16L1 promotes

CD is not known. However, both human and animal studies demonstrate that dysfunction

in ATG16L1 leads to increased inflammatory processes in the intestine and aberrant

Paneth cell function that mirrors what is observed in CD patients (Cho, 2009).

Furthermore, recent evidence indicates that the CD related polymorphism in ATG16L1

results in enhanced caspase mediated cleavage of ATG16L1 suggesting that a critical

level of ATG16L1 may be necessary to maintain intestinal homeostasis (Zhai et al.,

2013).

In addition to host genetic polymorphisms, miRNA can alter gene expression.

Furthermore, altered miRNA expression profiles have been identified in patients with

IBD. Thus, I hypothesized that dysregulated miRNA could target ATG16L1 mRNA,

reducing ATG16L1 levels to promote CD. Therefore, my project focused on elucidating

how a specific miRNA that is predicted to target ATG16L1 can modulate ATG16L1 and

autophagy.

In the current study, miR-142-3p was found to negatively regulate ATG16L1

mRNA and protein levels in both HeLa and HCT116 cell lines. miRNAs regulate cell

function by either targeting mRNA stability and/or mRNA translation (Sushila et al.,

2010). Both mechanisms ultimately lead to decreased protein expression and function.

mRNA degradation occurs if there is almost or perfect pairing between the miRNA and

the mRNA sequence whereas translational inhibition may occur if the complementary

sequences do not match well (Grimson et al., 2007). Evidence from my project suggests

that both mRNA and corresponding protein levels for ATG16L1 decrease in cells

!51!

transfected with the miR-142-3p mimic. These findings suggest that miR-142-3p

regulates ATG16L1 expression likely through mRNA degradation or translational

inhibition. Furthermore reduction of endogenous miR-142-3p in HeLa cells by

transduction with the miR antagonist reduced ATG16L1 mRNA levels providing further

support for the role of miR-142-3p in regulating ATG16L1 levels. We did not detect a

difference in ATG16L1 mRNA levels in HCT116 cells transfected with the miR-142-3p

antagonist. The difference in the effect of the miR-142-3p antagonist between these two

cell lines is likely due to variation in endogenous levels of miR-142-3p. It would be of

interest to perform qPCR to determine endogenous levels of miR-142-3p in both cell

lines.

The 3’UTR of ATG16L1 is approximately 1,323 nucleotides in length and

contains conserved binding site for hundreds of predicted miRNAs. In order to determine

if miR-142-3p directly targets the 3’UTR of ATG16L1 to regulate expression, a

luciferase reporter assay was employed. Our findings using the luciferase reporter assay

with the ATG16L1 3’UTR vector indicate that miR-142-3p directly targets and regulates

the gene.

We next determined if the miR-142-3p mediated reduction in ATG16L1 protein

had an impact on the autophagy pathway. We used several complimentary approaches to

assess autophagy including biochemical assessment of LC3-II by western blotting and

semi-quantitation of LC3-II positive autophagosome formation. In cells transfected with

the miR-142-3p mimic, a reduction in both LC3-II and autophagic puncta was detected in

the two epithelial cells lines. In addition, in cells transfected with the miR-142-3p

antogonist an increase in LC3-II levels and autophagosomes were detected. Taken

!52!

together these results suggest that miR-142-3p negatively regulates the autophagy

pathway by targeting and reducing expression of ATG16L1.

In contrast to endogenous autophagy, we did not detect a reduction in ATG16L1,

LC3-II or autophagosomes in miR-142-3p transfected cells in which autophagy was

stimulated by rapamycin. Although miR-142-3p may have an effect on basal autophagy

where lower levels of ATG16L1 may be required, our transfection efficiency may not be

sufficient to reduce ATG16L1 to critical levels under conditions of enhanced autophagy.

Future studies using cell lines with stable expression of miR-142-3p would eliminate

transfection efficiency as a variable. miR-142-3p activity in transfected cells could also

be validated by assessing levels of other confirmed downstream targets. Other target

genes that have been identified for miR-142-3p include Rho-associated coiled-coil

containing protein 2 (Rock2), adenylyl cyclase 9 (Adcy9), nuclear receptor subfamily 3

group C member 1 (NR3C1), interleukin 6 (IL-6) and a clock gene, ARNTL/BMAL1

(Nishiyama, 2012; Huang, 2009; Zhang, 2012; Tan, 2012). Alternatively, rapamycin may

have other off target effects on miR-142-3p. Therefore, additional studies using a variety

of relevant autophagic stimuli including enteric microbes are warranted. A recent paper

published by Zhai et al. demonstrated similar results to the work of this thesis. Although

rapamycin was not used, they found that overexpression of miR-142-3p in HCT116 cells

resulted in reduced autophagy under starvation conditions (Zhai et al., 2014).

The role of miR-142-3p in regulating ATG16L1 could also vary depending on the

endogenous and inducible levels of the miRNA in specific cell lines. The magnitude of

effect of the miR-142-3p mimic and antagonist would be based on whether endogenous

levels are high or low. For example, the effect of the antagonist on autophagy will be less

!53!

in cell lines with low endogenous levels of miRNA since it binds to endogenous miRNA

and represses function. Alternatively, upregulation of miR-142-3p with the mimic in cells

with endogenously low levels of the miRNA could produce greater increases in

ATG16L1 and autophagic activity in comparison to cells with higher endogenous levels

of miR-142-3p. miR-142-3p has been shown to be mainly expressed and functionally

active in the hematopoietic system. For example, studies have shown significant effects

on autophagy with transfection of the miR-142-3p antagonist in Jurkat T cells that have

higher endogenous miR-142-3p levels in comparison with HCT116 cells (Zhai, 2014).

From these results, miR-142-3p could fine-tune the amplitude of ATG16L1 gene

expression and overall autophagic activity. However, this seems to be dependent on

endogenous and inducible levels of miR-142-3p in different cell populations. In

conclusion, the findings brought here suggest a novel mechanism by which altered

miRNA expression leads to altered expression of autophagy genes.

The phenotype of disease is determined by the interactions of genetics and

environmental factors, which are often mediated by epigenetic mechanisms such as

miRNA silencing (Wang and Cui, 2012). Different environmental factors such as drugs,

virus and bacterial pathogens, nutrition and cigarette smoke have been shown to

functionally interact and alter miRNA profiles. (Wang and Cui, 2012). Therefore, in

addition to the SNP mediated cleavage of ATG16L1 by Caspase 3, epigenetic regulation

of ATG16L1 can also provide another layer of evidence of how the autophagy pathway is

implicated in disease. Environmental factors can alter miR-142-3p expression and

through the miRNA silencing pathway, leads to a decrease in ATG16L1 levels, which

could result in defective xenophagy, increased pro-inflammatory cytokine production and

!54!

abnormal Paneth cell morphology as mentioned in previous studies (Figure 13). Evidence

from my project supports the hypothesis that miR-142-3p regulates autophagy through

direct interaction with the 3’UTR of ATG16L1. miR-142-3p negatively regulates

!55!

Figure 13: How the environment and epigenetics could alter expression of

ATG16L1. Aside from the SNP mediated Caspase 3 cleavage of ATG16L1, epigenetic

regulation through miRNA silencing could decrease expression of ATG16L1, which

leads to various consequences such as abnormal Paneth cell morphology and function,

defective xenophagy and increased pro-inflammatory cytokine production.

!56!

ATG16L1 and subsequent autophagic activity (Figure 14). This implicates a role for this

miRNA in intestinal inflammation and CD. Although the effects of miR-142-3p on

autophagy may be modest as it is able to reduce endogenous autophagy but not with

chemical induction using rapamycin treatment, further studies are warranted to

investigate its significance in intestinal inflammation and CD pathogenesis.

!57!

Figure 14: Proposed model of miRNA regulation of autophagy in IBD. Various

environmental factors such as inflammation, microbial products or vitamin D levels could

alter the expression profile of miR-142-3p, which through the miRNA silencing complex,

regulates and decreases ATG16L1 expression. The reduction in ATG16L1 inhibits

autophagy and may contribute to intestinal inflammation.

!58!

Future Directions:

My project has focused on the effect of miR-142-3p on ATG16L1 in two specific

cell lines in vitro. The use of an intestinal organoid system would allow the study of

autophagy in more disease relevant primary cells that recapitulate the intestinal crypt

units. Our laboratory is currently working with an intestinal organoid model where we

hope to transduce a lenti-miR-142-3p construct and investigate changes in autophagy and

Paneth cell morphology that have been identified with decreased ATG16L1.

It would be clinically relevant to study what happens not only to the expression of

miR-142-3p but to broadly assess changes in miRNAs in patients with IBD. We have

already begun to collect patient tissue samples to determine whether the expression

profile of miR-142-3p and other miRNAs predicted to target ATG16L1 differs between

healthy and affected pediatric patients. We are currently collecting biopsy samples from

newly diagnosed Caucasian pediatric patients with inflammatory bowel disease age 6 and

older with informed consent from subjects or their legal guardians. Patients were

excluded from the study if they did not meet age and ethnic criteria as well as if they

were on medications other than 5-ASA for treatment of IBD. These exclusion factors

were put into place in order to minimize confounding variables. At the time of

colonoscopy for patients with IBD, 2 biopsy samples from the terminal ileum, 2 from the

hepatic flexure and 2 biopsy samples anywhere in the colon with opposite affection status

as compared to the hepatic flexure was collected. In addition, one biopsy sample from the

terminal ileum and hepatic flexure were collected and snap frozen in liquid nitrogen and

stored at -80°C. For control patients, only samples from the terminal ileum and hepatic

flexure were collected as described above. Blood was also collected from each patient for

!59!

genotyping. From the patient intestinal samples, we will isolate RNA and study miRNA

expression profile using the nCounter Human v2 miRNA expression assay from

Nanostring Technologies. The assay allows for accurate and sensitive expression

profiling of 800 human miRNAs without amplification.

Another interesting avenue to pursue is the role of environmental factors in

epigenetic regulation of the autophagy pathway. One environmental factor that has

garnered much attention for its potential role in IBD is vitamin D (Cantorna, 2006).

Vitamin D deficiency is common in adults and children with IBD as compared to healthy

controls, correlating with poorer health-related quality of life. Factors such as

malabsorption due to mucosal disease, reduced sunlight exposure and surgical resection

can contribute to this deficiency (Garg et al., 2012). Several lines of evidence through

epidemiological, physiological, genetic and clinical studies demonstrate the role of

vitamin D in immunomodulation of IBD. There is a correlation between the incidence

and prevalence of IBD and potential exposure to sunlight as indicated by distance from

the equator (Khalil et al., 2012). Studies have also shown that dendritic cells,

macrophages and intestinal epithelial cells express vitamin D receptor and that vitamin D

promotes the transcription of NOD2 gene (Wang et al., 2010).

Mouse models have shown that the active form of vitamin D and its receptor is

able to regulate susceptibility to experimental colitis. A study conducted by Coorona et

al. show that vitamin D deficient mice had more bacterial colonization compared to those

that were vitamin D sufficient (Cantorna, 2000). Vitamin D treatment of mice that were

deficient decreased colitis severity and reduced bacterial numbers as compared to

untreated mice. Furthermore, vitamin D promotes induction of autophagy. Therefore,

!60!

Vitamin D deficiency may result in altered miRNA that regulate the autophagy pathway.

To address this hypothesis we have collected tissue from the ileum and distal colon

female C57BL/6 mice that were either diet induced vitamin D deficient or sufficient. We

will use qPCR to quantify the expression levels of miR142-3p. However, as qPCR results

are based on crude tissue biopsies and could simply reflect differences in cellular

composition rather than true differences we will also conduct in-situ hybridization using

labeled probes to identify specifically levels of the miRNA in intestinal cells of interest.

The proposed future directions each look at the regulation of autophagy in IBD

from different angles. The organoid model will hopefully further elucidate how

autophagy is altered by miRNAs in more disease relevant cell types while collection of

patient biopsy samples will show which miRNAs are altered between inflamed and non-

inflamed tissue. Finally, the mouse study will identify how environmental changes such

as vitamin D levels could possibly alter miRNA expression profiles and affect the

autophagy pathway. In culmination, the aforementioned proposed future directions will

provide multiple perspectives in the study of miR-142-3p regulation of autophagy in the

pathogenesis of IBD.

!61!

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