5. Research Design and Methods, 5.1 Research Area: This application addresses the broad challenge area
“(15) Translational Science” and specific challenge topic, “15-HL-102: Develop new therapeutic strategies
for heart, lung, and blood diseases based on microRNA technology”. The project title is “microRNA
Regulation of Human Airway Epithelial Cell Phenotype”.
5.2 Challenge and Potential Impact: It is estimated that >22 million people in the USA currently have asthma
and that >15 million people have chronic obstructive pulmonary disease (COPD), which is the fourth leading
cause of death (data from Centers for Disease Control and Prevention). As well, there are other less
prevalent, but individually devastating, obstructive lung diseases such as cystic fibrosis (CF), non-CF
bronchiectasis and primary ciliary dyskinesia (PCD). These conditions place massive burdens on health care
resources and immeasurably degrade human spirit and potential. All of them are focused on the lung airways,
which become characteristically inflamed and remodeled, causing functional impairment. The airway
epithelium is strategically located at the lung-environment interface. In addition to its well-known barrier and
protective functions, it is now widely appreciated that the epithelium initiates, integrates and orchestrates host
immune responses and airway remodeling via secretion of regulatory factors within complex feedback loops {}.
Phenotypic changes in the airway such as enhanced epithelial shedding and turnover, deficient repair, mucous
secretory (goblet) cell hyperplasia and squamous metaplasia are variably present and are integral to the
progression of inflammatory airway diseases. Despite recent advances, many basic mechanisms regulating
altered structure and function of hBE cells remain poorly understood, and there are no specific therapies
directed at preventing or reversing disease-related phenotypic changes in the airway epithelium.
In the last decade, “omics” have revolutionized our understanding of biology and disease, specifically
including numerous microarray studies of airway mRNA in asthma and COPD {14248} {14244}. We now
appreciate that non-coding, small RNA’s including microRNAs (miRNAs) are critical regulators of genomes and
genes. Major effects of miRNA’s are to destabilize mRNA and to inhibit protein translation, but there are also
examples of miRNA positive regulation of transcription. In any case, a regulatory network between miRNAs
and gene expression is clearly involved in almost every aspect of biology from stem cell maintenance and
embryonic development / cell differentiation to cancer (reviewed in {14264}). Recent, but limited, studies
provide insight that miRNAs are important in lung development {14253} {14293}, lung inflammation {14238},
the biological response to tobacco smoking {14287}, and lung cancer {14250}. However, we do not know the
full miRNA repertoire of the airway epithelium, nor do we have a comprehensive understanding the miRNAmRNA regulatory networks controlling hBE cell phenotype in health and disease. A unique combination of
resources and talents at the University of North Carolina is poised to fill this major lung biology knowledge gap.
The Randell laboratory is a leading provider of primary human bronchial epithelial (hBE) cells in models
recapitulating their normal structure and function and has extensive knowledge of airway epithelial cell biology
{}. The Hammond and Hayes laboratories provide cutting edge expertise in miRNA biology and bioinformatics,
respectively {14223} {14253} {14252}. Supported by provocative preliminary data that miRNA expression
changes dramatically as a function of hBE cell differentiation, these groups have combined forces with a plan
to rapidly create new data that will significantly advance our understanding of miRNA regulation of airway
epithelial phenotype during normal growth and differentiation and under a series of highly relevant injury/repair
conditions.
5.3 Approach, Strategy: Our basic
hypothesis is that miRNAs are critical
regulators of hBE cell differentiation and
the response to injury, that miRNAs
become altered in pathologic states,
and that modulating miRNAs can
change hBE cell phenotype for
Figure 1. Time course of hBE cell differentiation in a replicate of ALI
therapeutic benefit. This hypothesis is
cultures used for preliminary miRNA arrays. Formalin paraffin sections
based on initial studies showing
stained with H&E, or AB-PAS for glucoconjugates (blue), illustrate the
dramatic regulation of miRNAs as a
progression from poorly- to well-differentiated.
function of hBE cell differentiation in
vitro (Figures 1-3). In this experiment, primary hBE cells were harvested from explanted lungs of six individual
tissue donors. The initial primary cell culture stage was on plastic, where the cells predictably de-differentiated
and proliferated but were unable to polarize and re-differentiate. The cells were then passaged from plastic to
a porous support and were grown at an air-liquid interface (ALI) where they again proliferated but were now
able to fully differentiate, recapitulating the normal pseudostratified airway epithelial morphology (Figure 1).
Total RNA was harvested on days 5, 14 and 35 after seeding under ALI conditions, representing distinct
differentiation stages. The 3’ hydroxyl group of miRNA was fluorescently labeled with P-cytidyl-uridyl-Cy3 using
T4-RNA ligase and RNA was hybridized to home-spotted microarray slides, consisting of 527 unique
oligonucleotides complementary to putative human miRNAs in the Sanger miRBase {14252}. The slides were
scanned using the 532-nM channel on a Genepix scanner and raw expression data was extracted and
analyzed using GeneCluster, TreeView and Significance Analysis of Microarrays (SAM). Median centered and
normalized miRNA expression data of hBE cell cultures from 6 individuals at day 5, 14 and 35, subjected to
unsupervised hierarchical clustering (Figure 2), revealed complete separation of the day 5 group from the later
time points. The day 14 and 35 groups also tended to cluster but there was some
admixture. Significance Analysis of Microarrays (SAM) software called 49 genes as
significantly different at a 0% false discovery rate (Figure 3). The predominant
expression pattern was a steady increase in expression of several miRNAs from day 5
to day 35, but there were examples of equivalent increases at day 14 and 35. Three
miRNAs definitively decreased from day 5 to day 35. Manual observation of the data
revealed additional borderline and/or inconsistent changes in several miRNAs.
Furthermore, many miRNAs not reliably detected by array technology likely changed
but were not visible to us. Manual searching of the literature for functions of highly
regulated miRNAs indicated several associated with cellular differentiation in other
systems, tumor suppressors, and those with variable expression in different cancers.
However, other strongly regulated miRNAs with unknown function were present. These
results strongly indicate a key role for miRNAs in regulation of hBE cell proliferation and
differentiation and illustrate significant gaps in our
knowledge.
Figure 2.
The above results underlie the strategy for this
Unsupervised
proposal.
Namely, that: 1) miRNAs are important
hierarchical clustering
of hBE cell miRNA
regulators of the phenotype of the airway epithelium; 2)
expression data.
technology exists enabling comprehensive miRNA
Cultures from 6
analysis through both unbiased discovery using deep
individuals (1-6) at
sequencing, and via state-of-the-art microarray and
day (D) 5, 14 and 35
RT-PCR technology for known miRNAs, and 3) in vitro
were studied.
studies are not plagued by the uncertainty of variable
non-epithelial cell contributions to the expression pattern, and are amenable to
genetic manipulation for functional testing. Our team has the optimal
combination of resources and experience. The hBE cells themselves, which
are prohibitively expensive when purchased commercially, will be provided to
this project from our existing collection at no expense, which enables more
ambitious experimentation. It is important to note, based on our prior array
experience that there can be significant variability in miRNA and mRNA
expression in hBE cells from human to human, which can be compounded by
experimental variability. Our ability to simultaneously study cells from a
representative sample of 6 people minimizes one source of experimental
variability. Furthermore, we will use triplicate cultures from each donor within
each group and will pool RNA to minimize contributions from one untoward,
outlier culture well. The cells will be employed in state-of-of-the-art models of
key disease processes that are either already well established in our labs or
easily achievable. Our team includes world-class, expert co-investigators in
Figure 3. Expression map of
both miRNA molecular biology and bioinformatics, which are key to achieving
miRNAs called as significant by
the experimental goals.
SAM. Cultures from 6
We note our strategy to perform mRNA expression analysis in the
individuals (1-6) at day (D) 5, 14
identical set of cultures as for the miRNA analyses, which entails considerable
and 35 were studied. Genes
extra effort. Theoretically, we could have explored existing databases for the
(rows) are median centered and
clustered (tree not shown).
mRNA data. However, such data does not exist for all the experimental
Arrays (columns) are median
conditions, and it is likely that specific technical differences between different
centered but not clustered.
laboratories would inhibit direct comparisons. Thus, we think that using the
identical samples is the optimal strategy to determine the miRNA regulatory network governing phenotypic
changes in hBE cells in health and disease. Finally, we already have RNA samples for 2 of the seven sets of
experiments in hand, which is compatible with the goal of the ARRA to quickly increase the use of Core
facilities, create new hires and generate novel data that accelerates the pace of scientific discovery.
Specific Aim 1) Develop a comprehensive portrait of miRNA expression in hBE cells during normal
differentiation and in a spectrum of relevant injury/repair conditions.
Rationale: miRNAs play a vital regulatory role in many cell processes and their expression is highly cell typeand differentiation stage-specific. Human bronchial epithelial cells are central participants in the pathogenesis
of several extremely important lung diseases and, despite provocative early evidence in the literature and
preliminary data in this proposal, there is minimal miRNA data in this key cell type. In vitro model systems
recapitulating in vivo structure and function are critical milestone tests for therapeutic development and
translation. We are uniquely poised to provide a comprehensive picture of the miRNA repertoire in hBE cells
and their expression patter and cell localization during normal differentiation and in a series of highly relevant
disease models. We will also perform mRNA arrays from the identical cell cultures in order to elucidate
miRNA:gene expression networks.
Aim 1A. Identify novel hBE cell miRNAs by creating libraries and performing deep sequencing from: 1) the ALI
hBE cell differentiation time course; 2) the response of well differentiated cells to wounding; 3) the response to
acute and chronic exposure to bacterial products; 4) exposure to the Th2-type cytokine IL-13, which induces
mucous secretory cell hyperplasia; 5) exposure to cigarette smoke gas phase; 6) simulated ambient ozone
exposures and 7) squamous metaplasia induced by retinoic acid deficiency.
Methods: We begin this section with a brief general discussion of the cell culture model and overall
experimental design and then a short piece on each of the 7 experimental conditions. Since the downstream
analytical methods are common to each of the groups, we follow with a single section with methods for
unbiased miRNA discovery by deep sequencing.
The ALI hBE cell culture methods have been described in detail previously {}. For all experiments, cells
from 6 different tissue donors will be employed. Within each donor, each experimental group (time point,
exposure etc.) will consist of 3 replicate wells for RNA harvest, which results in excess RNA, but prevents one
potentially bad well from ruining a data point and allows for replicates in downstream quantitative analyses
(qRT-PCR). Each well is inspected at each media change, culling any that are unusual. In experiments
involving potentially toxic exposures, we assess lactate dehydrogenase release into the apical media and
basolateral inflammatory cytokine (IL-8, Gro) production. Replicate wells are also prepared for histology
(H&E, other stians and in situ hybridization) using fixation with 4% formaldehyde followed by paraffin
embedding and blocks for frozen sections. RNA is extracted using a guanidinum:chloroform/phenol protocol
optimized for hBE cells to prevent glycoconjugate (mucous) contamination of RNA. An aliquot is assessed by
spectrophotometry (Nano Drop) and using LabChips on an Agilent 2100 Bioanalyzer.
The ALI hBE cell differentiation time course. As illustrated above in the Strategy section (Figure 1), the
time course of hBE cell growth at an ALI recapitulates a process similar to wound repair in vivo in which poorlydifferentiated cells undergo a phase of rapid proliferation, followed by polarization, and ultimately differentiation
into the characteristic airway epithelial cell types, namely basal, secretory
and ciliated cells. As noted above, we have chosen 3 time points, days 5,
14 and 35 that represent distinct stages in the process. Cells at day 5 are
almost uniformly squamoid and highly proliferative with little expression of
differentiation markers, whereas by Day 14 apical membrane polarization
and mucous secretory cell development are evident. By Day 35 large
numbers of ciliated cells are present as well as fully mature basal and
secretory cells. There is a single study of mRNA expression during the ALI
hBE cell time course in the literature {} but no miRNA data. Cell culture,
RNA harvest and preparation of histology sections of triplicate specimens
Figure 4. Wound repair model. A)
Complementary pair of probes to
from an n = 6 different tissue donors is already complete and ready for
wound ALI cultures. B) Probes
uniform downstream processing as described below.
placed in 12 mm diameter Millicell
The response of well differentiated cells to wounding. The normal
inserts. C) hBE cells 24 hours after
airway epithelium in vivo is mitogenically quiescent but responds
wounding, repairing cells (left of
dramatically to wounding via cell de-differentiation, migration, proliferation,
dash) showing nuclear localization
and re-differentiation to ultimately restore an intact epithelium, which is
of EGR-1 transcription factor.
critical to prevent airway obliteration. We have studied hBE cell wound
repair {} and have recently developed a unique device for wounding ALI cultures that enables subsequent
harvest of just the non-wounded and wound-repairing cell compartments (Figure 4). To our knowledge there
are no miRNA or mRNA gene array studies of hBE cell wound repair. To fill this knowledge gap we will collect
RNA for analysis of miRNA and mRNA expression at time 0, and in both cell compartments at 8, 24 and 48
hours after wounding, and subject them to uniform analysis as below.
The response to acute and chronic exposure to bacterial products.
Repeated and/or chronic infection is characteristic of COPD, CF, non-CF
bronchiectasis and PCD. The airway epithelium occupies the interface
between the body and luminal infectious agents and is strategically positioned
to detect and respond to danger and to orchestrate the host response. There
are several studies of the hBE cell transcriptional response to pathogens but
many are in cell lines and/or in poorly differentiated cells on plastic.
Furthermore, no studies have assessed adaptation to chronic infection,
although this is undoubtedly an important feature of the host response. We
performed mRNA arrays following acute and chronic exposure of ALI hBE cells
to P. aeruginosa bacteria and detected potent induction of inflammatory
cytokines and other genes but, interestingly, the cells exhibited partial tolerance
(decreased responsiveness) after repeat challenges (Figure 5). We will now
perform unbiased detection using deep sequencing and arrays to test the
hypothesis that miRNAs are involved in the ALI hBE cell response to acute and
chronic bacterial product exposure. We will study cells 4 hours after an initial
Figure 5. hBE cell mRNA
challenge (acute response) and 24 hours after three challenges (the new
expression after P.
aeruginosa. A) The acute
baseline at 72 hours, representing adaptation to chronic exposure) and 4 hours
response of naive cells- 372
after a fourth challenge (76 hours, representing modified responses in adapted
probesets changed > 1.5 fold
cells). As with the other experimental protocols, the cells will be subjected to
with p<0.01. B) The response
uniform downstream analysis as below.
of adapted cells- only 72
Exposure to the Th2-type cytokine IL-13, which induces mucous secretory probesets met these criteria.
cell hyperplasia. Increased mucus production is a feature of multiple lung
diseases and is thought to contribute to airway obstruction, especially in acute asthma exacerbations and fatal
asthma. Inefficient mucus clearance due to imbalanced, excessive mucous glycoprotein production and
deficient ion and water transport creates mucus stasis and infection characteristic of CF and COPD. However,
there are no specific therapies directed at preventing or reversing excess mucus production. To understand the
role of miRNAs in mucus hyper-production, we will expose ALI hBE cells to 10 ng/ml 1L-13, a Th2-type
cytokine known to induce mucous secretory cell hyperplasia. IL-13 treatment will take place every 48 hours
beginning at day 25, for up to 10 days, which is a standard protocol in this field, and RNA and histology
samples will be harvested at 8, 24, 48 and 240 hours.
Exposure to cigarette smoke gas phase. The vast majority of COPD is due to cigarette smoking, and it is
vital to understand mechanisms of smoke toxicity. There have been many
mRNA array studies of broncho-alveolar lavage cells (mostly macrophages)
and airway brushings (epithelium enriched) from smokers and former smokers
versus non-smokers, and numerous in vitro studies of smoke exposure of cell
lines on plastic to various tobacco preparations, ie. extracts and condensates
versus whole smoke. A previous study examined the acute mRNA, but not
miRNA, transcriptional response of ALI hBE cells to 1 hour of apical exposure
to whole, mainstream smoke and 6 and 24 hours after discontinuation of the
single dose {14291}. We will use the identical smoke exposure system, which
we posit is the best possible model (see letter from Dr. Tarran) and will extend
these studies to include both miRNAs and multiple exposures. These
experiments are modeled after the P. aeruginosa experiments (above) and the
time points are- 4 hours after exposure to 3 cigarettes over a 4 hour interval
Figure 6. Cigarette smoke
(acute exposure), 72 hours after 2 additional days of smoke exposure (new
model. ALI hBE cells exposed
baseline, 18 hours after the end of previous smoke exposure) and again 4
to air or smoke for 3
hours after the fourth day of smoke exposure (altered response in adapted
consecutive days modestly
cells). As shown in Figure 6, pilot experiments reveal modest LDH and IL-8
released IL-8 and LDH,
release under this exposure regimen, suggesting it is appropriate, sustainable,
indicating appropriate dosing.
and not overtly toxic, which will be verified by histology.
N=4 wells/group, mean + SD,
*p< 0.05.
Simulated ambient ozone exposures. Environmental ground level ozone
represents one of the most enduring human air pollution challenges. Especially during the summer, many US
municipalities routinely exceed the National Ambient Air Quality Standard of 0.075 ppm over an 8-hour period,
which is associated with increased respiratory symptoms and hospitalizations. There have been many mRNA
gene array studies after ozone exposure, mostly in plants and in whole animal models, with fewer studies in
cells and, to our knowledge, none in ALI hBE cells or examining miRNAs
(except one limited study of the arabidopsis response to ozone).
Interestingly, laboratory ozone has been associated with technical problems
with gene arrays. To examine the interaction of ozone exposure with host
defense against S. aureus bacteria, we performed pilot studies of ozone
exposure of a novel hBE cell line (UNCN3T cells, {}) capable of differentiation
at an ALI (Figure 7). Based on these results, we will perform additional
preliminary ozone-exposure studies, now using primary ALI hBE cells, to
determine if they have similar toxicity and cytokine induction profiles. We will
then expose ALI hBE cells both acutely and chronically to low (~0.2 PPM)
and high dose (~0.8 PPM) ozone for 5 hours on 3 consecutive days. The
chamber system has been previously well described and will be made
available to us by collaborators at the USEPA facility in Chapel Hill (see letter
from Dr. Jaspers). Again, samples for RNA and histology will be collected at
Figure 7. Ozone model. ALI
4 hours after the completion of the first acute exposure of naive cells, then
UNCN3T cells were exposed to
again 18 hours after repeated exposures (the new baseline), and finally 4
0.2 and 0.8 PPM O3 for 5 hours
hours after the last of 3 exposures (altered responses in adapted cells).
on 3 consecutive days and then
Squamous metaplasia induced by retinoic acid deficiency. Ongoing
challenged with S. aureus
injury from cigarette smoking or other stimuli such as chronic infection results
filtrates. These doses of O3 did
in squamous metaplasia of the bronchial epithelium, a characteristic
not cause LDH or cytokine (not
phenotypic alteration associated with disease severity, that may become
shown) release on their own, but
inhibited the normal response to
independent of the inciting cause and whose reversibility in vivo remains
the bacteria, which may be
unknown. Squamous metaplasia is likely a precursor lesion to carcinoma in
immunosuppressive. N=4
situ and invasive lung cancer. There are several reports of miRNA
wells/group, mean + SD, *p<
expression profiles in non-small cell lung cancer, including the squamous
0.05.
subtype {14276}, and one study of progressive pre-cancerous lesions
{14273}. To our knowledge, there is one relevant mRNA, but not miRNA, array study of an in vitro model of
squamous metaplasia induced by prolonged culture on plastic {13600}. We will employ the well-known model
of retinoic acid (RA) deficiency to induce squamous metaplasia in ALI hBE cells. RNA and sections for
histology will be harvested at days 5, 14 and 35 in the continuous
presence or absence of RA and also at 2 time points (24 and 96
hours) after re-addition of RA to squamous cultures, which reverses
the phenotype.
Creation of small RNA libraries. Since there is not yet a
comprehensive hBE cell miRNA expression database, and array
technology biases for detection of abundant miRNAs, we will perform
novel identification of miRNAs (and potentially other small regulatory
RNAs) using small RNA library deep sequencing. Since it is
impracticable to create libraries from each of the groups used for
Figure 8. Improved miRNA cloning
miRNA and mRNA arrays, we will equally pool RNA from all 6 of the
strategy. Mature miRNAs are size
tissue donors per experimental treatment and time point/condition into fractionated by gel isolation, tailed with
poly(A) polymerase and ligated to the RNA
one sample. This will result in approximately 60 deep sequencing
oligo R1. Reverse transcription is performed
runs, anticipating 40 runs in year one and 20 in year 2.
using the anchored DNA primer D2 (N refers
Cloning was originally described by the Bartel, Tuschl and
to any nucleotide, and V refers to any
Ambros labs {14299} {14300} {14301}. Their well-established method
nucleotide except T; this anchors the primer
to the beginning of the tail). PCR
is based on ligation of RNA/DNA oligonucleotides to each end of the
amplification is performed with primers D1
mature miRNA, followed by PCR amplification and sequencing. We
and D3. Sequencing tags are added by a
have modified this protocol, eliminating one ligation step (Figure 8).
second PCR step, and the population is
Instead of ligating an oligonucleotide to the 3' end of the miRNA, we
sequenced using a Solexa GAII.
use poly(A) polymerase to add a poly(A) tail, providing a primer
binding sequence for reverse transcription. This eliminates the cloning background caused by direct 5' and 3'
oligonucleotide ligation and reduces the gel purification from three steps to one. Since gel purification is a
major source of yield loss, our modification improves the discovery rate of rare miRNA species.
Total RNA (up to 10 ug) will be size fractionated on a 15% polyacrylamide/Urea/TBE gel. Isolated RNA
will be tailed with poly(A) polymerase, followed by phenol extraction. The tailed small RNAs will be ligated to
the 5' oligonucleotide R1. This RNA oligonucleotide lacks a 5' phosphate, therefore cannot concatamerize or
circularize, but can only be ligated to an existing 5' phosphorylated small RNA. This product is phenol
extracted and reverse transcribed with the anchored oligo d(T) primer D2. The DNA product will be amplified
with the primers D1 and D3. Cycle number will be empirically determined, using the minimum to produce a
SYBR-gold visible product. The PCR product will be re-amplified to add sequencing tags for Solexa
sequencing.
Deep sequencing. We will employ the UNC Lineberger Comprehensive Cancer Center (LCCC) High
Throughput Genomics Sequencing Facility directed by Dr. Poitr Mieczkowski. The protocols for PCR product
sample preparation, submission, prioritization, analysis and data transfer are dictated by facility policy and
practice and are routine. Sequencing is via the Genome Analyzer II (GAII)-Illumina (Solexa) platform, enabling
>50 million 35 base pair reads per flowcell, 8 lanes per flowcell, 5-7 mil reads per lane and thus generating
>1.5 GB of data per single read flowcell,
Sequencing data handling. A suite of programs called the “Illumina Genome Analysis Pipeline” is used for
data analysis. The data download includes five files: 1) “Raw” sequence file; 2) “ELAND” sequence alignment
results file; 3) “Filtered” sequence file; 4) Sequence “Export” file; and 5) “Sorted” sequence file. These files
enable compiling and analysis of the sequencing data and bioinformatics analysis including, clustering of
sequence similarity groups, construction of clone count tables, comparison of miRNAs between different
samples, and hierarchical clustering of samples.
Aim 1B. Map new miRNAs to the genome to validate stem-loop potential and vertebrate conservation, and
verify expression of bona-fide ~21 nucleotide small RNAs by Northern blotting.
Sequencing data analysis. Output from the “Export” file will be mapped to the UCSC genome assembly as a
custom track. Reads that correspond to known miRNAs, mRNAs exons, large and small noncoding RNAs
(tRNAs, rRNAs, snRNAs, etc) will be removed. The read sequence plus 80 nucleotide flanking genomic
sequence (each side separately) will be tested for hairpin folding potential (mFOLD). Candidate miRNAs that
pass these filters will be ranked by sequencing clone count and vertebrate conservation. Most miRNAs exhibit
a distinctive conservation pattern with high conservation in the mature strand, low conservation in the loop, and
moderately high conservation in the second strand of stem (star strand). It is formally possible that we identify
novel miRNAs that are not conserved outside of primates, therefore,
nonconserved candidates with >5 clone count will be validated.
Northern blotting. We will verify that novel sequences are derived from a
~21 nucleotide small RNA, rather than a fragment of a larger RNA species.
Total RNA will be analyzed by 12% acrylamide/Urea/TBE northern blot, using
an antisense oligonucleotide probe based on the candidate mature miRNA. If
necessary, large RNAs will be depleted by precipitation in 12.5% PEG 5000/
12.5 mM NaCl.
Aim 1C. Determine expression of known, annotated miRNAs and mRNAs in
the same seven conditions above using Agilent microarrays, facilitating
analysis of miRNA regulation of hBE cell gene expression.
Methods: Microarrays. The Hayes and Hammond labs have extensive
experience in the generation and analysis of microarray data {14223} {14252},
and the UNC Genomics Core is highly qualified to perform the microarray
experiments described in this application. This Core is 1 of 3 sites chosen for
the pilot phase of The Cancer Genome Atlas (TCGA) for which Dr. Hayes is a
co-Investigator {14223}, and also serves as the profiling center for the Cancer
and Leukemia Group B project. Figure 9 documents recent productivity,
showing classification of malignant brain tumors by expression profiling, for
which there is also paired miRNA data (see below). In this example, we
Figure 9. Gene expression data.
Analysis of 173 glioblastoma
samples from the Cancer
Genome Atlas project performed
at UNC identifies four gene
expression subtypes. Samples
were ordered based on subtype
predictions.
document the power of gene expression analysis in our hands to detect molecular subtypes (and the
associated genes), which in large part reflect abnormally differentiated cancer cells. In the current proposal,
we will use a similar approach to capture miRNA and mRNA
expression patterns associated with normal hBE cell
differentiation and the response to injury.
miRNA profiling. We will use 3rd generation Agilent Human v12
miRNA arrays containing 866 human and 89 viral miRNAs that
are regularly updated according to the Sanger miRBase
database. Briefly, 400 ng of total RNA are labeled per the
manufacturer protocol and samples are processed according to
standard protocols in the Genomics Core. Data is stored in the
UNC Microarray Database and we favor Quantile normalization
for miRNA data. Using this platform and associated protocols,
the Hayes lab has analyzed ~1000 arrays In support of the
TCGA, and we are the only site in the pilot phase profiling
miRNAs.
mRNA profiling. Gene expression profiling employs 180,000
feature, custom-designed Agilent long oligonucleotide arrays that
contain 25,000 unique human protein-coding gene probes, a
Figure 10. miRNA expression data. A)
second unique probe for almost all of these protein-coding genes miRNAs define four glioblastoma subtypes. B)
miRNA subtypes correlate with subtypes
as an independent measure of gene expression, and hundreds
derived by gene expression profiling in the
of control spots for normalization, mRNA quality and array
same sample set.
quality control. Probes are spotted in triplicate occupying
~150,000 features. The remaining ~30,000 features will
be left open initially, but will be used to include new
transcript and/or splicing content as it is discovered by
us and others, a primary feature for selection of the
Agilent technology over competing array products. The
basic protocol for the gene expression arrays has been
described in detail elsewhere {14223} and has been
performed 10’s of thousands of times in the UNC
Genomics Core. Briefly, 1 ug of total RNA is labeled
using the Agilent low input RNA linear amplification kit.
Microarray hybridizations are carried out using 2 ug of
Cy3-labeled common reference sample (Stratagene’s
Human Universal Reference RNA) and 2 ug of Cy5labeled experimental sample. Hybridizations are
performed using the Agilent hybridization kit and a
Figure 11. TMEFF1 gene expression is best predicted
Robbins Scientific hybridization oven. The arrays are
by hsa-mir-34a versus other genomic events. The 5
incubated overnight, washed and then scanned using an
diagonal plots represent histograms of the indicated
Agilent Microarray scanner and Agilent Feature
data types from ~200 brain tumors (TCGA data). From
Extraction and Analysis software. All raw data are then
top left to bottom right: TMEFF1 gene expression,
loaded into the UNC Microarray Database where a
TMEFF1 gene copy number, TMEFF1 locus
Lowess normalization procedure is performed.
methylation, hsa-mir-34a expression (targets TMEFF1),
and hsa-Mir-34a gene copy number. The lower offmiRNA target gene analysis. We document the power
diagonal plots graphically illustrate the relationship
of pairing microRNA profiling with mRNA gene
between the parameters and the corresponding upper
expression profiling in a cancer example. First, we
off-diagonal plots give the Pearson correlation
demonstrate that clustering algorithms of miRNA array
coefficient. TMEFF1 gene expression in glioblastoma
data define variants (subtypes) of tumors analogous to
varies by 4 orders of magnitude (row 1, column 1). The
gene expression arrays (compare Figure 10 to Figure
influence of TMEFF1 copy number on gene expression
is modest (correlation=0.31, row 2, column 1 and row 1,
9), and that these subtypes overlap with tumor subtypes
column 2), while there is robust linear anti-correlation
defined by gene expression arrays. This is strong
between TMEFF1 and hsa-mir-34a expression
evidence that shared programs of gene and miRNA
(correlation=0.53, row 4, column 1 and row 1, column
expression integrate to define fundamental
4).
characteristics of the tumors. Since tumor formation
frequently includes abnormal cell differentiation, the paired approach will be similarly useful to study normally
differentiating hBE cells and the response to injury. In Figure 11, we extend the integrated analysis of gene
expression data and miRNA data, focusing on microRNA-gene target pairs. In this cancer example, we
examine a gene known to be important in brain differentiation, and document a weak impact of common
cancer-specific genomic alterations such as gene loss and gene methylation. In contrast, using paired miRNA
and gene expression data, we detect a strong association between miR-has- 34a and its predicted gene target
TMEFF1. In the current proposal, we will similarly measure, in a genome wide manner, miRNA-gene target
interactions, determining experimentally which of the computationally predicted interactions are measurable in
our model systems.
Aim 1D. Perform confirmatory qRT-PCR and in situ hybridization localization for select highly regulated
miRNA’s identified above.
Methods: qRT-PCR. It is conventional to verify accuracy of array data using independent methods.
Accordingly, within the scope of this proposal, we will quantitate expression of approximately 5 miRNAs
identified on arrays per each of the 7 projects described in Aim 1A. We may assay more than 5 miRNAs in a
particular experiment, for example if we discover more than 5 highly regulated, unique or otherwise
scientifically provocative miRNAs in a specific project. qRT-PCR may also be used to further characterize
novel miRNAs discovered by sequencing. qRT-PCR will be performed exactly as per manufacturers’
instructions (miRNA reverse transcription kit and TaqMan miRNA assay kit, Applied Biosciences). PCR is
performed on 96 or 384 well format thermocyclers in the CF Center or in the LCCC, respectively, and analysis
is by conventional comparative Ct to 5S rRNA and/or miRNAs reported to not vary significantly across a wide
group of specimens {14298}. All assays are performed in duplicate using non-pooled RNA’s from 3 replicate
wells from 6 different tissue donors. Results are assessed with descriptive statistics and t-test or ANOVA with
post-hoc analysis, or using non-parametric tests when required.
In situ hybridization. Since miRNA expression may occur in a cell-type-specific spatial and temporal pattern,
we will perform in situ hybridization in the cultures at specific time points or conditions, prioritized based on the
sequencing and array results. Since this method is more consumptive than qRT-PCR, we anticipate being
able to study ~3 miRNAs per experimental group and will not necessarily examine all subgroups within the
project. Briefly, in situ hybridization of formalin paraffin and whole mount culture preparations will be
undertaken using specific and control probes (LNA probes, Exiqon) and colorimetric detection as previously
described {14253}. Recent studies indicate that frozen sections specifically cross-linked to preserve small
RNAs combined with fluorescence detection may be much more sensitive {14237} and frozen blocks for
sectioning will be prepared and retained for this purpose. Analysis of staining patterns will be primarily
qualitative and complementary to the qRT-PCR data above although antibody immuno-staining for cell-typespecific markers (keratin 5 &14 for basal cells, acetylated tubulin for ciliated cells and MUC5AC or 6 for
mucous secretory cells) combined with in situ hybridization will enable correlation of miRNA expression with
cell type.
Aim 1. Data Analysis/Limitations/Alternatives/Future Directions. A major, novel deliverable from Aim 1 is
a comprehensive hBE cell miRNA expression database based on unbiased discovery through deep
sequencing as well as arrays and qRT/PCR for known, annotated miRNAs. Studies of this type, by their
nature, are hypothesis generating, as opposed to the paradigm of testing specific hypotheses. Nevertheless,
the data will be comprehensive and novel, using state-of-the-art cultures, models and analytic methods. It will
enable selection of a subset of highly regulated miRNAs for functional analyses as proposed in Aim 2, which is
a more mechanistic approach.
As described above, we will develop a standardized analytical pipeline for ALI hBE cells using library
construction and deep sequencing. We think this is extremely valuable since it is widely appreciated that many
miRNAs are highly cell type and differentiation stage specific. For example, the miRNA lsy-6 is expressed in a
single neuron in C. elegans. We believe highly restricted cell-specific miRNAs remain to be discovered, and to
our knowledge unbiased discovery HAS NOT yet been performed in hBE cells. Drs. Hammond and Hayes
have prior experience in unbiased exploration of miRNA and mRNA expression via sequencing. As well as
adding to the miRNA repertoire and providing a basis for further quantitation of highly regulated miRNAs, it is
conceivable that we may find specific miRNA markers of hBE cells, cell subtypes and functional stages, which
will be an important contribution as such markers could be used to direct expression, ablate or purify cells as
was recently done for the pri-miR-375 promoter in endocrine pancreas {14254}, but this would be a future
direction.
We will initially evaluate the time course of normal hBE cell growth and differentiation in vitro and will
then exploit the data generating system to efficiently probe highly relevant injury/repair conditions. We
anticipate no difficulties regarding the ability to produce the requisite hBE cell cultures, to employ the injury
models or to purify adequate RNA and histology samples since the Randell lab has extensive experience and
resources for hBE cell ALI culture- the cells already exist in our collection and the models have already been
accomplished (wounding, bacteria, smoke, ozone, RA-deficiency) or are quite simple (IL-13).
The specific steps for data generation and analyses, including sample size and an outline of technical
and statistical considerations for specific assays, have been presented above. These methods have been
extensively used and published by members of our group. Many, such as the mechanical generation of
libraries, sequencing and, especially, arrays and qRT-PCR are now quite routine. Dr. Hayes has been
involved in massive array and sequencing efforts related to cancer for many years. Dr. Hammond has been in
the miRNA field for over 10 years and has abundant experience in the analysis of primary sequence and array
data and its translation via mapping and, processing, expression and functional assays. Based on the
preliminary studies, and the track record/complementary expertise of the team, we are confident that the
experiments can be mostly accomplished as planned. Realistically, the plan is highly ambitious in terms of the
number of samples to be generated and analyzed. The experiments all follow the same paradigm and should
be easier to accomplish as we gain specific experience with the systems employed. However, if experiments
must be dropped by necessity we will prioritize experiments in their order, ie. by eliminating the last planned
experiment (RA-deficiency) first.
In any event, the studies will certainly provide abundant, state-of-the-art profiling of the miRNA
repertoire of hBE cells, revealing highly regulated and potentially novel miRNAs almost certainly involved in the
dynamic regulation of their growth and differentiation and response to injury. This data will be useful from the
perspective of both enhancing our basic understanding of regulation of hBE cell phenotype in health and
disease and to prioritize subsequent functional testing as in Aim 2 of this proposal. We expect that this data will
be a beginning, a sound foundation, for the design of many experiments and analyses, by others and us, to
more fully understand the role of miRNAs in airway biology.
Correlative miRNA and mRNA expression analysis will enable exploration of gene expression
regulatory networks in these important cells. We expect to reveal many specific relationships between
miRNAs and gene expression of their predicted downstream targets, as illustrated in the cancer example
above. A commonly employed technique for verifying such relationships is to express a marker gene
(luciferase, GFP) fused to the 3’UTR of the putative target gene, sometimes in the cells of interest, but usually
in simple conventional culture models. We contemplated using this reporter approach in well-differentiated ALI
hBE cells, but like the functional studies in Aim 2 below, it would also require the development and use of
lentiviral vectors and infection of the cells while poorly differentiated. Alternatively, adenoviral vectors, which
can transduce mature hBE cells, could be used for reporter gene studies. These experiments are possible, but
are resource and time (especially adenovirus) consumptive. Based on the anticipated lower payoff of the
reporter approach versus manipulation of miRNAs as proof-of-concept for novel therapies (Aim 2), we have
prioritized the latter. However, such reporter vectors will be created to screen miRNA mimic and knockdown
strategies (Aim 2) and these may be employed in ALI hBE as resources allow.
An uncertainty of these studies is that they employ an in vitro model, which may not precisely replicate
the in vivo expression pattern. This is certainly true, but the model does allow for more precise cell-typespecific analysis than in vivo studies where cell populations are heterogeneous and variable. Since the model
recapitulates many of the functional properties of the in vivo epithelium, it allows for testing of specific
hypotheses regarding miRNA participation in normal growth and differentiation, injury and repair. The
experiments will clearly complement ongoing in vivo studies by others. A future direction is correlative in vivo
studies in humans (measurements in excised human cells and tissues, in situ hybridization of normal and
diseased human lung) and also the creation of genetically manipulated mice, but those are beyond the scope
and time frame of this already ambitious 2-year proposal and are more realistically considered a future
direction.
Specific Aim 2) Determine functional consequences of manipulating expression of candidate miRNAs
in hBE cells.
Rationale: Novel miRNA discovery by sequencing, high throughput screening of miRNA arrays, qRT-pCR and
robust northern blots as described above will allow for rigorous identification and quantitation of hBE cell
miRNAs, but they must be complemented by over-expression and inhibition in order to determine functional
effects. These studies are challenging in ALI hBE cells but are key for establishing whether modulating
miRNAs can change phenotype for potentially beneficial therapeutic effects.
Aim 2A. 2A. Use an inducible, titratable viral vector system to express miRNA mimics and inhibitors to
determine functional consequences of manipulating specific miRNAs in hBE cells.
Methods: Manipulating miRNAs in ALI hBE cells. miRNA
mimics or inhibitors for gain-of-function and loss-of-function
experiments, respectively, enables study of the biological role of
specific miRNAs and there are numerous commercial products for
this purpose. Lipid transfection or electroporation are typically
used to introduce oligonucleotides, modified oligonucleotides or
plasmids into tissue culture cells, although some have been
applied in vivo via systemic or local administration in animals, with
variable success. Well-differentiated ALI hBE cells are notoriously
difficult to transfect in vivo or in vitro, precluding typical plasmid
and oligonucleotide approaches for genetic manipulation
commonly used in routine tissue culture cells. We initially
developed lentiviral vectors with constitutive expression to infect
hBE cells while poorly differentiated on plastic, followed by
Figure 12. Inducible expression in ALI hBE
selection and passage to porous supports where they can
cells. Primary hBE cells on plastic were
differentiate. A potential downside of this approach is that
infected with pSLIK lentiviral vectors, selected
constitutive expression of inhibitors or genes that alter function,
with puromycin, then subcultured at an ALI
and treated with doxycycline as indicated.
for example that inhibit cell growth would be self-defeating. Thus,
The cells were harvested for mRNA (A), and
in more recent experiments we have used an inducible system,
protein (B), and were also studied in Ussing
pSLIK {14302}. Initial studies (Figure 12) examined the epithelial
chambers (C) and by confocal microscope
sodium channel (ENaC) with shRNA and illustrate knockdown at
imaging of airways surface liquid (ASL, D).
the RNA, protein and functional (decreased amiloride-sensitive
Significant knockdown and corresponding
ENaC electrical current and increased airway surface liquid due to changes in function are apparent (see
decreased Na absorption). This model enables well-controlled
adjacent text). Ctl GFP = pSLIK vector
containing GFP only as a control. shENaC
functional assessment of the ability of miRNA mimic or inhibitor
= pSLIK vector containing GFP and shRNA to
expression to modify hBE cell phenotype.
ENaC. Dox = doxycycline, which induces
miRNA mimic and inhibitor expression in ALI hBE cells. The
expression of GFP and shRNA.
method for manipulating gene expression in ALI hBE cells
involves design and cloning of experimental and control vectors and virus production as well as the
downstream characterizations, which are collectively fairly consumptive and will practically limit the number of
genes that we can express or knockdown. We anticipate accomplishing at least one example from each of the
seven experimental protocols, targeting a highly regulated and/or novel miRNA. This will be selected not only
based on expression but also looking for scientifically provocative candidates such as miRNAs highly
correlated with cell-type-specific panels of gene expression- for example secretory proteins characteristic of
goblet cells or the ciliated cell machinery detected in the mRNA arrays. This approach will be combined with
manually scanning the still manageable, but rapidly evolving miRNA biological function database. Ideally, we
will target two complementary genes per experimental treatment for both expression and knockdown- one that
increases and one that decreases.
There has been excellent recent progress, particularly from the Naldini lab, illustrating methods to
design and assess the function of introduced natural and chimeric/artificial miRNAs {14823}. They showed that
lentiviral vetors enabled exogenous/artificial miRNAs to reach the concentration and activity typical of highly
expressed natural miRNAs without perturbing endogenous miRNA maturation or regulation. Similarly to the
protocols described in that report, we will initially screen for functional effects of candidate miRNAs in a simple
cell line culture system on plastic, using co-transfection of the putative mimic and constructs containing 4
copies of its perfect target cloned into the 3’ UTR of a reporter gene (luciferase and/or GFP). Promising
candidates will then be sub-cloned into the pSLIK virus system and viral particles will be produced by triple
transfection of 3T3 producer cell lines using routine procedures. hBE cells will be infected and selected as
described above, and half of the cells will be exposed to doxycycline at specific time points as dictated by the
experimental goals. The loss of function, miRNA knockdown experiments involve a similar approach but now
over-expressing miRNA target sequences {14282}, which act as “decoys”. The ideal experiment requires 8
tandem imperfect, bulged matches, which appear to be more effective due to slower RISC complex
processing. Furthermore, strong promoters and high vector dosing are needed to result in multiple copy
integration and strong expression. The inhibitors will also be tested for knockdown efficiency in simple cell line
on plastic model systems to select the optimal candidates for expression in ALI hBE cells as above. In these
experiments, we will measure mRNA levels of the endogenous miRNA targets in hBE cells, which are
expected to rise.
Phenotypic characterization of ALI hBE cells. The Randell laboratory has many years of experience
evaluating airway epithelial cell structure and function. A primary method for assessing effects of manipulation
of miRNA expression and knockdown will be histology. The simple evaluation of H&E and Alcian blue-PAS
stained, routine formalin-paraffin sections is highly informative and cost effective. Semi-quantitative
morphological scoring of a series of specific characteristics (epithelial thickness, nuclear density, percent
ciliated surface, amount of stored mucosubstances) by multiple viewers blinded to sample identity is
concordant with the basic morphometric principle of “do more less well”, but fully quantitative morphometry, will
be used in the event of more subtle effects. Additional sections of the same histology blocks will be
immunostained with routine airway epithelial cell-type-specific markers (keratin 5 &14 for basal cells,
acetylated tubulin for ciliated cells and MUC5AC or MUC5B for mucous secretory cells). Gene (mRNA) and
protein expression of the putative target genes and proteins will be assessed using qRT-PCR and western
blot/immunostaining, respectively. The function of the putative (and/or verified) target genes as determined in
the literature and/or by gene annotation will be used to decide specific, contingent downstream assessments.
For example we would logically test the response to inflammatory stimuli for target genes implicated in
inflammatory processes. Similarly, targets regulating ion transport would be evaluated as described in Figure
12. We will assess hBE cell proliferation and apoptosis when target genes are involved in cell cycle regulation
and/or if hyper- or hypoplasia was seen morphologically. We do not, a priori, plan on performing mRNA (or
miRNA) arrays after miRNA modulation, but that is certainly possible, given a sufficiently compelling
phenotype.
Aim2 Data Analysis/Limitations/Alternatives/Future Directions: The studies in Aim 2 logically extend from
Aim 1 and are directed at understanding if ALI hBE cells in vitro can be used as a platform for functionally
testing the effects of manipulating highly regulated and/or novel miRNAs. An immediate benefit will be a
greater basic understanding of the role of miRNAs in the molecular regulation of airway epithelial cell structure
and function, which is still poorly understood. Another ostensible goal is proof-of-concept testing that altering
hBE cell phenotype via manipulating miRNA status would be of therapeutic benefit. For example, if we find
certain miRNAs to be specifically associated with mucous secretory cell hyperplasia after IL-13 treatment, we
will determine whether expression of mimics induced mucous cells and whether inhibition of these miRNAs
reduced mucous cells. Results indicating miRNA regulation of mucous secretory cell proliferation and
differentiation would suggest a new therapeutic approach to mucus hyper-production. A similar process would
be relevant to discovery of anti-inflammatory agents after P. aeruginosa treatment, cyto-protective therapies
after smoking or ozone, reversal of squamous metaplasia, and so forth. There are likely to be many examples
of highly regulated miRNAs in Aim 1 and we will only be able to test a small subset of the most compelling
candidates, but the development of the testing system in the most relevant differentiated cells will clearly
facilitate more extensive future studies.
There is a certain level of uncertainty in the Aim 2 experiments since regulated and inducible lentiviral
manipulation of miRNAs is a new and evolving technology. There are concerns about efficiency, although
these appear surmountable based on the literature {14283} {14282}. Similar to siRNA and shRNA, there are
concerns regarding off target effects, which may be exacerbated in the case of miRNA due to general effects
on related family members or the processing machinery. We will control for these as well as possible using
scrambled and irrelevant controls. There are concerns about gene silencing of integrated viral sequences,
which we have observed in earlier generations of retroviral vectors (data not shown). We may have to accept
reduced sensitivity to see effects if less than 100% of then cells are expressing at the time of analysis. If
silencing is profound (ie. >50%), which we do not expect with lenti- versus retroviruses, we could use
adenovirus in an up to 7-day expression timeframe. However adenovirus production is more cumbersome and
expensive and will limit the number of miRNAs we could study. Alternatively, we could test other lentiviral
backbones and promoters or adeno-associated virus to find minimally silencing vector strategies. Finally,
since the pSLIK system coordinately expresses a marker gene (GFP) we can employ single cell assays within
cultures containing a mixed population of expressing and silenced well-differentiated ALI hBE cells.
It will ultimately be important to test the effects of miRNA mimics and antagonists in vivo in genetically
manipulated mice, as has been done previously using a simple transgenic lung epithelial over-expression
approach {14253}. However, the two-year time frame of the RC1 format does not reasonably leave enough
time for the triple transgenic or ES cell knock-in strategies that would allow inducible, airway epithelial-only
expression that would prevent the neonatal lethal respiratory phenotypes so common in constitutive lung
expression transgenics {14253}. This would be a clear goal for future studies based on positive results from
Aim 2. Finally, the ultimate obstacle to miRNA manipulation as a human therapy for airway diseases, perhaps
via vector inhalation or instillation, is the long-standing problem of poor transduction of the human airway
epithelium in vivo. This challenge is beyond the scope of the current proposal, but novel vectors and/or
nanoparticles may be useful in the future. Alternatively, results showing positive phenotypic manipulation of
disease-related hBE cell phenotypes will likely spur the development small molecule therapies directed at
manipulating specific miRNAs.
5.4 Timeline and Milestones: We are poised to immediately make progress on the studies in this proposal,
which is consistent with the goals of the ARRA. We already have RNA in hand for the normal ALI hBE cell
time course as well as the RNA from the P. aeruginosa exposure protocol. These will be immediately
subjected to size fractionation, library construction, deep sequencing and arrays as described above.
Simultaneously, new ALI hBE cells cultures will be sequentially initiated for each of the other
exposure/treatment regimens, staggering those more highly demanding (smoke and ozone exposure) to
facilitate the workflow. The initial sequencing and array technical procedures and data analysis algorithms will
serve as test cases for evaluation of protocols and routines and whether they require modification in
subsequent experiments. We anticipate accomplishing 2/3 of the hBE cell cultures, sequencing and arrays in
year 1. Simultaneously in year 1, we will begin qRT-PCR and northern blot confirmation of miRNA expression
and in situ hybridization for cell localization in specimens from the initial experiments. Also in year 1, we
anticipate creating lentiviral vectors for miRNA manipulation and using them to begin to assess functional
changes due to modulation of miRNA expression in ALI hBE cells. Additional time in year 1 will be spent on
preparation of initil publications. In the beginning of year 2 we will perform the final 1/3 of the cell cultures,
sequencing and arrays, and the rest of year 2 will be dedicated to completing the downstream analyses and
preparation of publications.