MicroRNA expression in relation to different dietary habits: a comparison in stool and plasma samples
MicroRNA expression in relation to different dietary habits: a comparison in stool and plasma samples
Sonia Tarallo 2 3
Barbara Pardini 1 2 3
Giuseppe Mancuso 2 3
Fabio Rosa 1 2 3
Cornelia Di Gaetano 1 2 3
Floriano Rosina 0 2
Paolo Vineis 2 3 4
Alessio Naccarati 2 3
0 Division of Gastro-Hepatology, Ospedale Gradenigo , Corso Regina Margherita 8, 10153 Turin , Italy
1 Department of Medical Sciences, University of Turin , via Santena 19, 10126 Turin , Italy
2 The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions , please
3 Human Genetics Foundation , via Nizza 52, 10126 Turin , Italy
4 School of Public Health, Imperial College , Norfolk Place, London W2 1PG , UK
†These authors contributed equally to this work.
MicroRNAs (miRNAs), a class of small non-coding RNAs, are
fundamental for the post-transcriptional regulation of gene
expression. Altered expression of miRNAs has been detected
in cancers, not only in primary tissue but also in easily
obtainable specimens like plasma and stools. miRNA expression is
known to be modulated by diet (micro and macronutrients,
phytochemicals) and possibly by other lifestyle factors;
however, such influence has not yet been exhaustively explored
in humans. In the present study, we analysed the expression
levels of a panel of seven human miRNAs in plasma and stool
samples of a group of 24 healthy individuals characterised by
different dietary habits (eight vegans, eight vegetarians and
eight subjects with omnivorous diet, all groups with similar
age and sex distribution). The dual aim of the study was to
identify possible differences in miRNA expression due to diet
(or other lifestyle factors recorded from questionnaires) and
to compare results in both types of specimens. miR-92a was
differentially expressed in both plasma and stool samples and
with the same trend, among the three groups with different
diets (P = 0.0002 and P = 0.02, respectively, with expression
levels of vegans>vegetarians>omnivores). miR-92a was also
associated with low body mass index (P = 0.04 and P = 0.05,
respectively) in both types of specimens, and with several
dietary factors. Other analysed miRNAs (miR-16, miR-21,
mir34a and miR-222) were associated with dietary and lifestyle
factors, but not consistently in both stool and plasma. Our
pilot study provides the first evidence of miRNA modulation
by diet and other factors, that can be detected consistently in
both plasma and stools samples.
MicroRNAs (miRNAs) are a class of evolutionarily conserved,
small non-coding RNAs of 19–24 nucleotides in length that regulate
gene expression mostly at the post-transcriptional level (
miRNAs were thought to be involved in the regulation of development
and cell proliferation, but more recently it has been discovered that
they participate in a broad range of processes including cell cycling,
apoptosis, cell differentiation, tumour development, invasion,
metastasis and angiogenesis (
). The discovery that few miRNAs
are involved in sensing nutrient stress in plants (
) opens the way to
new epidemiological research on their roles. In fact, they seem to be
key factors in the response to environmental stress as they represent
one of the mechanisms deployed to reprogram gene expression so
that cells can adapt to changing environments (
For these reasons the interest for miRNAs has grown in the
last decades, and from being considered a curiosity of the model
organism Caenorhabditis elegans, they have been recently
described as promising biomarkers of disease (
particular, miRNAs have been observed to be aberrantly expressed in
several human cancers (
) and they are promising alternative
biomarkers for the detection and prognosis of cancer, and
monitoring response to treatment, as well as crucial players in cancer
initiation, development and metastasis (
). From the
biological point of view, miRNAs may be better predictive and
prognostic markers than DNA or messenger RNA (mRNA). A single
miRNA, in fact, may regulate hundreds of target mRNAs,
frequently grouped in specific biological pathways. Consequently,
a miRNA signature may provide prognostic information that
is several orders of magnitude greater than mRNAs. Besides,
miRNAs are more stable than other biomarkers during sample
processing, and are thus more suitable for analysis in plasma,
urine and stool (9), which is a key point in the search for cancer
markers. In the case of large scale screening for cancer
detection, for instance, the direct analysis in target tissue/biopsies is
often unfeasible, while the possibility of studying markers from
easily accessible and inexpensive surrogate tissues like plasma
and stool could be ideal, especially for repeated measurements.
It has been hypothesised that the observed differential
expression of miRNAs in cancer may be attributed also to
exposure factors, such as diet, generally not considered in
biomarker studies. Diet may modulate circulating miRNA profiles
(for a review see ref. (
)). The indirect influence of dietary
substances on endogenous miRNAs is well established:
vitamin A, zinc and various nutrients affect miRNA expression and
). On the other hand, a connection between diet
and disease has been repeatedly hypothesised for cancer (
The present study has measured and compared the expression
of six selected miRNAs in plasma and stool samples from healthy
volunteers (n = 24) following different dietary habits (vegan,
vegetarian and omnivorous diets). The aim was to assess whether the
different regimes may induce differences in miRNA expression
and whether the investigated specimens (plasma and stool)
consistently reflect these differences. Additionally, we have evaluated
the influence of demographic factors, specific food items and other
lifestyle factors on miRNA expression levels. The panel of
miRNAs was selected from previous studies in the literature reporting
their detection in plasma or stool samples in relation to colorectal
cancer, or indicated to be linked to diet in vivo and in vitro (
Material and methods
Sixteen subjects with differential dietary habits were enrolled in this study (eight
vegans and eight vegetarians) between June–August 2013, through online contact
with an Italian society of vegetarian nutrition, or by direct acquaintance. An
additional eight volunteers with similar age and sex distribution, but with an
omnivorous diet were also enrolled. Their characteristics are summarised in Table I. The
subjects, all living in the Turin city area, were invited to attend a meeting where
the aims of the study and the requirements to participate were briefly explained.
All participants received a kit containing detailed information about the study,
two questionnaires from the European Prospective Investigation into Cancer
and nutrition (EPIC) study (one about the dietary habits and one about lifestyle
habits, specific for men and women) (
), an additional short questionnaire
about changes in their dietary habits, medical history and additional questions not
present in the EPIC questionnaires, and finally a disposable 30 ml polystyrene
screw cap container with spoon for stool collection (AsOne). A written informed
consent form to participate in the study was signed by all volunteers.
The study was conducted according to the guidelines in the Declaration of
Helsinki. The protocol of the study was approved by the Gradenigo Hospital
Ethics Committee in Turin.
Collection of faecal samples
Naturally evacuated stool samples were obtained at home from all patients. All
participants were instructed to bring the sample to the laboratory. The faecal
samples were prepared for the next step immediately after they were brought to
our laboratory and excess faeces were stored at −80°C.
Collection of plasma samples
Plasma samples were collected according to standard phlebotomy procedures at
the moment when volunteers brought the stool samples to the laboratory. Blood
sample (5 ml) was collected into ethylenediaminetetraacetic acid tubes and
immediately placed on ice. Tubes were centrifuged at ×1000g for 10 min at room
temperature. Plasma was collected and distributed in cryovial tubes. One tube
was immediately used for RNA extraction while the other aliquots were labelled
and stored at −80°C. The time from sample procurement to storage at −80°C was
less than 3 h.
Extraction of total RNA from faeces and plasma
Before extraction of total RNA, faecal samples were solved in phosphate buffered
saline and then homogenised by a Silent Crusher (Heidolph Instruments, USA).
Homogenised samples (500 µl) were used for RNA extraction. For plasma
samples, the volume used for RNA extraction was 300 µl. Total RNA was extracted
using the miRvana MiRNA Isolation Kit (Ambion) using the protocol
recommended by the manufacturer.
cDNA synthesis and miRNA expression analysis by quantitative
Complementary DNA (cDNA) was synthesised using the TaqMan®
MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA,
USA), in accordance with the manufacturer’s instructions. cDNA synthesis
was conducted by incubation at 16°C for 30 min and 42°C for 30 min, 85°C for
5 min. The reaction mixture for the real-time PCR analysis consisted of 2 μl of
template cDNA, 10 μl of TaqMan® Universal PCR Master Mix II without UNG
(Applied Biosystems) and 1 μl of 20× primer/probe mixture in a total reaction
volume of 20 μl. Real-time PCR was conducted in the Applied Biosystems
7900HT Fast Real-Time PCR System with PCR initial activation at 50°C for 2
min, followed by enzyme activation step at 95°C for 10 min and then 40 cycles
of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. For
miRNA expression analysis, we targeted 7 miRNAs: hsa-miR-16, hsa-miR-21,
hsa-miR-34, hsa-miR-92a, hsa-miR-106a, hsa-miR-146 and hsa-miR-222. For
all miRNAs, the commercially available TaqMan MicroRNA Assay (Applied
Biosystems) was used (reference number available upon request).
The miRNA expression analysis was conducted using GenEx
(http://genex.genequantification.info/). A key issue in miRNA detection is the selection of
endogenous controls, which are RNA or miRNAs found to be largely invariant in a sample
set. Although several small RNA species (like RNU6B) have been recommended
for normalising miRNA expression, they are not particularly indicated for
specimens with low RNA yields such as stool or plasma, as they are rapidly degraded
and the detection rate is relatively low (
). As there is no consensus on suitable
controls for stool testing, quantification was performed adopting two different
approaches for normalisation: (i) each miRNA expression data was normalised by
the ΔΔCt method using the average of global measure of the miRNA expression
data, or alternatively (ii) using miR-222 as normaliser (identified by Normfinder
) as the most stable miRNA among the ones investigated). Since the
results were similar, we have opted for the former normalisation. Normalised data
were converted into relative quantities and transformed to log2 scale.
To evaluate the association with the dietary items and lifestyle factors, we
modelled the data from the questionnaires both as categorical (yes/no) and as a
continuous (weekly consumption) variables.
Student’s t-test, analysis of variance (ANOVA) and univariate linear
regression were used to compare means and to calculate bivariate correlations.
Statistical significance was set at 5% level.
To model the modulatory role of dietary, lifestyle and demographic factors
on miRNA expression, we have adopted the structural equation model (SEM)
statistical framework (
). The rationale behind SEM is that multiple linear
equations are used to specify causal relationships between variables, some of
which are manifest variables (i.e. observed and collected by the researchers),
while others are latent variables (i.e. derived from the observed variables by
specifying their relations using equations). Considering the heterogeneous
source of our measures (consisting of both categorical and continuous
variables), we have computed the Pearson product–moment correlations in our data
to fill the heterogeneous correlation (HC) matrix on which we have based the
development of the structural models. By using a cut-off of |R| > 0.5 for the
values of HC, we have obtained a network representation of the relevant relations/
factors. This has allowed us to further estimate the causal relations of selected
factors on individual miRNAs, both by graphical models and by conditional
independence tests (do-calculus) (
). Each model was represented in the form
of SEM by using the lavaan package (
) and was evaluated by taking into
account multiple fitting indexes. A comparative finding index (CFI) > 0.90, a
All (n = 24)
Vegans (n = 8)
Vegetarians (n = 8)
Omnivorous (n = 8)
Basic (<8 years)
Average (8–13 years)
High (>13 years)
Only gastrointestinal cancer
Mean ± SD
Mean ± SD
Short (<1 year)
Average (1–3 years)
Long (>3 years)
21.9 ± 2.5
root mean square error of approximation (RMSEA) < 0.05, suggest good fitting
performances, as described in (30).
All analyses were performed using R (R 2.15.2) (http://www.rproject.org).
Study population characteristics
The study was carried out in 24 healthy volunteers belonging
to 3 different dietary categories, each group with similar age
and sex distribution. The median age of the volunteers was
39 years (range spanning from 21 to 60 years), with vegans
being slightly older than vegetarians on average (mean 42 vs.
37 years). Males represented approximately one-third of the
study group. Subjects following an omnivorous diet displayed
a non-significantly higher body mass index (BMI) compared to
the other groups (23.2 ± 2.0 vs. 21.1 ± 2.0 and 21.5 ± 3.1 kg/m2).
The duration of a specific dietary habit was shorter for
vegans (3.1 ± 3.8 years) in comparison with vegetarians (7.7
± 9.7 years). The majority of vegans had adopted the diet less
than 1 year before recruitment, while the majority of
vegetarians since more than 3 years before. One of the volunteers
reported having been vegetarian for 30 years, while some of
the vegans were former vegetarians.
From the questionnaire, 13 subjects out of 16 reported to
have adopted the specific diet for ethical issues, 4 also for the
protection of the environment and 6 as a preventive measure.
The main characteristics of the study population are
summarised in Table I. Dietary and other lifestyle characteristics
are described in Supplementary Tables 1 and 2, available at
miRNA expression and different diets
In the present study, we have analysed the expression levels
of seven miRNAs: miR-16, miR-21, miR-34, miR-92a,
miR106a, miR-146 and miR-222 in plasma and stool samples of
all investigated subjects. Since qPCR for miR-146 did not
provided any results in both specimens, this miRNA has not
been included in the analyses.
After ANOVA analyses, we observed that miR-92a was
differentially expressed among different dietary groups in plasma
samples (P = 0.0003) with a trend: both vegans and
vegetarians showed higher levels in comparison to omnivores (P =
0.0002 and P = 0.02, respectively). Box plots for the results are
reported in Figure 1A.
In stool samples, we observed similar results with increased
miR-92a levels in vegans as compared to vegetarians and
omnivores. The trend was the same, but the difference was
statistically significant only between vegans and omnivores (P =
0.04). Box plots for results are reported in Figure 1B.
Among the other investigated miRNAs, no significant
differences were found in either plasma or stool samples. miR-106a
was differentially expressed in faeces (P = 0.04), but no clear
trend was observed, while in plasma the difference was not
significant. Individual data are reported in Supplementary Table 3,
available at Mutagenesis Online.
For each individual miRNA, expression levels were not
significantly related between plasma and stool samples, or with
duration of the diet (vegan and/or vegetarian).
miRNA expression and demographic characteristics
We further investigated the associations between miRNA
expression levels and covariates included in the study. In plasma
samples, we observed a decreased miR-16 expression with
increasing age of the subjects (P = 0.05), and in stool miR-34
expression levels decreased with age (P = 0.05). Only miR-222
was differentially expressed according to gender stratification:
women showed higher levels of this miRNA in plasma samples
(P = 0.0004) but not in faeces. Interestingly, higher BMI was
associated with decreased miR-92a in both plasma and stool (P
= 0.04, and P = 0.05, respectively, Figure 2A and B). Conversely,
in stool, higher BMI was related to higher expression levels of
miR-222 (P = 0.03) (data not shown).
miRNA expression and dietary characteristics
From the long list of items included in the questionnaires, we
have analysed only few and most representative for the dietary
regime differences (consumption of meat, processed meat, fish,
cheese and dairy products, bread and pasta, total vegetables
and fruit consumption; Supplementary Table 4, available at
Mutagenesis Online). Among the main findings, we observed
that miR-92a in plasma and in stool was less expressed in
meat/processed meat/fish consumers in comparison to
nonconsumers (P = 0.002 in plasma and P = 0.089 in stool),
reflecting the same pattern of dietary regimes. Interestingly,
a decrease in miR-92a levels was inversely related to weekly
consumption of meat/processed meat/fish in plasma samples
(P = 0.001), while a non-significant trend was observed in stool.
The same patterns can be observed when individual categories
of the above foods are separately analysed in each specimen.
miR-92a also decreased according to cheese
consumption (vs. none: P = 0.004 in plasma, and P = 0.01 in stool) or
weekly cheese consumption (significant in plasma P = 0.0093,
and same trend albeit not significant in stool). In particular for
dairy products, miR-92a was less expressed in both plasma
and stool (P = 0.0006, P = 0.02, respectively), when
comparing consumers versus non-consumers. miR-92a was also more
expressed in plasma samples according to bread/pasta intake
(P = 0.05).
miR-21 was related to weekly consumption of vegetables
(raw/cooked) in stool (P = 0.01) with the same but
non-significant trend in plasma. In contrast, in stool but not in plasma
miR-16 was inversely related to weekly intake of vegetables
(P = 0.01).
miR-34a expression levels were related to weekly
consumption of fruit in stool (P = 0.005).
miRNA expression and lifestyle characteristics
We did not observe any differential expression of the analysed
miRNAs in association with smoking habits or physical
activity, as derived from the questionnaires. About alcohol
consumption (categories: total alcohol consumers, wine or
beer, or liquor consumers, both yes/no or weekly intake), we
observed that in plasma miR-92a expression levels were lower
among liquor consumers and according to weekly estimated
intake. On the other hand, miR-21 was less expressed in wine/
liquor consumers, also according to weekly intake. Higher
miR16 expression levels were detected in both plasma and stool
of subjects consuming liquors (P = 0.0137 and P = 0.0292,
Supplementary Table 4, available at Mutagenesis Online).
Integrating modulatory effects of dietary, lifestyle and
demographic factors on miRNA expression levels
To integrate the contribution of previously identified relevant
(dietary, lifestyle and anthropometric/demographic) factors
on miRNA expression, we have initially performed a
correlation analysis. The resulting HC matrix reporting all
outcomes is shown in Figure 3: the majority of results have
confirmed the previous findings from ANOVA or univariate
regression analysis. All significant correlations (|R| > 0.500)
among investigated factors and analysed miRNAs have been
represented as a network that has provided the basis to detect
causal relationships and test different models by SEM analysis.
Among all investigated models, we have identified those related
to miR-92a in plasma and in stool samples providing the best
fitting indexes (CFI = 1, RMSEA = 0 for both). The relative
path diagrams are shown in Figure 4.
Genetic and epigenetic alterations may explain observed
aberrant miRNA expression in cancer. Specific dietary/
environmental factors may be responsible for these alterations,
since they have been shown to modulate miRNA expression
and their mRNA targets in various processes (such as apoptosis,
cell cycle regulation, angiogenesis and inflammation) (
In this respect, dysregulated miRNAs detected in surrogate
specimens such as plasma and stool samples may represent a
powerful non-invasive and sensitive tool and might aid in the
early detection of gastrointestinal cancer (
). However, at
present, scanty information is available—in epidemiological
studies—on how miRNAs are modulated by external factors
that are known to be relevant for the risk of gastrointestinal
diseases, such as diet and other lifestyle habits (12).
In the present pilot study, we aimed at exploring the possible
modulatory effect of different dietary habits on a panel of
miRNAs by evaluating their expression levels in stool and plasma
samples simultaneously, in order to compare in the same
subjects any relationship with dietary factors. Specifically, we have
recruited a group of 24 healthy individuals belonging to 3
different diet groups with comparable sex and age distribution.
miRNAs were selected on the basis of their role in cancer
(particularly in colorectal cancer) in previous studies (
The main and novel finding is the simultaneous detection in
both stool and plasma of differences between dietary groups
according to miR-92a expression levels. In those specimens,
subjects adopting a vegan diet had higher expression levels of
this miRNA in comparison with vegetarians and people
following an omnivorous diet, with a similarly decreasing trend.
miR-92a is part of the miR-17–92 cluster, which is located at
the 13q22 region and encodes for seven miRNAs processed
from a single polycistronic primary transcript (
cluster expression has been linked to the pathogenesis of several
malignancies, including lung cancer, leukemia, hepatocellular
carcinoma and colorectal cancer (
). miR-92a is the last
and least described member of the cluster. It has been observed
to be frequently overexpressed in different types of tumours
) and only recently it has been demonstrated to be highly
expressed in endothelial cells and to regulate their angiogenic
). In addition, some studies suggested that
miR92a may regulate the immune functions by controlling
lymphocyte and monocyte proliferation (reviewed by ref. 36) .
mir-92a has been studied in stool or in plasma samples in
association with colorectal cancer, where increased levels have
been observed, mostly in studies on Asian populations (
However, in other studies on Caucasians results were
discordant, with no difference in miRNA expression levels among
cancer cases and controls (43). Such contradictions may be due to
differences in dietary habits, as observed in the present study.
In fact, it is possible that populations with different dietary
habits may have different expression profiles of miRNAs, such as
miR-92a, also in relation to pathological conditions.
The adoption of a vegan/vegetarian diet has been for long
considered to lead to a decreased risk of cardiovascular disease and
). However, these diets may also be associated with
an increase of other disease risks: the complete omission of meat
and fish from the diet increases the risk of nutritional
deficiencies and the effect of vegetarianism on the haematological
system has been clearly described (46). The role of specific dietary
changes in miRNA expression has been so far only investigated
extensively in animals or in vitro. For instance, folate deficiency
in rats has been associated with up regulation of several
miRNAs and the development of hepatocarcinoma (
to complicate the scenario, the potential of food-derived
miRNAs (XenomiRs) to affect host gene expression has also been
). Witwer and colleagues (
) proposed the
existence of miRNAs that could be absorbed with the diet and would
contribute directly or indirectly to the apparent expression of
circulating miRNAs, although there is still no convincing evidence
on the putative effect of food-derived miRNAs (
We further investigated other possible associations with
recorded anthropometric/demographic data as well as with the
intake of selected food items and lifestyle factors. miR-92a
expression was inversely related to BMI in both types of
samples. BMI was only moderately associated with dietary groups:
a non-significant trend was observed comparing omnivores to
vegetarians and to vegans. In contrast, the expression of
miR222 was associated with higher BMI, but only in stool, and
with gender in plasma (increased levels in females). Despite a
limited age range (20–60 years) in our study, miR-16 and
miR34 showed decreased levels with age in plasma and in stool,
respectively. A general age-related down regulation of
miRNAs has been repeatedly observed in blood samples in the few
studies available so far with a broad age-range design (
though still with non-consistent results.
We have taken advantage of the food frequency
questionnaire adopted in the EPIC study (
) to relate expression
levels of analysed miRNAs with specific food items, that could
either reflect the different specific dietary habits of each group
of volunteers or, independently, those of all study participants
at a global level. In this way, we could detect more subtle
associations related to specific food components. Interestingly,
miR-92a expression was strongly inversely related to cheese
consumption (including dairy products), in both stool and
plasma samples. This miRNA was also inversely related to
meat (including processed meat) and fish consumption but only
in plasma, while in stool the trend was consistent but did not
reach statistical significance. In stool, miR-16 expression was
associated with increased meat/fish consumption while miR-21
with a decreased consumption of processed meat; the opposite
trend was observed in relation to weekly intake of vegetables.
Moreover, miR-34 levels were related to fruit intake.
Individual miRNAs can regulate many target genes while
a group of miRNAs may modulate specific pathways. Thus,
we additionally investigated the target genes of miR-92a (n =
36), miR-16 (n = 387) and miR-21 (n = 164) by TargetScan+
WebGestalt to understand whether any biological process was
commonly enriched. Interestingly, among the several
metabolic processes and molecular functions in which miRNA
target genes were involved, a subset of them was involved in
the metabolism of nitrogen compounds (GO: 0051 171, GO:
0051 172 and GO: 0034 641). This finding confirms that the
observed differences in miRNA expression according to
differential intake of meat/processed meat/fish or based on different
dietary groups, reflect the effect of foods consumed by
vegetarians and omnivores on the nutritional status.
We have observed additional associations with alcohol and
coffee consumption, but not with smoking habits and physical
activity. Since only few of the subjects were active smokers we
cannot draw any firm conclusion on this important exposure,
that might also modulate miRNA expression in relation to
). Physical activity in relation to changes of miRNA
levels in blood samples has recently received attention; however,
studies were based on short-term changes in miRNA
expression immediately following active aerobic exercise (
The main hypothesis of our study was that the diet and other
lifestyle factors of an individual may significantly influence
miRNA expression levels, and this modulation is detectable in
plasma and stool. This is presumably not a straightforward
process and to globally elucidate causal relationships from
observational studies remains challenging. Thus, we have adopted
the theory behind SEM, a statistical technique for testing and
estimating the complex structure of causal relations among
selected relevant factors (
). The potentiality of SEM in
epidemiological studies has still not been fully explored: this largely
confirmatory technique may help researchers to determine
whether a certain proposed model is valid (
). In our hands,
the models involving dietary factors and individual’s
anthropometric/demographic data on miR-92a levels in plasma and in
stool have provided the best performances. This analysis has
not only confirmed above reported findings, but has provided
a precise framework to elucidate the role of different factors
and their simultaneous effect. This represents a promising tool
for evaluating complex models such as the miRNA modulation.
In the present investigation, we were also particularly
interested in examining if both plasma and stool reflected the same
associations. In fact, miRNAs detected in these specimens may
represent different environmental/physiological conditions,
considering their origin. miRNAs in plasma represent
circulating molecules either included in vesicles or in lipid associated
), with a contribution from different tissues, but
mainly from blood cells. In contrast, miRNAs in stool are
originated mainly from exfoliated colonocytes daily shed from colon
crypts (16). In this sense, since miRNAs may also exhibit complex
tissue-specific patterns of expression, different expression trends
could be expected following different dietary/lifestyle habits
). However, we have observed quite good agreement between
findings in both specimens. Generally, the observed trends were
consistent in both specimens (with a particular evidence for
miR92a), with none of the statistically significant findings being in
disagreement between the two types of samples. Our observation
seems particularly interesting in view of a possible use of blood/
stool miRNA-based markers, which may consistently
complement each other in detecting small but significant miRNA
modulations, ultimately influencing the expression of genes relevant
for healthy status. As diet has been found to modulate miRNA
expression, profiling of these molecules could be useful in the
assessment of the nutritional status in dietary intervention studies.
We are aware of some limitations of the present study,
including a small sample size and a restricted number of
miRNAs analysed. On the other hand, this exploratory investigation
has some strengths and peculiarities never adopted in previous
studies: (i) we focused on an homogenous group of healthy
volunteers matched by sex and age from the same area, recruited
in the same time interval; (ii) two different samples (blood and
stool) were simultaneously collected from the same individual
and immediately processed, (iii) miRNA analysis was
performed simultaneously on both specimens in the same
experiment, (iv) the dietary regime differences secured some of the
specific answers retrieved by the questionnaires giving more
strength also to the analysis of food consumption.
In conclusion, we reported the influence of different dietary
habits on individual miRNAs from the concomitant analyses of
plasma and stool samples of healthy subjects. Our findings clearly
indicate the need to investigate modulating dietary and lifestyle
factors, along with the clinical/diagnostic data, for the evaluation
of miRNAs expression as stool/plasma-based disease markers.
Supplementary Tables 1–4 are available at Mutagenesis Online.
The study was funded by Compagnia di San Paolo, Torino,
The authors are very thankful to all volunteers that participated with enthusiasm
to the present study. Design of the study: S.T., B.P., G.M., A.N.; experiments:
S.T., B.P., G.M., A.N.; analysing the data: F.Rosa, B.P., C.D.G.; formulating
the research question(s): A.N., F.Rosina, G.M., P.V.; writing the article: B.P.,
G.M., S.T., A.N., P.V.
Conflict of interest statement: None declared.
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