Original Research Article—Basic Science

miR-206 as a Biomarker for Response to Mesalamine Treatment

in Ulcerative Colitis

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Carlos D. Minacapelli, MD,* Manisha Bajpai, PhD,*,† Xin Geng, MD,* James Van Gurp, MD,‡
Elizabeth Poplin, MD,† Peter S. Amenta, MD, PhD,‡ Steven R. Brant, MD,* and Kiron M. Das, MD, PhD,*,†,‡

Background:  MicroRNAs (miRNAs) are important post-translational regulators. Elevated levels of miR-206 in ulcerative colitis (UC) were
associated with suppression of anti-inflammatory A3 adenosine receptor (A3AR) expression. However, the relationship of miR-206 to histologic
remission in UC patients remains unknown. This study correlates expression levels of miR-206 with histologic remission in patients treated via
long-term mesalamine treatment to identify a possible mode of action for this mainstay drug for UC.
Methods:  Expression of miR-206 and its target A3AR were analyzed in HT29 cell line before and after mesalamine treatment (2 mM) at differ-
ent time points (0, 4, 12, and 24 hours) by qRT-PCR and western blot analysis. Expression of miR-206 and pathological scores of colonoscopic
biopsy specimens were studied in 10 UC patients treated with mesalamine treatment for 2 to 6 years.
Results:  miR-206 transcripts decreased 2.23-fold (P = 0.0001) 4 hours after 2 mM mesalamine treatment in HT29 colon cells compared with
untreated controls. However, the mRNA/protein levels of A3AR increased by 4-fold (P = 0.04) and 2-fold, respectively, in same cells. miR-206
relative expression decreased significantly in patients treated with 4.8 g of mesalamine (P = 0.002) but not with 2.4 g (P = 0.35). Tissue assess-
ment of sequential mesalamine-treated colonoscopic biopsies indicate a strong correlation between downregulation of miR-206 and histologic
improvement (R = 0.9111).
Conclusion:  Mesalamine treatment has an effect on epithelial miRNAs. Downregulation of miR-206 by long-term mesalamine treatment may
confer a protective effect in inducing and maintaining histologic remission. Thus, miR-206 expression levels can be utilized as a possible bio-
marker for therapeutic response to mesalamine treatment.
Key Words: microRNA, mesalamine, ulcerative colitis

INTRODUCTION chronic, relapsing, and remitting inflammatory disease limited

Chronic ulcerative colitis (UC) is 1 of the 2 major types
of inflammatory bowel diseases (IBD) and presents as a
Received for publications March 15, 2018; Editorial Decision August 9, 2018.
From the *Division of Gastroenterology, Department of Medicine, Rutgers
Robert Wood Johnson Medical School, New Brunswick, NJ, USA; †Rutgers Cancer
Institute of New Jersey, New Brunswick, NJ, USA; ‡Department of Pathology
and Laboratory Medicine, Rutgers Robert Wood Johnson Medical School, New
Brunswick, NJ, USA
Conflicts of interest: No conflicts of interest, financial or otherwise, are declared
by the authors.
Author Contributions: CDM, MB, XG, and KMD conceived and designed
research; CDM performed experiments; CDM, MB, XG, JVG, and KMD analyzed
data; CDM, MB, SRB, and KMD interpreted results of experiments; CDM prepared
figures; CDM and KMD drafted the manuscript; CDM, MB, XG, JVG, EP, PA,
SRB, and KMD edited and revised the manuscript; CDM, MB, XG, JVG, EP, PA,
SRB, and KMD approved the final version of manuscript.
Supported by: This work was supported in part by Gastrointestinal Divisional
funds and a fellowship grant from Takeda Pharmaceuticals to CDM.
Address correspondence to: Kiron M.  Das, MD, PhD, Professor Emeritus,
Division of Gastroenterology and Hepatology, Department of Medicine, The
Crohn’s and Colitis Center of New Jersey, Rutgers-Robert Wood Johnson Medical
School, 1 Robert Wood Johnson Place, MEB 478, New Brunswick, NJ 08901, USA.
E-mail: daskm@rwjms.rutgers.edu.
© 2018 Crohn’s & Colitis Foundation. Published by Oxford University Press.
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
doi: 10.1093/ibd/izy279
Published online 11 September 2018
to the mucosal layer of the rectum and colon. Ulcerative colitis
is characterized by mucosal ulceration, which generates symp-
toms of abdominal pain and bloody diarrhea.1 An increas-
ing incidence of UC has been reported worldwide in the last
20  years.2 However, the pathogenesis of UC remains largely
unknown.3
Gene expression studies have found a significant number
of genes differentially expressed in patients with UC compared
with healthy individuals.4–7 However, it is still unclear how such
a differential expression of genes is regulated. In recent decades,
new insights into the functions of microRNAs (miRNAs) in
the pathogenesis of UC have emerged with a potential to be
identified as UC biomarkers and therapeutic targets.8, 9
miRNAs comprise noncoding RNAs that are 18–25
nucleotides long. Mature miRNAs combine with the RNA-
induced silencing complex and bind to complementary
sequences in the 3′-untranslated region (3′-UTR) of target
mRNAs. miRNA binding results in target-mRNA degra-
dation, or translation inhibition, thus regulating the expres-
sion of multiple protein-coding genes.10 miRNAs have been
reported to function as key regulators of various pathophys-
iological processes including cell proliferation, differentiation,
homeostasis, and inflammation.11–13
A recent study by Wu et al described how the microRNA
miR-206 is significantly expressed in mucosal tissues of UC

Inflamm Bowel Dis • Volume 00, Number 00, Month 2018 1

Minacapelli
 et al
patients compared with normal controls and demonstrated
that miR-206 has a pro-inflammatory role in UC by downreg-
ulating A3 adenosine receptor (A3AR) expression both in vivo
and in vitro.14 A3AR is a subtype of the adenosine-receptor
family. A3AR activation inhibits adenylyl cyclase and cAMP
formation through the Gi protein.15 Furthermore, A3AR has
been reported to play a protective role in certain pathophysio-
logical processes such as rheumatoid arthritis and myocardial
ischemia/reperfusion injury, and hence, its activation could pro-
tect against colonic inflammation.16–19 Therefore, these reports
suggest that reversal of the expression levels of miR-206 and
A3AR may provide a protective effect in the intestinal mucosa
in UC patients.
Despite several decades of research, the mode of action
of mesalamine (5-ASA) for both induction and maintenance
of remission in UC is unclear. Various mechanisms have been
suggested which include altering the bacterial flora, provid-
ing antioxidant effects, modulating the immune functions (ie,
PPAR-gamma, prostaglandins, etc.), and possibly modulating
epithelial mucosal genetic and epigenetic profiles.20–24 To date,
no studies have explored the effect of 5-ASA on specific miR-
NAs, such as miR-206, or correlated miRNA expression levels
with achievement and maintenance of histological remission in
UC patients.
In the present study, we hypothesize that 5-ASA targets
miR-206 expression levels in colonic mucosa, and its downregu-
lation confers a protective effect by achieving and/or maintain-
ing histologic remission in patients with mild to moderate UC.
We postulate that 5-ASA downregulates miR-206 which modu-
lates its anti-inflammatory target gene A3AR. Thus, this study
provides insights on a possible mode of action of 5-ASA via
modulation of miRNAs, offers an understanding of UC disease
progression, and identifies predictors of therapeutic response.
MATERIAL AND METHODS
In Vitro Cell Culture
Colon cancer cell line HT29 (ATCC) was utilized. Cells
were grown to confluency in RPMI-1640 medium (Gibco, MD,
USA), supplemented with 10% fetal bovine serum (Gibco), 100
IU/mL penicillin, and 100 ug/mL Streptomycin at 37ºC in a 5%
CO2 incubator. Cells were trypsinized, washed with phosphate
buffered saline, counted, and reseeded in 6 well plates (2x10^6
cells/well).
5-ASA Treatment
The intraluminal concentrations of 5-ASA in patients
with inflammatory bowel disease receiving mesalamine main-
tenance therapy have been reported as approximately 7.0 to
14.0 mM.25, 26 In a series of initial in vitro experiments using 0
to 20 mM 5-ASA, we have observed that 5-ASA at the molar-
ity of 10 mM or greater is toxic to cells in culture. Thus, after
a series of titration experiments, a more conservative final
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Inflamm Bowel Dis • Volume XX, Number XX, XX 2018
concentration 2 mM 5-ASA was used for all of the subsequent
in vitro experiments.27 Treatment with 5-ASA was performed at
0, 4, 12, and 24 hours in serum free medium.
Colonic Biopsy Specimens
A cohort of 10 established UC patients, each with a diag-
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nosis of active (mild to moderate) UC and seen in the outpa-
tient clinic at the Crohn’s and Colitis Center of New Jersey, were
started on a 5-ASA maintenance therapy. Patients consented
to participate in an IRB-approved pilot study protocol from
May 2006 to June 2013. Patients were treated with oral doses
of either 2.4 g (n = 4) or 4.8g (n = 6) of 5-ASA. The first set of
biopsy specimens were collected during an initial colonoscopy
from the rectosigmoid area. Follow-up biopsy specimens were
collected from the same patients and from the same location
during subsequent colonoscopies at 2 to 6 year intervals. No dis-
crimination was made based on age, sex, extent of disease, or the
dose of 5-ASA administered. Biopsy specimens were collected
during the colonoscopy and stored immediately in formalin for
routine histology, and the specimens were also snap frozen in
RNA later for RT-PCR analysis. The tissue was stored at −80ºC
until processed for RNA extraction. Only initial, intermediate (2
to 3 years), and last follow-up colonoscopic specimens (6 years)
were utilized for this study. Assessment of histologic disease
activity was analyzed by 2 pathologists (PSA and JVG), with-
out clinical knowledge, and estimated using a modified Nancy
index score system.28 The pathologists’ evaluations of disease
activity in UC was blinded and was histologically categorized
as follows: (A) chronic inflammatory infiltrate, (B) acute inflam-
matory infiltrate (cryptitis and/or lamina propria), (C) crypt
distortion/destruction (graded 0–3 for each attribute), and (D)
erosion/ulceration (graded 0–1). The total score was additive
and included 0.5 intervals for gray areas between integer scores.
A  higher score indicates greater inflammation. A  response to
5-ASA therapy was considered if a patient’s histology activ-
ity scores dropped by at least 1 point. Some patients did not
show any improvement in pathology and were classified as “no
change” or “nonresponders.” In addition, biopsy tissue from
UC patients untreated with 5-ASA due to 5-ASA allergy (n = 5)
and normal colon (n = 5) were collected and used as controls.
Gene Analysis
Total RNA was extracted using the Qiagen RNAse Mini
Kit per manufacturer’s protocol (Qiagen, Germantown, MD,
USA). The RNase-Free DNase set (Qiagen, USA) was used to
remove any genomic DNA contamination. cDNA was gener-
ated using the High Capacity cDNA Reverse Transcription kit
(Applied Biosystems, CA, USA) on the Gene Amp PCR System
9700 (Applied Biosystems). Real-time RT-PCR underwent 40
cycles of denaturing at 95˚C for 15 seconds, annealing at 60˚C
for 20 seconds, and extending at 72˚C for 20 seconds on the
Roche Light cycler 2.0 Instrument using the QuantiTect SYBR
green PCR kit (Qiagen, Germantown, MD, USA). Expression
Inflamm Bowel Dis • Volume XX, Number XX, XX 2018 miR-206 Targeted by Mesalamine in UC

of target microRNA and mRNA was normalized with respect

to β-actin using the ∆∆-CT method. Primers were as follows: TABLE 1.  Clinical Characteristics of Patients
for miR-206 forward (F)-5’-TGG AGA CTT CTG AAC GAG UC untreated UC treated
AG-3’ and reverse (R)- 5’-CTT GAA GAT GGC GTT GGG- Normal with 5-ASA* with 5-ASA
3’, and for A3AR forward (F)- 5’-GGC TGC CCT CAA ATA
ACA TC-3’ and reverse (R)- 5’-CTC CAC CTC TTC TTC No. of patients 5 5 10

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ACT TCT G-3’. Reproducibility was examined in triplicates. No. of male, (%) 3 (60%) 3 (60%) 5 (50%)
No. of female (%) 2 (40%) 2 (40%) 5 (50%)
Protein Analysis Age (years.) — — —
Thirty micrograms of total cell extracts from HT29 cells Mean 40.8 38.2 43.5
were resolved on 4%–20% gradient SDS-polyacrylamide gel elec- Range 42–65 23–62 40~64
trophoresis (Invitrogen Corp., Carlsbad, CA, USA). The blots Disease duration (years) — — —
were then incubated with the respective primary antibodies (poly- Mean — 14.6 11.5
clonal antibody A3AR, ADORA3, ThermoFisher Sci, IL, USA, Range — 6~19 7~19
and β-actin, Abcam, MA), and then subsequently with HRP- Extent of disease — — —
conjugated anti-rabbit IgG secondary antibody (Santa Cruz Left sided — 5 4
Biotechnology, Santa Cruz, CA, USA). Blots were then devel- Pancolitis — — 6
oped by ECL reagent (Perkin-Elmer). The same blots were also Medication — — —
incubated with an HRP-conjugated anti-β-actin antibody (Sigma 0.0 g 5 5* —
Corp., St. Louis, MO, USA) to determine the amount of protein 2.4 g — — 4
loading. Target protein was quantified by ImageJ (NIH public 4.8 g — — 6
domain) processing software using β-actin as the internal control.
*Treated with 6-mercaptopurine only

Statistical Analysis
The results of qRT-PCR were compared between treated
and untreated cells using the Student t test. A P value of <0.05
was considered statistically significant. The Pearson correlation
coefficient was used to measure the strength of a linear associ-
ation between the 2 variables (ie, miR-206 vs pathology scores),
where the value r = 1 means a perfect positive correlation.
RESULTS
Colonic Biopsy Specimens
A total of 5 males and 5 females, in the age range of 40
to 64 years, consented to the study (Table 1). The median age
was 43.5  years. The duration of disease in these individuals
ranged from 7–19 years, and the median disease duration was
11.5  years. The UC involved was either pancolitis (n  =  6) or
only left-sided colitis (n = 4).
miR-206 and Its Target A3AR Expression Level in
5-ASA Treated HT29 Colon Cells
The expression levels of miR-206 and its putative anti-
inflammatory target A3AR (14) were quantitatively assayed using
reverse transcription and real-time RT-PCR and Western Blot
Analysis, before and after 2 mM 5-ASA treatment for 4, 12, and
24 hours (Fig. 1). The viability of cells was greater than 90% after
each incubation period. We observed that miR-206 significantly

FIGURE 1.  Expression and correlation of miR-206 (A) and A3AR (B) in HT29 cells treated with 2 mM 5-ASA for 4, 12, 24 hours and assayed by
RT-PCR. miR-206 was significantly decreased by 4 hours (2.23-fold), and although we observed suppression at 12 hours (1.73-fold) and 24 hours
(1.39-fold), the expression of miR-206 increased after 4 hours towards baseline. At the same time, there is a significant increase in A3AR mRNA
transcript (4-fold) at 4 hours followed by return to baseline by 12 hours. (*P < 0.05)

3
Minacapelli
 et al
FIGURE 2.  A, Representative Western Blot analysis of A3AR after 0, 4, 12, and 24 hours of 5-ASA treatment of HT29 cells (n = 4) (14). B, At 4 hours
of 5-ASA treatment, we observed significant increase of A3AR by 2-fold, and levels were suppressed by 12 hours and 24 hours. Target protein was
quantified by ImageJ (NIH public domain) processing software using β-Actin as the internal control. All samples were derived at the same time and
processed in parallel. (*P < 0.05)
decreased at 4 hours by 2.23-fold (P = 0.0001) compared with the
untreated control cells, and although there was a suppression at 12
hours (1.73-fold decrease, P = 0.002), the levels began to increase
toward baseline at 24 hours (1.39 decrease, P = 0.02). Inversely,
after 4 hours of 5-ASA treatment, we observed significant increase
of A3AR by 4-fold (P = 0.04) in the same cells, although these
levels returned to baseline by 12 hours (Fig.  1A, B). Similarly,
A3AR protein profiles also showed a significant increase at 4
hours (2-fold) with a more gradual decrease afterwards, as shown
in a representative Western Blot analysis (Fig. 2).
Expression Levels of miR-206 in the Colonic
Biopsy Tissue From Normal, Active UC (5-ASA
Untreated), and UC Patients Treated With 5-ASA
qRT-PCR results indicated that expression levels of miR-
206 were significantly higher (P = 0.0006; Fig. 3) in the histo-
logically active UC tissues (5-ASA untreated) compared with
FIGURE 3.  qRT-PCR analysis of miR-206 expression levels in normal
(n = 5), UC (5-ASA untreated, n = 5), and UC 5-ASA treated (n = 10) biopsy
tissues from colon (last follow-up colonoscopy). Higher expression levels
of miR-206 was observed in UC tissue (5-ASA untreated) compared with
normal controls, while UC 5-ASA treated tissues show a significantly
lower expression compared with the UC 5-ASA untreated biopsy tissues.
Expression levels of miR-206 between normal controls vs 5-ASA-treated
tissues showed no significant difference.
4
Inflamm Bowel Dis • Volume XX, Number XX, XX 2018
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the normal controls. Expression levels of miR-206 between
normal controls vs 5-ASA-treated tissues showed no significant
difference. Interestingly, 5-ASA-treated tissues (last follow-up
colonoscopy at 6  years) show significantly lower expression
levels of miR-206 compared with the UC tissue that was not
treated with 5-ASA (P = 0.0007).
4.8 g of 5-ASA Has Greater Effect on miR-206
Expression Levels and Pathology Improvement
Global analysis of the 5-ASA treated tissues (n = 10) indi-
cated that expression levels of miR-206 significantly decreased
(P = 0.04) from the initial to the last follow-up colonoscopies
with pathological improvement (Fig.  4A and B, respectively).
Furthermore, patients treated with 4.8 g of 5-ASA (n = 6) had
a significantly greater miR-206 relative expression decrease
(AVG  =  53% decreased change, P  =  0.01, Fig.  4C) with sig-
nificant pathological improvement as compared with patients
treated with 2.4  g of 5-ASA (AVG  =  21% decrease change,
P = 0.61, Fig. 4C). Intermediate (2–3 years) biopsy specimens
show a decrease in miR-206 expression that is not statistically
significant (P = 0.33) compared with the time of initial scope
(Fig. 4A).
Correlation of miR-206 and Pathology Scores
qRT-PCR expression levels of miR-206 and pathology
scores (based on modified Nancy index score system28) were
analyzed from each tissue sample (Table 2). We observed that
there is a significant global (n = 10) miR-206 expression level
decrease from the initial to the last follow-up biopsy specimens
with an improvement in pathology scores. This observation
has a strong correlation (r = 0.9111). Data stratification indi-
cates that 6 of 10 or 60% of samples had significant decrease in
expression of miR-206 (P = 0.02) with improvement of patho-
logical scores, which supports a strong correlation (R = 0.8214).
In addition, 3 of 10 or 30% of samples had no change in expres-
sion of miR-206 and no pathological change or improvement,
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FIGURE 4.  qRT-PCR expression of miR-206 (A) and improvement in pathology scores (B) in UC biopsy tissues treated with 5-ASA from initial, inter-
mediate biopsy of 2–3 years, and final follow-up (6 years) colonoscopy values (n = 10). Further analysis of the changes from initial to final colonos-
copy specimens in miR-206, and improvement in pathology scores from patients receiving 2.4 g (n = 4) or 4.8 g (n = 6) of 5-ASA treatment is shown
in (C). Intermediate evaluation at 2–3 years demonstrates a decrease of miR-206 expression compared with the time of initial scope; however, this
was not statistically significant (P = 0.33). There is a significant decrease in relative expression level of miR-206 (P = 0.04) when comparing initial to
final colonoscopies in 10 patients treated with 5-ASA (A and B). In addition, these data indicate that biopsies from UC patients treated with 4.8 g of
5-ASA have a significant relative expression decrease of miR-206 (AVG = 53% decreased change, P = 0.01) and a significant pathological improve-
ment compared with biopsy specimens from UC treated with 2.4 g of 5-ASA (AVG = 21% decreased change, P = 0.61).

and 1 of 10 sample had no change in expression of miR-206 DISCUSSION

and a worsening of pathological score. Although technically
there is a positive correlation between miR-206 and pathology
scores at the interval analysis at 2–3  years, the relationship is
weak (Pearson Correlation Coefficient R  =  0.287) due to the
limited amount of samples (data not shown).
After the discovery of miRNAs, the number of
publications regarding their possible functions have been
increasing exponentially. Several of these studies have
examined the potential role of microRNAs dysregulation in
UC, which might facilitate the inflammatory process and the

TABLE 2.  Correlation Between miR-206 and Pathology Scores in UC Tissue Treated With 5-ASA
miR206 Pathology
Initial scope Final scope Fold Change P Initial Scope Final Scope Correlation Coefficient

All patients treated with 23.24 12.7 −1.83 P = 0.04 2.3 1.25 →R = 0.9111 strong
5-ASA n = 10 positive correlation
Patients treated with different amounts of 5-ASA 0
10
ΔmiR206

-6 -4 -2 -10 0 2
2.4 g 5-ASA n = 4 24.98 19.15 −1.3 P = 0.61 2.25 1.62 -20
-30
4.8 g 5-ASA n = 6 22.08 8.4 −2.62 P = 0.01 2.3 1 ΔPath
-40

Patients responding to 31.59 15.39 −2.85 P = 0.02 2.66 0.75 →R = 0.8214 strong
5-ASA 6/10 (60%) positive correlation
2.4 g 5-ASA 2/6 (33%) 33.72 30.39 −1.32 P = 0.35 2.5 1.16 -5 -4 -3 -2 -1
0

-10
0
ΔmiR206

4.8 g 5-ASA 4/6 (66%) 27.52 8.05 −3.41 P = 0.002 2.75 0.75 -20

-30

-40
ΔPath

5
Minacapelli
 et al
time to relapse.9, 29–32 Recent in vitro and in vivo studies suggest
that miR-206 is significantly upregulated in UC and that miR-
206 acts as a pro-inflammatory factor in the inflammatory
response by directly suppressing A3AR expression.14, 18, 32, 33 This
observation indicates that a direct reversal of the expression
levels of miR-206 and A3AR may provide biomarkers of disease
activity in UC prognosis and help monitor response to therapy.
As of now, there is no report in the literature comparing miR-
206 expression patterns in UC patients treated long-term with
5-ASA and its correlation to induction of remission state and/
or maintenance of remission.
There are several instances of poor correlation between
indices of clinical activity for UC and histologic findings where
clinical and endoscopy reports indicate remission, but histol-
ogy reports indicate abnormal findings in a large number of
patients.34–36 Microscopic features of inflammation may persist
in macroscopically inactive disease, suggesting that endoscopy
may underestimate the activity and sometimes the extent of the
disease.36–39 For these reasons, histologic healing may be a better
predictor of time to relapse than macroscopic appearance or
clinical criteria,40, 41 and histologic healing may provide a poten-
tial to guide treatment decisions.
Our study supports evidence that miR-206 is signifi-
cantly elevated in active UC biopsy tissue and treatment
with 5-ASA downregulates its expression in colonic mucosa.
Downregulation of miR206 exhibits a strong correlation with
histologic improvement in UC patients. Moreover, we observed
that patients who are refractory to 5-ASA treatment exhibit no
change in miR-206 expression levels from their first to last fol-
low-up colonoscopies, and their histologic scores either stayed
the same or have a worsening outcome. This observation sug-
gests that unaltered levels of miR-206 may be used as an indi-
cator of nonresponse to 5-ASA.
Previous studies have demonstrated possible therapeutic
potentials with miRNA-mimics or miRNA-inhibitors in differ-
ent types of autoimmune diseases that lead to cancer progres-
sion.42 In our in vitro studies using HT29 colon cancer cells, we
demonstrated that downregulation of miR-206 is possible to
achieve by employing a low dose 5-ASA treatment that upreg-
ulates its downstream anti-inflammatory target, A3AR, at the
gene and protein level. This observation provides a possible
mechanism of action, by which 5-ASA drug therapy confers
a protective role in colonic mucosa by reducing miR-206; this
merits further investigation.
Accumulating evidence indicates that histologic healing
may be associated with a better clinical outcome in UC,36–40, 43–45
such as a reduced risk of relapse and a reduced risk of develop-
ing cancer. The main areas of uncertainty with use of 5-ASA
in patients with UC center on the optimal dose for induction
of response. We demonstrate that patients treated with 4.8  g/
day of 5-ASA have a significant and strong correlation between
downregulation of miR-206 and histologic improvement from
initial to last follow-up colonoscopy. On the other hand, while
6
Inflamm Bowel Dis • Volume XX, Number XX, XX 2018
we see a reduction of miR-206 and histologic improvement
in patients treated with 2.4  g/day of 5-ASA, there was not a
strong statistical correlation. For example, as seen in Fig. 4C, 3
out of the 4 patients treated with 5-ASA 4.8 g/day that showed
improved pathology scores had a relative expression decrease
of miR-206 greater than 15, whereas the 2 patients that showed
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improved pathology scores treated with 5-ASA 2.8 g/day had
a relative expression decrease in miR-206 of less than 4. These
observations suggest that miR-206 may be affected in a more
direct manner by 5-ASA and may possibly be affected in a
dose-response manner. Additionally, these observations may
identify a group of patients who will benefit and experience
a more favorable disease course with higher doses of 5-ASA.
Moreover, intermediate (2–3  years) biopsy specimen results
show a decrease of miR-206 expression compared with that of
initial scope with a weak pathological scores correlation, sug-
gesting that there is an induction phase and that it does require
at least 6 years of adherent treatment with 5-ASA. This study
further supports our previous reports that suggested the impor-
tance of the clinical use of higher concentrations of 5-ASA
due to its continuous anti-inflammatory effects and its ability
to prevent further progression into neoplasia.27, 46, 47 However,
while these results are encouraging, we clearly need further val-
idation of our data with a larger cohort at both doses.
In summary, our data provides a strong correlation
between downregulation of miR-206 and histologic improve-
ment in UC patients treated long-term with 5-ASA. More inter-
estingly, we found a possible mechanism of action of 5-ASA by
suppressing expression levels of miRNA in colonic mucosa and
inversely inducing anti-inflammatory targets in vitro. Therefore,
we propose that miR-206 be utilized as an important biomarker
for the assessment of therapeutic response and disease progres-
sion in UC patients treated with 5-ASA.
ACKNOWLEDGEMENTS
We thank Dr. Bing-bing Wang and Ms. Nataliya
Parobchak from the Rutgers Robert Wood Johnson Medical
School Department of Surgery for providing technical
assistance. We would also like to express great appreciation
to Mr. Todd Weakley and Ms. Lauren Seem for their valuable
suggestions in writing this manuscript.
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