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Received: 22 February 2017
Revised: 30 August 2017
Accepted: 27 September 2017
DOI: 10.1002/dc.23838
Aberrant single-minded homolog 1 methylation as a potential
biomarker for cervical cancer
Hyun-Jung Kim1,2 | Chan Young Kim1,2 | Jinghui Jin1,2 | Moon Kyoung Bae1,2 |
Yun Hee Kim1,2 | Woong Ju1,2 | Yun Hwan Kim1,2 | Seung Cheol Kim1,2
Innovative Research Center for Control and
Prevention of Women’s Cancer, Ewha
Womans University Mokdong Hospital,
Yangcheon-gu, Seoul Korea
Division of Gynecologic Oncology,
Department of Obstetrics and Gynecology,
School of Medicine, Ewha Womans
University, Seoul, Republic of Korea
Seung Cheol Kim, MD, PhD and
Yun Hwan Kim, MD, PhD,
Division of Gynecologic Oncology,
Department of Obstetrics and Gynecology,
Ewha Womans University School of
Medicine, 1071, Anyangcheon-ro,
Yangcheon-gu, Seoul 07985,
Republic of Korea.
Emails: [email protected];
[email protected]
Funding information
The Korean Health Technology R&D
Project (Ministry of Health and Welfare,
Korea), Grant/Award Number: HI12C0050;
Ewha Womans University, Grant/Award
Number: RP-Grant 2017
Background: The aim of this study is to evaluate the possibility of using the methylation status of
single-minded homolog 1 (SIM1) as a diagnostic biomarker for cervical cancer.
Methods: All the patient and normal specimens including the normal cervix (n 5 10), cervical cancer tissues (n 5 45), blood (n 5 45), and cervical brush specimens (n 5 110) were retrospectively
obtained. Quantitative methylation-specific PCR was performed to detect SIM1 methylation in primary tumors, cervical brush specimens, and plasma circulating cell-free DNA (ccfDNA). SIM1
expression was detected by western blot analysis.
Results: We found that SIM1 was highly methylated in the majority of the cervical cancer tissues
that we tested, but not in any of the normal tissues. Hypermethylation of SIM1 led to a pronounced reduction in SIM1 expression in cervical cancer tissues compared with normal cervix.
SIM1 methylation status on cervical brush specimens also distinguished cervical cancer from normal, cervical intraepithelial neoplasia (CIN) 1 and 2. The degree of SIM1 methylation was
significantly associated with the severity of the disease (Ptrend < .0001). We also investigated the
possibility of detecting methylated SIM1 in plasma ccfDNA from cervical cancer patients. Methylated SIM1 was detected in 36.6% (15/41) of ccfDNA samples, and concordance rate with the
matched cancer tissues was 41.5% (17/41) with sensitivity 38.5% and specificity 100%.
Conclusion: This study has shown that SIM1 is frequently hypermethylated in cervical cancer,
compared with normal cervix tissue, CIN1 and 2 samples, suggesting that the methylation status of
SIM1 could be a potential diagnostic biomarker for cervical cancer.
cervical cancer, circulating cell-free DNA, diagnostic biomarker, DNA methylation, single-minded
homolog 1(SIM1)
benign inflammatory conditions contributed to a significant number of
false-positive results. To complement its insufficiencies, HPV DNA
Cervical cancer is one of the most commonly diagnosed types of gyne-
testing is now widely used in most developed countries because its
cological cancers worldwide, with especially high incidences in develop-
oncogenic role in cervical cancer is well established.2 Despite high sen-
ing countries. The introduction of the Pap smear has led to dramatic
sitivity of DNA testing, HPV DNA testing shows limited specificity
decreases in the mortality and morbidity rates for patients with inva-
because many infected women harbor transient viral infections that are
sive cervical cancers.1 Although the Pap smear as a cervical cancer
not associated with cervical cancer development, leading to unneces-
screening method is highly successful, a substantial number of cases of
sary referrals for colposcopy and overtreatment. For this reason, the
cervical cancer have still been missed which can be attributed to false-
identification of additional biomarkers that can be used in isolation or
negative test results due to sampling errors, interobserver and intraob-
in combination with the Pap smear and HPV DNA testing for cervical
server variability. Also, the varied composition of normal epithelia or
cancer is still needed.
Diagnostic Cytopathology. 2017;1–7.
C 2017 Wiley Periodicals, Inc.
Disease-associated epigenetic modifications such as aberrant DNA
medium tube provided by the manufacturer and were stored at 2808C
methylation hold particular promise for the identification of biomarkers
prior to being used to extract the DNA. Plasma was collected by cen-
associated with cancers. Aberrant DNA methylation contributes to the
trifugation (1800g, 20 min) within 2 h of the blood samples being col-
neoplastic process by transcriptional silencing of tumor suppressor
lected. All the samples were stored at 2808C prior to being analyzed.
genes and by increasing the rate of genetic mutations, implicating that
Blood samples collected before surgery were used to analyze ccfDNA
methylated genes can serve as biomarkers for early detection of can-
in the plasma. All the specimens including brush specimens tested in
cers.3,4 Epigenetic gene silencing via the hypermethylation of CpG
this study were classified based on the final diagnosis made by tissue
islands occurs in many cancer types, including cervical cancer and is
biopsy-based pathology. This study was approved by The Institutional
often associated with the down-regulation of tumor suppressor genes
Review Board of Ewha Womans University Mokdong Hospital, Repub-
such as p16, CDH1, DAPK1, and APC.5,6 CDH1 is observed in the major-
lic of Korea and written informed consent was obtained from all the
ity of primary cervical tumors and a substantial number of high-grade
patients and controls.
CIN3. Several other genes such as DAPK1 and RARB have also been
reported to be hypermethylated in cervical cancer, suggesting that differences in the epigenetic statuses of specific genes could be used as
2.2 | Genomic DNA isolation and circulating cell-free
DNA isolation
biomarkers for cervical cancer.7,8 However, there are currently no
methylation biomarkers used clinically for the screening or diagnosis of
Genomic DNA (gDNA) was isolated from tissue and cervical brush
specimens using a DNA mini kit (Qiagen, Hilden, Germany) according
cervical cancer.
The single-minded family basic helix–loop–helix (bHLH) transcrip-
to the manufacturer’s instructions, and the concentrations of DNA
tion factor 1 (SIM1) is a member of the bHLH PER-ARNT-SIM (bHLH-
were subsequently measured using a Nanodrop system (Thermo Fisher
PAS) family of transcription factors, which is involved in both develop-
Scientific, Waltham, MA). gDNA from peripheral blood leucocytes
ment and function of the paraventricular nucleus (PVN) of the
(PBL) was extracted from buffy coat. Plasma ccfDNA was prepared
hypothalamus. SIM1 plays also a key role in obesity because haploin-
using a Maxwell® RSC Instrument with a Maxwell® RSC ccfDNA kit
sufficient Sim11/2 mice developed hyperphagic early-onset obesity.10
(Promega, Madison, WI), and the ccfDNA was quantified using a Quan-
Genome-wide studies for identifying epigenetic biomarkers indicated
tiFluor dsDNA system (Promega) according to the manufacturer’s pro-
significant increases in SIM1 methylation and its association with
tocol. Plasma samples of 200–500 lL were used to isolate the ccfDNA.
increased risk of developing metastasis in lung and breast cancers.
These recent studies strongly suggest that SIM1 might be involved in
neoplastic process. However, the roles of SIM1 in tumor development
and progression have not been elucidated yet.
In this study, we investigated the possibility of aberrant SIM1
hypermethylation as a biomarker for distinguishing cervical cancer from
noncancer. The methylation status of SIM1 was also evaluated by analyzing ccfDNAs found in plasma of cervical cancer patients. Our study
therefore suggests that hypermethylated SIM1 could be used as a possible biomarker for cervical cancers.
2.3 | Bisulfite conversion and quantitative
methylation-specific PCR (qMSP)
gDNAs (0.5–2 lg) were subjected to a bisulfite treatment process using
an EZ DNA Methylation-LightningTM Kit (Zymo Research, Orange, CA).
The resulting bisulfite-modified samples were quantified using a Nanodrop system and stored at 2808C. TaqMan-based qMSP was performed after the bisulfite treatment on the denatured gDNA samples.
The primers and probe were specifically designed to bind to bisulfitemodified methylated SIM1. CpG sites within 22000 to 21001 promoter region of SIM1 are targeted for selecting specific primers and
probe for methylated SIM1. The forward and reverse primers used in
this study were as follows: 50 -GTTGGATTGAGCGTTTAATTATTC-30
2.1 | Specimens
All the patient and normal specimens—including the normal cervix
probe was 50 -NED-CGTCGATTCGGTATTTT-MGB-30 . ACTB (b-actin)
(n 5 10), cervical cancer tissues (n 5 45), blood (n 5 45), and cervical
was used as an internal control to normalize the DNA input and con-
brush specimens (n 5 110)—were retrospectively obtained from Ewha
firm the quality and quantity of the bisulfite-treated DNA. Notably, the
Biospecimen Bank of Ewha Womans University Mokdong Hospital and
primers and probe used for ACTB did not cover any of the CpG sites of
Korean Gynecologic Cancer Bank, Republic of Korea. Forty-five cervi-
ACTB. The forward and reverse primers used for ACTB were as follows:
cal cancer tissues (squamous cell carcinoma, n 5 37; adenocarcinoma,
n 5 7; adenosquamous carcinoma, n 5 1) were composed of stages I
CACAAT-30 , respectively. The probe used for ACTB was 50 -VIC-
(n 5 29), II (n 5 14), and III (n 5 2). The cervical brush specimens
TGGATTGTGAATTTGTGTTTG-MGB-30 . qMSP was conducted on a
(n 5 110) used in this study included normal (n 5 17), CIN1 (n 5 20),
QuantiTect Multiplex Mix (Qiagen). For the SIM1 amplification reaction,
CIN2 (n 5 19), CIN3 (n 5 34), and cervical cancer (n 5 20). The brush
2 lL of bisulfite-treated DNA (10 ng) was added to 18 lL of a amplifi-
specimens were separately collected from the patients for DNA extrac-
cation mix containing 13 Multiplex mix, 300 nM of the forward and
HC2 DNA collection Device (Qiagen, Hilden,
reverse primers for SIM1, and 200 nM of the probe. One hundred and
Germany). The brush samples immediately were placed in a transport
fifty nanomoles of the primers and probe were used for the
tion using the digene
SIM1 methylation increased considerably in cervical cancer. (A) The PMR values of SIM1 in normal cervix and cervical cancer
tissues. Individual PMR values are represented with dots. Normal, n 5 10; cancer, n 5 45. (B) Comparison of the PMR values of SIM1
according to cervical cancer stages. Normal, n 5 10; stage I, n 5 29; stage II, n 5 14; stage III, n 5 2. (Ptrend < .0001). (C) SIM1 expression
levels in normal cervix and cervical cancer tissues. (D) The PMR values of SIM1 on cervical brush specimens. Normal, n 5 17; CIN1, n 5 20;
CIN2, n 5 19; CIN3, n 5 34; cancer, n 5 20 (Ptrend < .0001). Horizontal lines indicate the median for each group
amplification of ACTB. The amplification and real-time measurements
with multiplex, followed by qMSP), which is known as quantitative mul-
were performed on a 7500 Fast ABI system (Applied Biosystems, Fos-
tiplex MSP (QM-MSP). This technique allowed us to enhance the meth-
ter City, CA), using the following conditions: 15 min at 958C followed
ylation signal, as previously described by Fackler’s group.14 In the first
by 45 cycles of 30 s at 948C and 1 min at 608C. The SIM1 was consid-
PCR reaction (the multiplex step), 5 lL of bisulfite-treated ccfDNA was
ered to be methylated when the Ct of at least two of three PCR repli-
used to co-amplify SIM1 and ACTB using a cocktail of gene-specific
cates for each sample was amplified and was considered to be
primer pairs that operated independently of the DNA methylation sta-
unmethylated when the Ct was not measurable. The percentage of
tuses of these genes. This process afforded 227 bp SIM1 and 77 bp
methylated reference (PMR) was defined as the percentage of fully
ACTB amplicons containing the target region required for qMSP.
methylated molecules at a specific locus of the SIM1 gene. The PMR
The forward and reverse primers for SIM1 in the first step of the PCR
was calculated by dividing the SIM1/ACTB ratio of a specific sample by
50 -
the SIM1/ACTB ratio of M.SssI-treated HeLa DNA. The PMR for the
CRCCCTCCRCRCRCCCAAAC-3 , respectively. The same primer pair as
SIM1 gene in each sample was determined using a comparative Ct
mentioned above for qMSP was used for ACTB. The PCR conditions
method instead of the relative standard curve method. The PMR value
were as follows: 958C for 5 min, followed by 30 cycles of 958C for
was calculated using the following formula13: PMR 5 22ddCt 3 100,
30 s, 608C for 30 s, and 728C for 30 s, with a final extension cycle of
ddCt 5 [(Ct(SIM1) 2 Ct(ACTB))sample] 2 [(Ct(SIM1) 2 Ct(ACTB))HeLa].
728C for 5 min. The PCR products were diluted 1:200 with water and
For PMR values > 100%, the methylation was considered to be 100%.
stored at 2208C. The second step in the PCR process was conducted
In the absence of any methylation, the PMR value was considered to
in the same way with other bisulfite-treated samples.
be 0%.
2.5 | Western blot analysis
2.4 | Quantitative multiplex MSP (QM-MSP)
Tissues were homogenized and protein lysates were prepared using a
For the ccfDNAs, all the purified material was used for the bisulfite
PRO-PREP protein extraction solution (iNtRON, Seoul, Korea) accord-
conversion process because of low levels of DNA in the samples. To
ing to the manufacturer’s protocol. Equal amounts of protein were
allow for the methylation analysis of small amounts of ccfDNA, we
resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis
conducted two sequential PCR reactions (i.e., a preamplification step
(SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF)
SIM1 methylation in normal and cervical cancer tissues
PMR values of the normal and cancer tissue samples were 0–3.3 (median
0) and 0–100 (median 80.9), respectively. A comparison of the PMR val-
ues for stage I and stage II/III samples revealed that there was no signifi-
(n 5 10)
(n 5 45)
P valuea
cant difference in the SIM1 methylation status of these samples (P 5 .27),
Median (range)
0 (0–3.3)
80.9 (0–100)
but we did observe a trend towards increasing SIM1 methylation accord-
Positive for methylation,b
n (%)
0 (0)
43 (95.6)
ing to the tumor stage (Ptrend < .0001) (Figure 1B). We subsequently
investigated whether the hypermethylation of SIM1 in cervical cancer
correlated with a decrease in SIM1 expression. Western blot analysis
Abbreviation: PMR, percentage of methylated reference.
Cutoff value for positivity of methylation: >7.03.
P values, two-tailed Student’s t tests.
showed that there was a considerable decrease in the expression of
SIM1 in cervical cancer tissues compared with normal cervix tissues
(Figure 1C). Taken together, these results show that SIM1 hypermethyl-
membrane. After blocking with 5% nonfat skimmed milk, the mem-
ation is a common occurrence in cervical cancer compared with normal
brane was blotted with designated primary and secondary antibodies,
cervical tissue, indicating that the methylation status of SIM1 could be
before being developed using the enhanced chemiluminescence
used as a potential biomarker for detecting cervical cancer.
method (ClarityTM Western ECL Substrate, BIO-RAD, Hercules, CA),
and visualized with an ImageQuant LAS4000 mini system (GE Healthcare, Chicago, IL). ACTB was used as the protein loading control. Anti-
3.2 | SIM1 methylation status on cervical brush
bodies against SIM1 and ACTB were purchased from LSBio (Seattle,
WA) and Santa Cruz Biotechnology (Dallas, TX), respectively.
As the detection of methylation biomarkers can be performed on the
same samples collected for HPV DNA test, methylation-based methods
using cervical brush specimens can become a promising noninvasive
2.6 | Statistical analysis
molecular test for detecting or monitoring cervical cancer, compared
Statistical analyses and data plotting were performed with GraphPad
with other cancer types requiring some invasive biopsy methods. So,
Prism (GraphPad Software Inc., San Diego, CA). For determination of
we examined the PMR values of SIM1 on cervical brush specimens
optimal cutoff values, receiver-operating characteristics (ROC) curve
(n 5 110), including normal (n 5 17), CIN1 (n 5 20), CIN2 (n 5 19), CIN3
method was used and cutoff value with the highest sum of sensitivity
(n 5 34), and cervical cancer (n 5 20). Fifteen of the 20 cervical cancer
and specificity was determined depending on the population of sam-
of cervical brush specimens were collected from matched patients with
ples for discriminating positive and negative. P values of <.05 were
cervical cancer tested for Figure 1A. The relationship between SIM1
considered significant and differences between the two groups were
methylation and disease severity was evaluated quantitatively, and the
analyzed using two-tailed Student’s t tests. Post-test for linear trend
results are shown in Table 2 and Figure 1D. While the median PMR
(Ptrend) was used as part of the one-way ANOVA for trend analysis.
value was zero in the normal, CIN1, CIN2, and CIN3 samples with
ranges of 0-0.4, 0-0.6, 0–4, and 0–56.9, respectively, the median PMR
in the cervical brush specimens collected from cancer patients was
27.95 with a range of 0–100. The methylation positivity for the individ-
3.1 | Hypermethylation of SIM1 in cervical cancer
ual stages was determined to be 0% (0/17), 0% (0/20), 5.3% (1/19),
41.2% (14/34), and 85% (17/20) for the normal, CIN1, CIN2, CIN3, and
To determine whether the methylation status of SIM1 could be used to
cancer samples, respectively. Although the median PMR value of CIN3
distinguish cervical cancer tissues from normal tissues, we compared the
was determined to be zero because of the low PMR values, the number
SIM1 methylation status of cervical cancer tissues with that of normal
of SIM1 methylation-positive samples increased significantly in CIN3
cervix tissues. As shown in Figure 1A and Table 1, the SIM1 promoter
(P < .05), compared with normal, CIN1, and CIN2 samples. As shown in
region was found to be highly methylated in the majority of the cancer
Figure 1D, the increase in SIM1 methylation in the cervical brush speci-
tissues tested, but not in the normal tissues (P < .0001). SIM1 methyla-
mens was significantly associated with the severity of the disease
tion was observed in 43 (95.6%) of the 45 cancer tissue samples tested
(Ptrend < 0.0001). Furthermore, the SIM1 methylation statuses of all 15
in this study, compared with 0 (0%) of the 10 normal cervix tissues. The
of the paired samples collected from the same cervical cancer patients
SIM1 methylation on cervical brush specimens
Median (range)
Positive for methylation, n (%)
Normal (n 5 17)
CIN1 (n 5 20)
CIN2 (n 5 19)
CIN3 (n 5 34)
Cancer (n 5 20)
0 (0–0.4)
0 (0–0.6)
0 (0–4)
0 (0–56.9)
27.95 (0–100)
0 (0)
0 (0)
1 (5.3)
14 (41.2)
17 (85)
Cutoff value for positivity of methylation: >0.8.
Ptrend, post-test for linear trend as a part of the one-way ANOVA.
SIM1 methylation status of matched tissue and plasma
ccfDNA in cervical cancer patients (n 5 41)
To confirm that the methylated SIM1 observed in the plasma
ccfDNA was originated from cancer cells, we examined the SIM1 meth-
n (%)
n (%)
rate, n (%)a
ylation status in the gDNA of peripheral blood leucocyte (PBL) col-
Positive, n (%)
39 (95.1)
15 (36.6)
17 (41.5)
observed in none (0%) of the 19 PBL samples, compared with 17
Negative, n (%)
2 (4.9)
26 (63.6)
Concordance rate of SIM1 methylation status between cancer tissues
and ccfDNAs.
lected from matched cervical cancer patients. Methylated SIM1 was
(89.5%) of the 19 matched cervical cancer tissues (P < .0001, cutoff
value of PMR > 7.03) (Figure 2). The PMR values ranged from 0 to
6.26 (median 0) and 0 to 100 (median 56.6) for the paired PBL and cancer tissues, respectively. This result suggests that methylated SIM1
were identical (2 negative and 13 positive), indicating that the SIM1
methylation status of cervical brush specimens adequately reflects that
of the cancer tissues. Taken together, these results demonstrate that
the detection of SIM1 methylation on cervical brush specimens could
genes detected in plasma are derived from cancer cells. Taken together,
these results imply the possibility of using plasma ccfDNA for detecting
methylated SIM1 to diagnose or monitor cervical cancer even if it does
not meet expectations.
be useful for distinguishing cancer from noncancer including normal,
CIN1, and CIN2.
3.3 | Detection of methylated SIM1 in plasma ccfDNA
from cervical cancer patients
The goal of this study was to determine whether the methylation status
ccfDNA has recently attracted considerable interest in terms of poten-
detecting methylated SIM1 in plasma ccfDNA collected from patients.
of SIM1 could be used to differentiate cancer from noncancer as a potential biomarker for cervical cancer. We also investigated the possibility of
tial application to the diagnosis and monitoring of cancer as a relatively
This study revealed that SIM1 hypermethylation is very common in cervi-
noninvasive technique.15 Several studies have reported the possibility
cal cancer compared with normal cervical tissues, CIN1, and CIN2.
of detecting methylated genes in ccfDNA fragments from various can-
The aberrant hypermethylation of promoter regions of various
cer patients for early diagnosis and delivering a personalized treatment
genes is a common epigenetic event in many cancers including cervical
plan.16,17 To determine whether it is possible to use the methylated
cancer. Several studies have reported the hypermethylation of many
SIM1 found in ccfDNA fragments to diagnose cervical cancer, we
genes, including DAPK1, RARB, and TIMP3 in high-grade CINs and/or cer-
attempted to detect methylated SIM1 in plasma ccfDNA samples col-
vical cancer, which strongly suggest that DNA methylation could be used
lected from cervical cancer patients. Methylation status of SIM1 was
as a possible biomarker for the early diagnosis for cervical cancer.7,8,16
evaluated in 41 ccfDNA samples with matched cancer tissues (Table 3).
Recent studies have also shown that the combination of a primary
Unlike the high positivity of SIM1 methylation in cancer tissues of the
hrHPV DNA test with a gene methylation panel composed of JAM3,
same patients (39/41, 95.1%), SIM1 methylation in ccfDNA was found
EPB41L3, TERT, and C13ORF18 to triage patients resulted in an increase
in 15 of 41 samples (36.6%). The concordance rate between ccfDNA
in the identification of CIN31 and a higher percentage of correct refer-
and matched cancer tissue was 41.5% (17/41, 15 positive, and 2 nega-
rals compared with the use of an hrHPV DNA test in combination with
tive) with sensitivity 38.5% and specificity 100%.
the Pap smear.18 In this study, we showed that the methylation status of
SIM1 can distinguish cervical cancer cells from normal cells, CIN1, or
CIN2 using cervical brush specimens (Figure 1D and Table 2). Our results
suggest that if SIM1 methylation test combines with hrHPV DNA test, it
can also help to overcome the limitation of hrHPV DNA test and reduce
unnecessary colposcopy and biopsy. Although SIM1 methylation is highly
sensitive and specific for cervical cancer in this study, it is insufficient for
detecting high-grade CINs (CIN2/3) requiring treatments. The p16INK4a
(p16) protein has been shown to be a surrogate marker of high-risk HPV
oncogenic activity and p16 overexpression has been associated with
dysplastic/neoplastic cells but not seen in normal cervical epithelium,
and it is also related to degree of histologic dysplasia.19–21 The value of
p16 in the diagnostic process is important because the strong and diffuse
staining of p16 is highly sensitive for CIN2 and 3 but not for CIN1.21,22 If
the SIM1 methylation test and p16 immunostaining are combined in cer-
Comparison of SIM1 methylation in matched cancer
tissues and PBL from the same cervical cancer patients. The cancer
tissues and PBL were obtained from 19 cervical cancer patients.
Individual PMR values are represented with dots. The median of
each group is indicated by a horizontal line. P < .0001
vical cancer screening or diagnosis, it could better distinguish between
cervical low-grade CIN1, high-grade CIN 2/3, and cancer. Large-scale
cohort clinical studies will be required to demonstrate the clinical usefulness of combining these two tests.
A recent study indicated that the aberrant methylation of SIM1
together with DLX4 could be used a predictive marker for disease progression in cervical low-grade squamous intraepithelial lesions (LSILs
and CIN1).
Consistently with our data, the authors found that the fre-
plasma ccfDNAs from patients could be hopeful if experimental techniques including ccfDNA preparation and qMSP are further improved.
The SIM1 methylation observed in this study were specific to cervical cancer and not present in normal cervical epithelium, and the hyper-
quency of SIM1 methylation was highly elevated in cervical squamous
methylation of SIM1 in cervical cancer correlated with a decrease in
cell carcinoma compared with a normal control despite using primer set
SIM1 expression, suggesting that the downregulation of SIM1 might
detecting different CpG sites of SIM1 gene and semi-quantitative MSP
play a role in cervical cancer development. However, its role remains
method. They showed that the frequency of SIM1 methylation signifi-
unknown in carcinogenesis. Murine SIM factors (SIM1 and SIM2) have
cantly increased in LSILs with progress to high-grade SILs (HSILs and
been reported to be capable of binding mammalian hypoxia response
CIN2/3) or higher within 365 days after LSIL diagnosis, compared with
element sequences in combination with ARNT protein, resulting in the
nonprogressive LSILs. However, we observed very little SIM1 methyla-
attenuation of hypoxic reporter gene transcription by causing competi-
tion in the CIN1 (0/20) and CIN2 (1/19) samples evaluated in this
tion between the HIF-1 alpha and SIM proteins for binding to the ARNT
study. This discrepancy in the SIM1 methylation status may be due to
and DNA binding sites.26 In contrast to HIF-1 alpha, which rapidly accu-
the small number of cases of patients tested in our study and may also
mulates under low oxygen conditions, SIM protein levels decrease under
be due to differences in experimental conditions, including the primer/
hypoxic conditions, most probably as a result of translational inhibition in
probe design and specific CpG site. Despite these differences, it is clear
hypoxia-stressed cells.26 These results suggest that the decrease in SIM1
that the SIM1 methylation can effectively differentiate cancer from
under hypoxic conditions could enable cancer cells to adapt to these
normal, CIN1, and CIN2 under our experimental conditions.
harsh environmental conditions, and that the decrease in the expression
While SIM1 methylation was rarely detected in CIN1 and CIN2,
of SIM1 through translational inhibition or promoter methylation could
the PMR value of SIM1 in CIN3 was low but the positivity was as high
be involved in carcinogenesis or cancer cell survival. We are currently
as about 41% of the tested brush samples (Table 2). This suggest that
studying the role of SIM1 in cervical cancer to reveal the causal relation-
the proportion of SIM1 gene methylation in cells begin to increase
ship between SIM1 methylation and cervical carcinogenesis.
mainly at the stage of CIN3, and the suppression of SIM1 gene expres-
In conclusion, this study has shown that the methylation status of
sion by SIM1 methylation may play an important role in the progression
SIM1 could be used a possible biomarker for cervical cancer. The SIM1
from CIN3 to invasive cervical cancer. Therefore, the detection of SIM1
methylation biomarker needs to be evaluated in greater detail in larger
methylation at the stage of CIN3 could increase the opportunity of
cohorts of patients for its clinical application as a screening, diagnostic,
treatment for preventing the progression to invasive cervical cancer.
or monitoring tool for cervical cancer.
Circulating DNA fragments carrying tumor-specific gene alterations
can be found in the cell-free fraction of blood (plasma or serum). The
ccfDNAs in plasma or serum therefore represent a promising pool of
potential biomarkers for cancers. Furthermore, recent advances in
sequencing technologies have allowed for the rapid identification of
This study was supported by a grant from the Korean Health
Technology R&D Project (Ministry of Health and Welfare, Korea,
HI12C0050) and RP-Grant 2017 of Ewha Womans University.
genomic and epigenetic alterations in ccfDNA samples collected from
cancer patients.17,24 Notably, ccfDNA fragments have been reported to
exhibit similar alterations in their methylation status to those found in
primary cancer cells.17,25 In a recent report, SDC2 methylation detected
The authors declare that they have no conflict of interest.
in the serum DNA of colorectal cancer patients (stages I–IV) and healthy
individuals by qMSP demonstrated a high sensitivity and specificity for
detecting cancers, highlighting the huge potential of ccfDNA as a diag-
Seung Cheol Kim
nostic tool for the detection of cancer.17 In this study, we tried to detect
methylated SIM1 in plasma ccfDNA of cervical cancer patients to evaluate the feasibility of ccfDNA for detecting methylated SIM1. Our results
showed that the SIM1 methylation positivity was not so high with
36.6% (15/41) in plasma ccfDNA samples of the cervical cancer
patients, and the concordance rate of SIM1 methylation status between
matched cancer tissues and ccfDNAs was just 41.5% (17/41). The reason of low detection rate could be linked to several factors, such as the
relative scarcity of ccfDNA fragments derived from tumor cells in the
entire ccfDNA pool, contamination of genomic DNA, genetic heterogeneity of tumors, and the sensitivity of the qMSP method. Although low
detection rate of methylated SIM1 ccfDNA from cervical cancer patients
in this study is somewhat disappointing, SIM1 methylation test using
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How to cite this article: Kim H-J, Kim CY, Jin J, et al. Aberrant
single-minded homolog 1 methylation as a potential biomarker
for cervical cancer. Diagnostic Cytopathology. 2017;00:1–7.
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