Folate Deficiency and FHIT Hypermethylation and HPV 16 Infection Promote Cervical Cancerization

Abstract

Fragile histidine triad (FHIT) is a suppressor gene related to cervical cancer through CpG island hypermethylation. Folate is a water-soluble B-vitamin and an important cofactor in one-carbon metabolism. It may play an essential role in cervical lesions through effects on DNA methylation. The purpose of this study was to observe effects of folate and FHIT methylation and HPV 16 on cervical cancer progression. In this study, DNA methylation of FHIT, serum folate level and HPV16 status were measured using methylation-specific polymerase chain reaction (MSP), radioimmunoassay (RIA) and polymerase chain reaction (PCR), respectively, in 310 women with a diagnosis of normal cervix (NC, n

Full text

Asian Pacic Journal of Cancer Prevention, Vol 15, 2014 9313
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9313
Folate Deciency, FHIT Hypermethylation and HPV 16 Infection Promote Cervical Cancerization
Asian Pac J Cancer Prev, 15 (21), 9313-9317
Introduction
Cervical cancer is one of the most common cancers
in women globally and is responsible for the large
percentage of women’s death in developing countries.
Epidemiological and laboratory data have shown that
persistent infection with oncogenic human papillomavirus
(HPV), particularly HPV-16 and HPV-18, is the main risk
factor, but not solely responsible for the development
of cervical cancer. Other cofactors including genetic
and epigenetic factors, may facilitate the progression of
cervical cancer as well.
Previous studies have reported that DNA methylation
of genes is a frequent epigenetic factor in cervical cancer
(Piyathilake et al., 2010). Aberrant DNA methylation in
the CpG islands at the promoter region is an important
epigenetic mechanism underlying the inactivation
of tumor suppressor genes (Abouzeid et al., 2011).
1Maternal and Children Health care of Shanxi Province afliated to Shanxi Medical University, 2Department of Epidemiology,
School of Public Health, Shanxi Medical University, Taiyuan China, 3Department of Biomedical Science, Mercer University School
of Medicine, USA *For correspondence: wangjt59@163.com
Abstract
Fragile histidine triad (FHIT) is a suppressor gene related to cervical cancer through CpG island
hypermethylation. Folate is a water-soluble B-vitamin and an important cofactor in one-carbon metabolism. It
may play an essential role in cervical lesions through effects on DNA methylation. The purpose of this study was
to observe effects of folate and FHIT methylation and HPV 16 on cervical cancer progression. In this study, DNA
methylation of FHIT, serum folate level and HPV16 status were measured using methylation-specic polymerase
chain reaction (MSP), radioimmunoassay (RIA) and polymerase chain reaction (PCR), respectively, in 310 women
with a diagnosis of normal cervix (NC, n=109), cervical intraepithelial neoplasia (CIN, n=101) and squamous cell
carcinoma of the cervix (SCC, n=101). There were signicant differences in HPV16 status (χ2=36.64, P<0.001),
CpG island methylation of FHIT (χ2=71.31, P<0.001) and serum folate level (F=4.57, P=0.011) across the cervical
histologic groups. Interaction analysis showed that the ORs only with FHIT methylation (OR=11.47) or only
with HPV 16 positive (OR=4.63) or with serum folate level lower than 3.19ng/ml (OR=1.68) in SCC group were
all higher than the control status of HPV 16 negative and FHIT unmethylation and serum folate level more than
3.19ng/ml (OR=1). The ORs only with HPV 16 positive (OR=2.58) or with serum folate level lower than 3.19ng/
ml (OR=1.28) in CIN group were all higher than the control status, but the OR only with FHIT methylation
(OR=0.53) in CIN group was lower than the control status. HPV 16 positivity was associated with a 7.60-fold
increased risk of SCC with folate deciency and with a 1.84-fold increased risk of CIN. The patients with FHIT
methylation and folate deciency or with FHIT methylation and HPV 16 positive were SCC or CIN, and the
patients with HPV 16 positive and FHIT methylation and folate deciency were all SCC. In conclusion, HPV
16 infection, FHIT methylation and folate deciency might promote cervical cancer progression. This suggests
that FHIT may be an effective target for prevention and treatment of cervical cancer.
Keywords: Folate - FHIT - methylation - cervical cancerization - prevention
RESEARCH ARTICLE
Folate Deciency and FHIT Hypermethylation and HPV 16
Infection Promote Cervical Cancerization
Li-Xia Bai1, Jin-Tao Wang2*, Ling Ding2, Shi-Wen Jiang3, Hui-Jie Kang2, Chen-
Fei Gao2, Xiao Chen2, Chen Chen2, Qin Zhou2
The fragile histidine triad (FHIT) gene is a candidate
tumor suppressor gene located at chromosome 3p14.2
encompassing the FRA3B site, which is the most active
fragile site in the human genome. The reduced FHIT
expression was associated with the high CpG island
methylation (Al-Temaimi et al., 2013). The promoter
methylation status of the FHIT gene was observed in a
number of cancers, such as breast cancer (Jeong et al.,
2013), lung cancer (Tan et al., 2013), non-small cell lung
carcinomas (Haroun et al., 2014; Li et al., 2014), bladder
cancer (Han et al., 2011) and Nasopharyngeal Carcinoma
(Chen et al., 2013), and others as well. In cervical cancer
tissues, the methylation rate of the FHIT gene promoter
was also signicantly higher than in cervical intraepithelial
neoplasia and normal cervical tissues (66.0 vs. 59.1 vs.
25.0 %, P=0.0033) (Banzai et al., 2014).
Folate is a water-soluble B-vitamin and an important
cofactor in one-carbon metabolism in which folate

Li-Xia Bai et al
Asian Pacic Journal of Cancer Prevention, Vol 15, 2014
9314
participates in nucleotide synthesis and methylation
reactions (Wang et al., 2011; Pathak et al., 2012). The
relationship between folate deciency and cervical cancer
has not been consistent. Some evidence has suggested that
folate deciency may increase the risk of cancers and may
have other negative effects on human health (Tomaszewski
et al., 2011). However, in an India study, there was no
statistically signicant association between folate levels
and cervical carcinogenesis. This was observed in 136
control subjects, 92 low-grade squamous intraepithelial
lesions (LSIL) subjects, and 94 invasive cervical cancer
cases (ICC) in Kerala, South India (Ragasudha et al.,
2012). The previous ndings of our team indicated that the
low level of serum folate was signicantly associated with
cervical carcinogenesis (Jin-Tao et al., 2014), and the cell
proliferation decreased and the apoptosis increased with
the concentration of folate increasing in human cervical
cancer cell lines (Ding et al., 2013).
Above all, high FHIT methylation and low serum
folate and HPV infection may be risk factors for cervical
cancer independently. However, few studies reported how
they work together in cervical cancer progression. Hence,
the present study was conducted to study their interactions
by examining serum folate and DNA methylation of
tumour suppressor gene FHIT and HPV 16 in women
ranging from normal cervix to cervical intraepithelial
neoplasia to squamous cell carcinoma of the cervix.
Materials and Methods
In this study, after been approved by the institutional
Review Board (IRB) of the appropriate hospitals where
the participants came from and informed consent was
obtained from the patients, the author obtained blood and
tissues of participants.
Blood collection
Prior to surgery or other treatments, a 3 ml blood
sample was drawn in the morning after an overnight fasting
(12 hours) and centrifuged at 4000 rpm for 15 minutes at
room temperature within 10 hours of collection. Plasma
and serum were separated, and serum was drawn into an
EP tube and stored at -80℃ until analysis. Blood folate
(folic acid) levels were determined by radioimmunoassay
(RIA) (Dongya Institute of Biological Medicine, Beijing,
China).
Cervical tissues collection
Samples of squamous cell carcinoma of the cervix
tissues were derived from patients who experienced
primary surgery for cervical diseases at the Department
of Gynaecology and Obstetrics in Tumor hospital
of Shanxi Province (Taiyuan, China), and cervical
intraepithelial neoplasia tissues and normal cervix
underwent vaginoscope inspection from the Second
Affiliated Hospital of Shanxi Medical University
(Taiyuan, China) from June 2008 to August 2009. All
of the selected cervical tissues met the criteria including
no history of any other type of malignant tumor, without
neoadjuvant therapy prior to surgery and without using
vitamin B in the past three months. All the cervical tissues
were put into the nitrogen canister immediately and then
storaged in a refrigerator at -80℃.
Polymerase chain reaction (PCR)
Polymerase Chain Reaction (PCR) primers for
HPV16 E2 and E6 were purchased from Integrated DNA
Technologies (Shanghai, China). All primers are shown
in Table 1.
PCR was performed in 50µl reaction volumes
containing 10×PCR buffer. DNA was amplied using
the following protocol: 95ºC for 5 minutes followed by
30 cycles of 95ºC for 60 seconds, 55ºC for 60 seconds,
72ºC for 60 seconds, and nally of 72ºC for 10 minutes.
Methylation-specic polymerase chain reaction
Methylation-specic polymerase chain reaction (MSP)
primers for FHIT (Gene ID: 2272) were purchased from
Integrated DNA Technologies (Shanghai, China). All
primers are also shown in Table 1.
MSP was performed in 50µl reaction volumes
containing 10×PCR buffer. DNA was amplied using
the following protocol: 95ºC for 5 minutes followed by
40 cycles of 94ºC for 30 seconds, 56ºC for 30 seconds,
72ºC for 30 seconds, and nally of 72ºC for 7 minutes.
Statistical analysis
A comparison of categorical variables among histology
groups was examined by χ2 test; continuous variables were
tested by ANOVA analysis and if a signicant difference
was detected, further Post-Hoc test was used.
Results
Demographic characteristics
In the present study, there were three groups of
participants. Specically, 100 women were aged 31-
75 years (median±SD: 49.00±10.75 y) in squamous
cell carcinoma of the cervix group (SCC), 101 women
were aged 29-67 years (48.00±12.00 y) in cervical
intraepithelial neoplasia group (CIN), and 109 women
were aged 24-81 years (47.00±13.50 y) in normal cervix
Table 1. The Sequences and Length of HPV16 E2 and
E6 and FHIT
Product name Primer sequences Length
HPV16 E2 P1: AAG GGC GTA ACC GAA ATC GGT 351bp
P2: CAT ATA CCT CAC GTC GCA G
HPV16 E6 P1: CTT GGG CAC CGA AGA AAC AC 208bp
P2: TTG GTC ACG TTG CCA TTC AC
FHIT(M) P1: 5’-TTG GGG CGC GGG TTT GGG TTT TTA CGC-3’ 74 bp
P2: 5’-CGT AAA CGA CGC CGA CCC CAC TA-3’
FHIT(U) P1: 5’-TTG GGG TGT GGG TTT GGG TTT TTA TG-3’ 74 bp
P2: 5’-CAT AAA CAA CAC CAA CCC CAC TA-3’
Table 2. Correlation between HPV-16 Status and
Cervical Histologic Groups
Group HPV 16 positive, HPV 16 negative, χ2 P
n(%) n(%)
NC 22 (20.2) 87 (79.8) 36.64 <0.001a
CIN 38 (37.6) 63 (62.4) 36.29 <0.001b
SCC 61 (61.0) 39 (39.0) 7.34 0.005c
total 122 (39.0) 188 (61.0)
acomparison between three groups; b, comparison between CIN and NC groups;
ccomparison between SCC and NC groups

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Folate Deciency, FHIT Hypermethylation and HPV 16 Infection Promote Cervical Cancerization
(NC) group (control group). There were no signicant
differences in age, nationality, birthplace, city of residence,
and marital status among the SCC, CIN and NC groups.
Detection of HPV16
Table 2 shows the HPV 16 status of the women with
different cervical histology diagnoses. HPV 16 was
detected in 39.4% of the study participants. There was a
signicant difference in HPV16 status across the cervical
histologic groups (total χ2=36.64, P<0.001). The Post-Hoc
analysis showed that women with SCC were more likely
to be HPV 16 positive than women with normal cervix
(χ2=36.29, P<0.001), and that women with CIN also had
an increased risk of HPV 16 infection (χ2=7.34, P=0.005).
Detection of CpG island methylation of FHIT
Table 3 shows a statistically signicant difference
in the proportion of CpG island methylation of tumor
suppressor gene FHIT across the cervical histologic grades
(χ2=71.31; P<0.001). The rate of CpG island methylation
of FHIT in the SCC group was significantly higher
than that in the NC group (χ2=42.67; P<0.001), but the
signicant difference was not observed between the CIN
and NC groups (Fhisher χ2, P=1.000).
Serum folate levels
The level of serum folate among the three groups was
signicantly different which was tested by the ANOVA
(Table 4). Women with SCC had a signicantly lower
serum folate level than women with normal cervical
histology (P<0.0167, Adjusted test α=α/3=0.0167), but
there was no signicant difference between SCC and
CIN groups (P>0.0167) and between CIN and NC groups
(P>0.0167).
Interactions between HPV infection and FHIT methylation
and serum folate levels in SCC and CIN groups
Table 5 shows the three factors for interactions
between HPV infection and FHIT methylation and serum
folate levels in SCC and CIN groups respectively. The
odds ratios (OR) in different conditions were calculated
compared with the “control status” of HPV 16 negative
and FHIT unmethylation and serum folate level more than
3.19ng/ml. It was observed that women only with FHIT
methylation (OR=11.47) or only with HPV 16 positive
(OR=4.63) or with a serum folate level lower than 3.19ng/
ml (OR=1.68) were more likely to be SCC compared with
the “control status” (OR=1), and women only with HPV
16 positive (OR=2.58) or with a serum folate level lower
than 3.19ng/ml (OR=1.28) were more likely to be CIN
compared with the “control status” , but women only with
FHIT methylation (OR=0.53) in CIN group were lower
than the control status. HPV 16 positive was associated
with a 7.60-fold increased risk of SCC with folate
deciency and with a 1.84-fold increased risk of CIN.
The patients with FHIT methylation and folate deciency
or with FHIT methylation and HPV 16 positive were all
SCC or CIN, and the patients with HPV 16 positive, FHIT
methylation and folate deciency were all SCC.
Discussion
The key nding in this study is that the serum folate
level, CpG island hypermethylation of FHIT and HPV 16
positive are signicantly associated with the progression
of cervical lesions. Serum folate levels signicant differed
between the SCC and NC groups, but no signicant
difference was observed between SCC and CIN groups.
FHIT showed increased methylation with increasing
severity of cervical neoplastic changes. Interaction
analysis showed the odds ratios of HPV 16 infection and
FHIT methylation and folate deciency existed at the
same time or when two of them existed were higher than
one of them existed, the ORs of which were all higher
than the control status of HPV 16 negative and FHIT
unmethylation and serum folate level higher than 3.19ng/
ml in SCC and CIN.
Folate is important for normal cell division. Folate
deciency has many negative effects on health and can be
one of the risk factors of cancers which has been widely
documented. This study showed that folate level in SCC
group was statistically lower than that in the NC group,
but no signicant difference was found between the SCC
and CIN groups and between the CIN and NC groups. It
implied that folate deciency had more inuence on SCC
than CIN and NC. Previous studies reported that folate
status was associated with the natural history of HR-HPV
infection. An Indian study with 742 women found that
women with higher serum folate levels (>6ng/ml) were
at a lower risk of HR-HPV positivity compared to those
Table 3. Comparison of FHIT Methylation Rates
between Cervical Histologic Groups
Group FHIT methylation, FHIT unmethylation, χ2 P
n(%) n(%)
NC 3(2.8) 106(97.2) 71.31 <0.001a
CIN 3(3.0) 98(97.0) - 1.000b
SCC 39(39.0) 61(61.0) 42.67 <0.001c
total 45(14.5) 265(85.5)
acomparison between three groups; bcomparison between CIN and NC groups,
Fhisher χ2; c comparison between SCC and NC groups
Table 4. Comparison of Serum Folate Levels between
Cervical Histologic Groups(ng/ml)
Group n serum folate F P
NC 109 3.31±1.73 4.57 0.01
CIN 101 3.06±1.86a
SCC 100 2.59±1.60b, c
acomparison between CIN and NC groups, P>0.0167; bcomparison between SCC
and NC groups, P<0.0167; ccomparison between SCC and CIN groups, P>0.0167
Table 5. Interaction between HPV Infection and FHIT
Methylation and Serum Folate Levels in SCC and
CIN Groups
HPV FHIT serum folate NC SCC OR CIN OR
16 methylation level (ng/ml)
- - ≥3.19 43 10 1 27 1
- - <3.19 41 16 1.68 33 1.28
- + ≥3.19 3 8 11.47 1 0.53
- + <3.19 0 5 - 2 -
+ - ≥3.19 8 7 4.63 13 2.58
+ - <3.19 14 28 8.6 25 2.84
+ + ≥3.19 0 3 - 0 -
+ + <3.19 0 23 - 0 -

Li-Xia Bai et al
Asian Pacic Journal of Cancer Prevention, Vol 15, 2014
9316
with serum folate levels ≤6ng/ml (Piyathilake et al.,
2010). In a prospective study including 345 subjects, the
results showed that women with higher folate status had
a higher risk of becoming HR-HPV-positive (Piyathilake
et al., 2004). A research study in Turkey including 122
women also indicated that the means of serum folate level
in HSIL or LSIL or ASCUS group were lower than that of
the control group (P<0.05). Another report showed that
in all cervical dysplasia groups, folate levels were lower
in HPV-positive patients than in HPV-negative patients
(P<0.05) (Abike et al., 2011). A multicenter case control
study that involved 927 Korean women (440 controls,
165 patients with CIN1, 167 with CIN2/3, and 155 with
cervical cancer) reported that patients with cervical cancer
had signicantly lower median serum folate levels than
controls and the linear trend test showed significant
associations between higher serum folate levels and
lower cancer risks (P for linear trend=0.0058) (Tong et al.,
2011). However, a recent study including 322 women (136
control subjects, 92 low-grade squamous intraepithelial
lesions (LSIL), 94 invasive cervical cancer cases(ICC)) in
South India did not nd signicant associations between
folate levels and cervical carcinogenesis (Ragasudha et
al., 2012).
FHIT is a suppressor gene related to cervical cancer
through CpG island hypermethylation. In this study, the
SCC group had a signicantly higher rate of CpG island
methylation than the NC group, although no statistical
difference was observed between the CIN and NC groups.
A positive correlation was observed between methylation
rates and progression of cervical cancer. The results
showed that the vital role of FHIT methylation in cervical
carcinogenesis, which was supported by previous studies.
A case-control study found that FHIT methylation was
observed in 28.3% (17/60) of cervical cancer cases, while
FHIT was not methylated in any of the 23 healthy NCs
(Neyaz et al., 2008). Another case-control study in China
indicated that the rate of 5’CpG island methylation of the
FHIT gene was 40.0% (16/40) in cervical cancer groups,
while the rate of methylation in normal cervical tissue was
0% (0/10). The FHIT methylation rate in CIN1 (14.3%,
2/14) was statistically lower than its rate in CIN2 (56.3%,
13/23) (P<0.05) (Shi et al., 2005).
The present study shows in the beginning that
there was a significant interaction between HPV 16
infection and serum folate levels and FHIT methylation
in cervical tissue. The factors for HPV 16 infection and
FHIT methylation and folate deciency existed at the
same time or two of them existed have higher risks than
that of one of them existed or all absent. It is generally
known that HPV infection is the primary cause of SCC.
Aberrant DNA methylation is associated with cervical
pathogenesis (Flatley et al., 2012; Pathak et al., 2012).
The study demonstrated that women with a lower serum
folate level and HPV 16 infection had a greater risk of
SCC than those with a higher serum folate level and HPV
negative, which was also revealed in a previous study
(Piyathilake et al., 2007).
The study also found that folate deciency dramatically
increased the risk of cervical cancer patients with
high FHIT methylation for SCC. This could be due to
low serum folate levels which lead to decreased DNA
methyltransferase 1 (DNMT1) expression, which plays
a signicant role in maintaining DNA methylation status
and regulating expression of tumor suppressor genes in
cervical cancer cells (Zhang et al., 2011). Chromosomal
fragile site FRA3B at 3p14.2 located in the FHIT gene
may be particularly prone to forming gaps or breaks on
metaphase chromosomes under the conditions that inhibit
DNA replication or repair, such as folate deciency, which
is an essential cofactor in de novo synthesis of purines
and thymidylate-nucleotides necessary for DNA repair.
The interaction between FHIT and HPV 16 would be
caused by the fact that FRA3B fragile site of FHIT gene
is a candidate region for HPV 16 interaction (Wilke et
al., 1996).
The study has several strengths. Firstly, it observed
interactions between serum folate levels and HPV status
and FHIT methylation rates for the rst time. Second,
serum and tissue samples originated from different
stages of cervical cancer progression, including normal
cervix, CIN and cervical cancer, which document natural
cervical cancer progression. However, this study also has
limitation. We only showed the relationships between
folate and FHIT and HPV in cervical cancer progression,
but did not indicate mechanisms of action even though
other studies have indicated that folate may play an
essential role in cervical lesions through impacting on
DNA methylation (Flatley et al., 2009; Piyathilake et al.,
2011). Future studies should avoid these limitations.
Acknowledgements
This study was supported by National Natural Science
Foundation of China (Grant No. 30872166, No. 81273157)
and the Natural Science Foundation of Shanxi Province,
China (Grant No. 2008011075-1).
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