Retinoid X Receptor-¦Á Expression Suppression and its Promoter
Methylation Associates with ¦Â-catenin
Expression in Colorectal Cancer
Li-Li
Kang, Fen-Fen Zhang, Hui Li, Tian-Tian Zhen, Hui-Juan Shi, Yang Yang, Jian-Ming
Tang, Ying-Jie Liang, An-Jia Han
Li-Li
Kang, Fen-Fen Zhang, Hui Li, Tian-Tian Zhen, Hui-Juan Shi, Yang Yang, Jian-Ming
Tang, Ying-Jie Liang, An-Jia Han, Department of Pathology, the First Affiliated Hospital,
Sun Yat-Sen University, Guangzhou, China
Li-Li Kang and
Fen-Fen Zhang contributed equally to this work.
Correspondence to: Anjia Han, MD, PhD, Department of Pathology, The First
Affiliated Hospital, Sun Yat-Sen University, 58, Zhongshan Road II, Guangzhou,
510080, China.
Email: hananjia@mail.sysu.edu.cn
Telephone: +86-020-87332235
Fax: +86-020-87332235
Received: March 1, 2014
Revised: May 5, 2014
Accepted: May 11, 2014
Published online: June 18, 2014
ABSTRACT
AIM: Whether retinoid X receptor-¦Á (RXR¦Á) expression suppression is related to
epigenetic regulation in colorectal cancer (CRC) is unclear. Our current study
is to investigate the promoter methylation status of RXR¦Á and correlated the
results with ¦Â-catenin expression and clinicopathological characteristics in
CRC.
METHODS: Real-time PCR, Western blot, immunohistochemistry staining, methylation
special PCR, and methylation sensitive-high resolution melting (MS-HRM) curve
analysis were used to detect the expression of RXR¦Á and ¦Â-catenin and RXR¦Á
promoter methylation status in CRC cells and tissues.
RESULTS: Our study showed that RXR¦Á low expression rate was significantly higher
in 202 samples of CRC tissues than that in adjacent non-tumor colorectal mucosa
(ANM) tissues (P<0.001). CRC patients had a significantly shorter
overall survival rate in the low RXR¦Á expression group compared with that in
the high RXR¦Á expression group (p=0.038). There was a significant
correlation between RXR¦Á low expression and ¦Â-catenin abnormal expression in
CRC tissues. The promoter methylation of RXR¦Á was detected in HT29, HCT116,
SW480, SW620, and LoVo except SW116 and NCM460, all 8 pairs of fresh CRC and
ANM tissues, all 13 samples of paraffin-embedded ANM, 21 CRC, and 8 lymph node
metastatic CRC tissues by methylation special PCR. Further MS-HRM curve
analysis showed that the promoter methylation of RXR¦Á was detected in 76.92%
(10/13) of ANM, 85.71% (18/21) of CRC, and 75.0% (6/8) of lymph node metastatic
CRC tissues, respectively. The mean methylation degree of RXR¦Á was
significantly higher in CRC and lymph node metastatic CRC than that in ANM
tissues, respectively (P=0.038, P=0.035). Methylation degree of
RXR¦Á was decreased in SW620 and LoVo cells treated with 5-Aza-2'-Deoxycytidine.
Furthermore, RXR¦Á and ¦Â-catenin mRNA and protein expression dramatically
elevated and suppressed in LoVo cells treated with 5-Aza-2'-Deoxycytidine at
different concentrations and times compared with the control group,
respectively.
CONCLUSION: Our results first demonstrate that RXR¦Á low expression
associates with poor prognosis of CRC patients. The promoter methylation of RXR¦Á
might be related to RXR¦Á expression suppression in CRC. Targeting RXR¦Á promoter
methylation might be a novel therapeutic strategy for CRC patients.
© 2014 ACT. All rights reserved.
Key words: Retinoid X receptor-¦Á; Promoter methylation; ¦Â-catenin; Colorectal cancer
Kang LL, Zhang FF,
Li H, Zhen TT, Shi HJ, Yang Y, Tang JM, Liang YJ, Han AJ. Retinoid X Receptor-¦Á
Expression Suppression and its Promoter Methylation Associates with ¦Â-catenin Expression in Colorectal Cancer. Journal of
Tumor 2014; 2(6): 153-160 Available from: URL:
http://www.ghrnet.org/index.php/JT/article/view/732
INTRODUCTION
Colorectal cancer
(CRC) is one of the most common malignancies worldwide. The retinoid X
receptors (RXRs) are nuclear receptors and are members of the superfamily of
ligand-inducible transcriptional regulatory factors that mediates the
anti-cancer function of retinoids (natural retinoic acids and their synthetic
derivatives). Retinoid X receptors have three different isotypes-¦Á, ¦Â and ¦Ã. RXR could heterodimerise with retinoid acid receptor
(RAR) and other receptors such as thyroid hormone receptors, vitamin D
receptors and peroxisome-proliferator-activated receptors[1-3]. RXR
expression associates with a variety of malignant tumors and prognosis of
patients with these tumors[3-9].
The Wnt/¦Â-catenin signaling pathway is tightly regulated and has
important functions in development, tissue homeostasis, and regeneration. Oncogenic
activation of the Wnt/¦Â-catenin signaling
pathway by mutation in adenomatous polyposis coli or ¦Â-catenin, which results in the cytoplasmic and nuclear ¦Â-catenin accumulation and in ¦Â-catenin/T-cell factor (TCF) 4-regulated transcription of
TCF target genes such as cyclin D1 and c-Myc, is mandatory for the initial
neoplastic transformation of intestinal epithelium[10,11].
Recently, instances of
crosstalk between retinoid signaling pathways and the Wnt/¦Â-catenin signaling
have been reported[12,13]. Our previous study showed that RXR¦Á directly interacted
with ¦Â-catenin and that RXR¦Á directly inhibited ¦Â-catenin transcriptional
activity and protein expression in CRC cell lines[14]. Further study
showed that suppression of RXR¦Á and aberrant ¦Â-catenin expression
significantly associated with progression of CRC[8]. The mechanism
underlying the impaired expression of RXR¦Á in CRC remains unclear. Aberrant
methylation of promoter CpG islands is known to be a major mechanism for
inactivation of tumor suppressor genes[15]. Lee et al[9]
have reported that methylation of RXR¦Á, RXR¦Â, and RXR¦Ã is 5.7%, 4.3%, and
23.7% in lung cancer, respectively. In this study, we first investigated the
promoter methylation status of RXR¦Á and correlated the results with ¦Â-catenin expression
and clinicopathological characteristics in large series of CRC.
MATERIALS
AND METHODS
Cell lines and cell culture
Human CRC cell lines
LoVo and SW116 were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA). HT29
was cultured in Dulbecco's modified Eagle's medium (Invitrogen). SW480 and
SW620 were grown in Leibovitz¡¯s L-15 Medium (Invitrogen). HCT116 was maintained
in McCoy¡¯s 5A Medium (Invitrogen). The human colonic epithelial cell line
NCM460 was cultured in RPMI-1640 medium. All medium was supplemented with 10%
(v/v) fetal bovine serum (GIBCO, Carlsbad, CA), 1¡Áantibiotic/antimycotic (100
units/mL streptomycin, 100 units/mL penicillin, and 0.25 mg/mL amphotericin B).
All cell lines were cultured in humidified incubator at 37¡æ with 5% CO2. Inhibitor of DNA
methyltransferase-5-Aza-2'-Deoxycytidine (Sigma, St. Louis, MO) was dissolved
in dimethyl sulfoxide (DMSO) and diluted in a series concentration.
Patient information and tissue
specimens
Eight pairs of fresh
CRC tissues and adjacent non-tumor colorectal mucosa (ANM) tissues, 202 pairs
of paraffin-embedded, archived CRC and ANM tissues and 75 samples of lymph node
metastatic CRC tissues between 2002 and 2008 were collected from Department of
Pathology, the first Affiliated Hospital, Sun Yat-sen University, Guangzhou,
China. No patients had received chemotherapy or radiotherapy before surgery.
The histopathology of the disease was determined by two pathologists according
to the criteria of the World Health Organization. Clinical staging was done
according to the Dukes¡¯ classification. For the research purposes of these
clinical materials, prior patient's consents and approval from the
Institutional Research Ethics Committee were obtained. Follow-up information
was available for all patients. Detailed clinical information about these
patients including sex, age, histological differentiation, Dukes¡¯ classification,
TNM stage, T classification, N classification, and distant metastasis status,
is summarized in table 1.
RNA extraction and Real-time polymerase
chain reaction (PCR)
Total RNA from CRC
cell lines, fresh CRC tissues, and ANM tissues was extracted using the Trizol
reagent (Invitrogen) according to the manufacturer¡¯s instruction. Real-time PCR
was done according to the manufacturer¡¯s protocol from TIANGEN BIOTECH
(Beijing, China) CO., LTD. The primer sequences used for RXR¦Á were followed: forward: 5¡¯ GCA AGC TGG TGT GTC ATC AGC
AAA; reverse: 5¡¯ ACA GAG GGC AGC TCA TGT TCT CAT. The primer sequences used for
¦Â-catenin were followed: forward: 5¡¯ TTG
AAA ATC CAG CGT GGA CA; reverse: 5¡¯ TCG AGT CAT TGC ATA CTG TC. The geometric
mean of housekeeping gene GAPDH was used to normalize the variability at
expression levels.
DNA extraction and methylation special
PCR
DNA was extracted
from CRC cell lines and tissues using DNA isolation kits (Qiagen, Hilden,
Germany). To investigate the methylation status of RXR¦Á, methylation special PCR with primers specific for the
methylated and unmethylated alleles of each gene after treatment of the genomic
DNA with sodium bisulfite using EpiTect Fast Bisulfite Conversion Kits (Qiagen)
was performed. Two CpG islands were found in the promoter sequence of RXR¦Á, Island 1, size: 352 bp (622-973); Island 2, size: 894 bp
(1053-1946) (http://www.urogene.org/), thus enabling us to design methylation
special PCR primer around the CpG island site with MethPrimer computer
program(http://www.urogene.org/methprimer/and Methyl Primer Express v1.0 ). The
methylated primer sequences for RXR¦Á were followed: forward: 5¡¯ GAT TTT TTA TCG GAA TAG GTT ATC G; reverse:
5¡¯ GCG TAT AAA CAA CCA TAA CAC GAC. The unmethylated primer sequences for RXR¦Á were followed: forward: 5¡¯ TGT TTT TTG TTG TTG GGG TAT
ATT; reverse: 5¡¯ CCA TTA TCT ACA CAA CCA CAC A.
Methylation sensitive-high resolution
melting (MS-HRM) curve analysis
The designed
sequences for RXR¦Á primers were:
forward 5¡¯-GGG TAT TTT TGT TTT AAG TGG AAT-3¡¯, Reverse: 5¡¯-CAT AAC ACG ACC CAA
AAA TAA CA-3¡¯. MS-HRM was carried out on Light Cycler480 (Roche). PCR was
performed in 20 ¦ÌL volume containing
2 ¦ÌL 10¡ÁPCR Buffer , 25 mM MgCl2,
0.5 ¦ÌL 10 mM dNTPs, 10 ¦Ìm of each primer, 1 ¦ÌL 20¡Á Eva-Green dye, 1U FastStart Taq DNA Polymerase, 1 ¦ÌL of bisulfite modified DNA template, 12.3 ¦ÌL purified water. The cycling condition used was as
follows: the initial denaturation at 95¡æ for 5 minutes, followed by 50 cycles of denaturation at 95¡æ for 30 seconds, primer annealing at 60¡æ for 15 seconds, and the extension step at 72¡æ for 25 seconds, followed by an HRM step at 95¡æ for 1 minute, 40¡æ for 1 minute, 65¡æ for 1 second and continuous acquisition to 95¡æ at 25 acquisitions per 1¡æ, followed by the final step 40¡æ for 30 seconds. Methylated and unmethylated genomic
templates were used to calibrate the quantitative measurements of methylation. Bisulfite
converted human control DNA including methylated and unmethylated (Qiagen) was
used as 100% methylated control DNA and unmethylated control DNA, respectively.
Methylation standards were constructed by diluting 100% methylated control DNA
in a pool of unmethylated control DNA at levels of 100%, 75%, 50%, 25%, 5%, and
0%. These standards were included in each experimental run. Each reaction was
performed in duplicate.
Western blot analysis
As described
previously[8], RXR¦Á antibody and ¦Â-catenin antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) were used to probe the alteration of
protein at 4¡æ for 12 hours. Signal was detected by
enhanced chemoluminescence techniques (Amersham Life Science, Piscataway, NJ). GAPDH
(Sigma, St. Louis, MO) was used as loading control.
Immunohistochemistry
The sections were
deparaffinized, rehydrated in serially graded ethanol, and heated in citric
buffer (pH 6.0) once for 5 minutes in a microwave oven for antigen retrieval. They
were then washed with distilled water, blocked with 3% hydrogen peroxide and
incubated with the primary antibodies including RXR¦Á (dilution 1:200) and ¦Â-catenin (dilution 1:200, Santa Cruz Biotechnology) at 4¡æ for 12 hours. After washing with a 0.01 mol ⁄ L
concentration of PBS, the sections were incubated with EnVision-HRP secondary
antibody (Dako, Carpinteria, CA) for 30 minutes at room temperature, washed
with a 0.01 mol/L concentration of PBS, stained with 0.5% diaminobenzidine and
counterstained with Mayer¡¯s haematoxylin, then air dried, and mounted with
glycerol gelatin.
Evaluation of immunohistochemistry
staining
Immunohistochemistry
staining was independently assessed by two researchers who were unaware of the
specific proteins being assessed or their putative role in CRC. The degree of
RXR¦Á immunostaining was based on both the
proportion of positively stained tumor cells and intensity of staining. The
proportion of positively stained tumor cells was scored as follows: 0 (no
positive tumor cells), 1 (<10% positive tumor cells), 2 (10-50% positive
tumor cells), and 3 (>50% positive tumor cells). Staining intensity was
classified according to the following criteria: 0 (no staining); 1 (weak
staining=light yellow), 2 (moderate staining=yellow brown), and 3 (strong
staining=brown). Staining index was calculated as the staining intensity
score¡Áthe proportion score. Using this method, we evaluated the expression of
RXR¦Á in colorectal specimen by determining
the staining index, with scores 0, 1, 2, 3, 4, 6, or 9. The staining index
score of 4 (a cutoff point) was used to distinguish between low and high
expression of RXR¦Á. The staining of ¦Â-catenin was scored according to Maruyama¡¯s method[16].
When more than 70% of carcinoma cells were positively stained for membranous ¦Â-catenin, the cells was classified as ¦Â-catenin normal expression; if more than 10% of carcinoma
cells were positively stained for cytoplasm or nuclei was regarded as ¦Â-catenin abnormal expression.
Statistical analysis
Chi-square test and
Fisher exact test were used to compare the levels of RXR¦Á and ¦Â-catenin expression
with both different groups and various clinicopathological parameters. Overall
survival (OS) time of CRC patients was analyzed by Kaplan-Meier curve and
log-rank test. The correlation of ¦Â-catenin and RXRa expression was analyzed by Spearman¡¯s correlation
coefficients. All statistical analyses were performed using SPSS 16.0
statistics software. P<0.05 was set to be statistically significant.
RESULTS
RXR¦Á and ¦Â-catenin
mRNA and protein expression in CRC cell lines and fresh tissues
As shown in Figure
1, RXR¦Á mRNA expression was
higher in CRC cell lines including SW116, HT29, HCT116, SW480, SW620, and LoVo
compared with the human colonic epithelial cell line NCM460 by real-time PCR
analysis. However, RXR¦Á protein expression
was lower in NCM460, SW620, and LoVo cells than that in other CRC cell lines
including SW116, HT29, HCT116, and SW480 by western blot analysis. Furthermore,
RXR¦Á protein expression was dramatically
lower in LoVo and SW620 than NCM460 cells. The mRNA and protein expression of ¦Â-catenin were higher in such 6 CRC cell lines compared with
NCM460 cells by real-time PCR and western blot analysis, respectively. In
addition, RXR¦Á mRNA expression was
dramatically lower in 8 samples of fresh CRC tissues than that in their
corresponding ANM tissues except one sample (case 4) by real-time PCR analysis
(Figure 1D).
Relationship between expression of RXR¦Á and ¦Â-catenin and
clinicopathological features of CRC
Of 202 samples of
CRC tissues, 128 samples (63.4%) were RXR¦Á low expression, 74 samples (36.6%) were RXR¦Á high expression. However, of 131 samples of ANM tissues,
only 53 samples (40.5%) were RXR¦Á low expression, 78 samples (59.5%) were RXR¦Á high expression. RXR¦Á low expression rate was significantly higher in CRC tissues than that
in ANM tissues (P<0.001, figure 2A-B). Furthermore, RXR¦Á low expression was significantly associated with
histological differentiation (P=0.047), Dukes¡¯ classification (P=0.003),
N classification (P=0004), and M classification (P=0.002). However,
no significant association of RXR¦Á expression with gender, age, and T classification (P>0.05)
was found. Kaplan-Meier analysis showed that CRC patients had a significantly
lower OS rate in RXR¦Á low expression
group compared with that in RXR¦Á high expression group (p=0.038, figure 2C). The OS time for
patients with RXR¦Á low expression was
57.19¡À3.93 months (range: 49.49-64.90 months), which was significantly shorter
than 67.19¡À2.73 months (range: 61.84-72.54 months) of OS time for patients with
RXR¦Á high expression, respectively. The OS rate
for patients with RXR¦Á low expression at 3
years and 5 years was 69.86% and 67.12%, respectively, compared with 79.53% and
74.16% for patients with RXR¦Á high expression at 3 years and 5 years, respectively. 181 samples
(89.6%, 181/202) were ¦Â-catenin abnormal
expression, 21 samples (36.6%, 21/202) were ¦Â-catenin normal expression. However, of 115 samples of ANM tissues,
only 8 samples (7.0%) were ¦Â-catenin abnormal expression, 107 samples (93.0%) were ¦Â-catenin normal expression. ¦Â-catenin abnormal expression was significantly higher in
CRC tissues than that in ANM tissues (P<0.001, figure 2D). Furthermore,
¦Â-catenin abnormal expression was
significantly associated with poor histological differentiation (P=0.002),
high Dukes¡¯ classification (P=0.009), T classification (P=0048),
and N classification (P=0.022). However, no significant association of ¦Â-catenin abnormal expression with gender, age, and M
classification (P> 0.05) was found (Table 1).
Of 202 samples of CRC
tissues, 110 samples were ¦Â-catenin abnormal expression in RXR¦Á low expression group, 71 samples were ¦Â-catenin abnormal expression in RXR¦Á high expression group. There was a significant correlation
between RXR¦Á low expression and ¦Â-catenin abnormal expression in CRC tissues (P=0.028,
table 2).
Promoter
methylation status of RXR¦Á in CRC.
The promoter methylation
status of RXR¦Á was first
determined in 6 CRC cell lines and normal colonic mucosa cell line NCM460 by
methylation special PCR analysis. As shown in figure 3A, unmethylated bands
were detected in all cell lines. Methylated bands were detected in HT29,
HCT116, SW480, SW620, and LoVo cells except SW116 and NCM460. Moreover,
methylated bands were stronger in SW620 and LoVo cells than those in other CRC
cell lines. In addition, methylated bands were found in 8 pairs of fresh CRC
and ANM tissues (Figure 3B). Further study showed that methylated bands were
found in all 13 samples (100%) of paraffin-embedded ANM tissues, 21 samples
(100%) of CRC tissues, and 8 samples (100%) of lymph node metastatic CRC
tissues.
To confirm the above
results, MS-HRM analysis was performed to detect methylation status and
methylation degree in such 13 samples of paraffin-embedded ANM tissues, 21
samples of CRC tissues, and 8 samples of lymph node metastatic CRC tissues. As
shown in figure 3C and table 3, the promoter methylation of RXR¦Á was detected in 10 samples (76.92%, 10/13) of ANM, 18
samples (85.71%, 18/21) of CRC, and 6 samples (75.0%, 6/8) of lymph node
metastatic CRC tissues, respectively. Furthermore, the mean methylation degree
was 5% (95% confidence interval: -0.57-10.57) in ANM, 16.57% (95% confidence
interval: 6.81-26.33) in CRC, and 26.13% (95% confidence interval: -0.54-52.79)
in lymph node metastatic CRC tissues. The mean methylation degree of RXR¦Á was significantly higher in CRC and lymph node metastatic
CRC tissues than that in ANM tissues, respectively (P=0.038 and P=0.035,
figure 3D). Of 18 samples of CRC with promoter methylation of RXR¦Á, 12 samples (66.6%) were RXR¦Á low expression by immunohistochemistry staining. There was
no significant association between promoter methylation of RXR¦Á and clinicopathological features of CRC (P>0.05).
Furthermore, MS-HRM analysis
was performed to detect methylation status and methylation degree in NCM460 and
CRC cell lines. The methylation degree of RXR¦Á was 0%, 0%, 25-50%, 5-25%, 25-50%, 50-75%, and 50% in
NCM460, SW116, HT29, HCT116, SW480, SW620, and LoVo cells, respectively. The
results showed that methylation degree of RXR¦Á was higher in SW620 and LoVo cells than that in other CRC
cells and NCM460. Then, methylation degree of RXR¦Á was decreased in SW620 and LoVo cells treated with
inhibitor of DNA methyltransferase -5-Aza-2'-Deoxycytidine at 1 ¦ÌM, 5 ¦ÌM, and 10 ¦ÌM for 1 day and 3 days compared with the control group
(Table 4).
As shown in figure 4, RXR¦Á mRNA and protein expression dramatically elevated in LoVo
cells treated with inhibitor of DNA methyltransferase -5-Aza-2'-Deoxycytidine
at 1 ¦ÌM, 5 ¦ÌM, and 10 ¦ÌM for 1 day and 3 days compared with the control group by real-time PCR
and western blot analysis, respectively. However, ¦Â-catenin mRNA and protein expression dramatically
suppressed in LoVo cells treated with 5-Aza-2'-Deoxycytidine at 1 ¦ÌM, 5 ¦ÌM, and 10 ¦ÌM for 1 day and 3 days compared with the control group by
real-time PCR and western blot analysis, respectively.
DISCUSSION
RXRs are nuclear
receptors for retinoids that play a critical role in the regulation of growth
and differentiation in normal and tumor cells[3,5-9,17]. Our
previous study showed that RXR¦Á directly inhibited ¦Â-catenin transcriptional activity and protein expression in CRC cell
lines[14]. Further study showed that suppression of RXR¦Á and aberrant ¦Â-catenin expression significantly associated with progression of CRC[8].
Our current results showed that RXR¦Á protein expression was lower in NCM460, SW620, and LoVo cells than
that in SW116, HT29, HCT116, and SW480 by western blot analysis. Furthermore,
RXR¦Á protein expression was dramatically
lower in LoVo and SW620 than NCM460 cells. Further study showed that RXR¦Á low expression rate was significantly higher in 202
samples of CRC tissues than that in ANM tissues. ¦Â-catenin abnormal expression was significantly higher in
CRC tissues than that in ANM tissues. There was a significant association
between RXR¦Á low expression and ¦Â-catenin abnormal expression in CRC tissues. To our
knowledge, our results first showed that CRC patients had a significantly lower
OS rate in RXR¦Á low expression
group compared with that in RXR¦Á high expression group.
Lee et al[9]
have reported promoter methylation of RXR¦Á is 5.7% in lung cancer. To further investigate the mechanism
underlying the impaired expression of RXR¦Á in CRC, we detected methylation status of RXR¦Á in CRC cell lines and tissues using methylation specific
PCR analysis. Our result showed that promoter methylation of RXR¦Á was detected in HT29, HCT116, SW480, SW620, and LoVo
except SW116 and NCM460 cells. Moreover, methylated bands were stronger in
SW620 and LoVo cells than those in other CRC cell lines. Moreover, methylation
degree of RXR¦Á was higher in SW620
and LoVo cells than that in other CRC cells and NCM460 by MS-HRM analysis. Further
study showed that methylation degree of RXR¦Á was decreased in SW620 and LoVo cells treated with inhibitor of DNA
methyltransferase -5-Aza-2'-Deoxycytidine. The result was consistent with the
findings that RXR¦Á protein expression
was dramatically lower in SW620 and LoVo than NCM460 and other CRC cells. Consistent
with our previous report[8], methylation of RXR¦Á was found in 8 pairs of fresh CRC and ANM tissues. Further
study showed that methylation of RXR¦Á was found in all 13 samples of paraffin-embedded ANM tissues, 21
samples of CRC tissues, and 8 samples of lymph node metastatic CRC tissues. To
confirm the above results, MS-HRM analysis showed that promoter methylation of
RXR¦Á was detected in 10 samples (76.92%,
10/13) of ANM, 18 samples (85.71%, 18/21) of CRC, and 6 samples (75.0%, 6/8) of
lymph node metastatic CRC tissues, respectively. Furthermore, the mean
methylation degree of RXR¦Á was significantly
higher in CRC and lymph node metastatic CRC tissues than that in ANM tissues,
respectively. The result was consistent with the findings that RXR¦Á protein expression was lower in CRC and lymph node
metastatic CRC tissues than that in ANM tissues by immunohistochemistry
staining. In addition, this finding suggests that MS-HRM analysis is much
better than methylation specific PCR for detecting methylation status of RXR¦Á in CRC. MS-HRM analysis is much stable, reliable, and
sensitive compared with methylation specific PCR[18]. Most
importantly, MS-HRM analysis could provide not only methylation status but also
methylation degree for CRC samples. Pande et al[5] have
reported single nucleotide polymorphisms in RXR¦Á may contribute to breast cancer disease-free survival
(DFS) and predict for poorer or improved DFS in patients according to type of
systemic treatment received. Lee et al[9] have reported RXR¦Ãmethylation is associated with a significantly worse
survival in lung cancer patients with never-smokers. However, no significant
association between promoter methylation of RXR¦Á and clinicopathological features of CRC was found in our
series. The reason might be related to the limited CRC samples analyzed by
MS-HRM analysis. This issue needs further study in large samples of CRC.
To further investigate
whether RXR¦Á and ¦Â-catenin expression altered in CRC cells treated with
inhibitor of DNA methyltransferase, our results showed that RXR¦Á and ¦Â-catenin mRNA and
protein expression dramatically elevated and suppressed in LoVo cells treated
with 5-Aza-2'-Deoxycytidine at different concentrations and times compared with
the control group, respectively. Volate et al[19] have
reported a low concentration of green tea significantly decreased CpG
methylation of RXR¦Á, increased RXR¦Á expression, and inhibit intestinal tumorigenesis in the
Apc(Min/+) mouse. Xiao et al[20] have reported green tea
polyphenols inhibit colorectal aberrant crypt foci formation and prevent
oncogenic changes in dysplastic ACF in azoxymethane-treated F344 rats partially
by preventing the loss of RXR¦Á expression. Targeting truncated RXR¦Á by a natural product molecule (CF31) induces TNF¦Á-dependent apoptosis in various cancer cell lines[21].
Whether inhibitor of DNA methyltransferase treatment has an effect on cell
biological functions in CRC cells with RXR¦Á promoter methylation needs further study.
In summary, our results
first demonstrate that RXR¦Á low
expression associates with poor prognosis of CRC patients. The promoter
methylation of RXR¦Á might be related to
RXR¦Á expression suppression in CRC. Our data
suggest that targeting RXR¦Á promoter methylation might be a novel therapeutic strategy for CRC patients.
ACKNOWLEDGMENTS
This study was supported by National
Natural Science Foundation of China (81201582; 81272636).
CONFLICT OF
INTERESTS
There are no
conflicts of interest with regard to the present study.
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Peer reviewer: Abdul-Wahed Meshikhes,
FRCSI, Consultant Surgeon, Department of Surgery, King Fahad Specialist
Hospital, Dammam 31444, Saudi Arabia.
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