Companion Diagnostics and Cancer Biomarkers
MALAT1 Is Associated with Poor Response to
Oxaliplatin-Based Chemotherapy in Colorectal
Cancer Patients and Promotes Chemoresistance
through EZH2
Peilong Li
1
, Xin Zhang
1
, Haiyan Wang
2
, Lili Wang
1
, Tong Liu
1
, Lutao Du
1
,
Yongmei Yang
1
, and Chuanxin Wang
3
Abstract
A major reason for oxaliplatin chemoresistance in colorectal
cancer is the acquisition of epithelial–mesenchymal transition
(EMT) in cancer cells. The long noncoding RNA (lncRNA),
MALAT1, is a highly conserved nuclear ncRNA and a key regulator
of metastasis development in several cancers. However, its role in
oxaliplatin-induced metastasis and chemoresistance is not well
known. In this study, we aim to investigate the prognostic and
therapeutic role of lncRNA MALAT1 in colorectal cancer patients
receiving oxaliplatin-based therapy and further explore the poten-
tial transcriptional regulation through interaction with EZH2
based on the established HT29 oxaliplatin-resistant cells. Our
results showed that high MALAT1 expression was associated
with reduced patient survival and poor response to oxaliplatin-
based chemotherapy in advanced colorectal cancer patients. Oxa-
liplatin-resistant colorectal cancer cells exhibited high MALAT1
expression and EMT. LncRNA MALAT1 knockdown enhances
E-cadherin expression and inhibits oxaliplatin-induced EMT in
colorectal cancer cells. EZH2 is highly expressed and associated
with the 3
0
end region of lncRNA MALAT1 in colorectal cancer,
and this association suppressed the expression of E-cadherin.
Furthermore, targeted inhibition of MALAT1 or EZH2 reversed
EMT and chemoresistance induced by oxaliplatin. Finally, the
interaction between lncRNA MALAT1 and miR-218 was observed,
which further indicated its prognostic value in patients who
received standard FOLFOX (oxaliplatin combine with 5-fluoro-
uracil and leucovorin) treatment. In conclusion, this study illu-
minates the prognostic role of lncRNA MALAT1 in colorectal
cancer patients receiving oxaliplatin-based treatment and further
demonstrates how lncRNA MALAT1 confers a chemoresistant
function in colorectal cancer. Thus, lncRNA MALAT1 may serve
as a promising prognostic and therapeutic target for colorectal
cancer patients.
Mol Cancer Ther; 16(4); 739–51. 2017 AACR.
Introduction
Colorectal cancer is a leading cause of cancer-related deaths in
the world. It is the second- and third-most commonly diagnosed
cancer in females and males, respectively, and more than 1.2
million patients are diagnosed with colorectal cancer every year
(1, 2). Currently, oxaliplatin-based chemotherapy after surgical
resection is one of the most frequently used therapeutic strategies
(3). Its use in combination with 5-fluorouracil (5-FU) and leu-
covorin (FOLFOX) has led to response rates >50% and median
survival approaching 2 years for metastatic colorectal cancer (4).
However, a large proportion of patients receiving chemotherapy
finally become metastatic and chemoresistant, and this has been a
key barrier to the efficacy of colorectal cancer treatment (5). A
major reason for colorectal cancer chemoresistance is the
enhanced invasion and metastasis of cancer cells, such as the
cell acquisition of epithelial–mesenchymal transition (EMT;
refs. 6, 7). Revealing the underlying mechanism and finding new
therapeutic and prognostic targets are necessary for developing
effective therapies for colorectal cancer patients.
Long noncoding RNAs (lncRNA) are most commonly defined
as the RNA transcripts of more than 200 nucleotides (nt) and
located in nuclear or cytosolic fractions with no protein-coding
capacity (8). Recent studies have demonstrated that lncRNAs play
important roles in carcinogenesis and cancer metastasis, and some
lncRNAs functioned as oncogenes, tumor-suppressor genes, or
both, depending on the circumstances (9). The lncRNA metasta-
sis-associated lung adenocarcinoma transcript 1 (MALAT1) was
first demonstrated by Ji and colleagues as an oncogene in non–
small cell lung cancer through the promotion of cell metastasis
and invasion (10). LncRNA MALAT1 can interact with Pc2 (Poly-
comb 2), a component of the Polycomb Repressive Complex 1
(PRC1). This interaction contr ols the re-localization of growth
control genes between polycomb bodies and interchromatin
granules (11). Subsequent studies reported that lncRNA MALAT1
expression was an independent prognostic parameter and had a
role in cell metastasis and EMT processes in bladder cancer, renal
cancer, gastric cancer, and colorectal cancer (12–15). Xu and
1
Department of Clinical Laboratory, Qilu Hospital, Shandong University, Jinan,
Shandong Province, China.
2
Department of Clinical Laboratory, Women &
Children's Hospital of Linyi, Linyi, Shandong Province, China.
3
Department of
Clinical Laboratory, The Second Hospital of Shandong University, Jinan, Shan-
dong Province, China.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corrected online January 7, 2021.
Corresponding Author: Chuanxin Wang, The Second Hospital of Shandong
University, No. 247 Beiyuan Street, Jinan 250000, China. Phone: 86-531-
82166801; Fax: 86-531-86927544; E-mail: cxwang@sdu.edu.cn
doi: 10.1158/1535-7163.MCT-16-0591
2017 American Association for Cancer Research.
Molecular
Cancer
Therapeutics
www.aacrjournals.org
739
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colleagues demonstrated that a motif of the 3
0
end MALAT1 gene
played an important role in the biological processes of human
colorectal malignancies (16). Despite the research advances of
lncRNA MALAT1, the role of lncRNA MALAT1 in colorectal cancer
development and chemoresistance is still poorly understood, and
new research to uncover the potential mechanism is urgently
needed.
EZH2, a critical component of polycomb repressive complex 2
(PRC2), functions as a histone H3 Lysine 27 (H3K27) methyl-
transferase in target gene promoters and inhibits specific gene
expression (17). EZH2 has frequently been found to be over-
expressed in a variety of human cancers (13, 18). In addition,
EZH2 silenced E-cadherin during the EMT processes of cancer cells
and gave rise to cancer progression (19). More importantly,
studies demonstrated that MALAT1 interacted with EZH2 and
facilitated its recruitment to gene promoter in renal and gastric
cancer (13, 14), but it was not reported in colorectal cancer. Thus,
the study on the regulatory mode between lncRNA MALAT1 and
EZH2 in colorectal cancer is a meaningful work.
In this work, we focused on the prognostic role of lncRNA
MALAT1 in colorectal cancer patients receiving oxalipla tin-
based chemoth erapy. On this basis, we investig ated the under-
lying function of lncRNA MALAT1 on oxaliplatin-induced EMT
and chemoresistance of colorectal cancer cells. Moreover, we
revealed the potential mechanism involving the miR-218 inter-
action and special E-cadherin sile ncing by EZH2-indu ced gene
methylation.
Materials and Methods
Clinical samples
For clinical parameter analysis, 68 colorectal cancer tissues and
paired adjacent noncancerous tissues were collected at Qilu
Hospital of Shandong University between 2008 and 2011. For
chemoresponse study, 221 serum samples and 48 primary tissues
were collected from the patients who received standard oxalipla-
tin-based chemotherapy at Qilu Hospital of Shandong University
between 2008 and 2011. To explore the interaction between
MALAT1 and miR-218, an independent set of 46 primary cancer
tissue samples from patients who received standard FOLFOX
chemotherapy were also collected at Qilu Hospital of Shandong
University between 2011 and 2014. All the patients were path-
ologically confirmed, and the clinical samples were collected
before chemotherapy was started. Patients were classified accord-
ing to the World Health Organization criteria and staged accord-
ing to the tumor–node–metastasis classification. Tumor response
was confirmed through CT and evaluated according to the RECIST
criteria as complete response (CR), partial response (PR), stable
disease (SD), and progressive disease (PD). Overall survival (OS)
was defined as the duration from inclusion to death of any cause,
and progression-free survival (PFS) was defined as the duration
from inclusion to PD or death of any cause. All patients received a
standard follow-up with CT of the abdomen after surgery. Written
informed consent was obtained from all patients according to the
guidelines approved by the Ethics Committee of Qilu Hospital,
Shandong University.
Cell culture
The human colorectal cancer cell lines HT29, SW480, SW620,
and human fetal normal colonic cell (FHC) were obtained from
the Type Culture Collection of the Chinese Academy of Sciences in
2014. All colorectal cancer cell lines were maintained in RPMI
1640 (Thermo Fisher Scientific) containing 10% FBS (Sigma-
Aldrich), 100 U/mL penicillin, and 100 g/mL streptomycin (Life
Technologies) at 37
Cin5%CO
2
and 95% air. Normal colon
FHC cells were grown in DMEM/F12 medium with 10% FBS,
10 ng/mL cholera toxin, 5 mg/mL transferrin, 5 mg/mL insulin,
100 ng/mL hydrocortisone, and an extra 10 mmol/L of 4-(2-
hydroxyethyl)-1-piperazine
€
ethanesulfonic acid at 37
Cin5%
CO
2
and 95% air. The cell authenticity was determined by short
tandem repeat analysis technology (Cell ID System, Promega).
Development of oxaliplatin-resistant colorectal cancer cell
lines
The HT29 oxaliplatin-resistant (OxR) colorectal cancer cells
were established as described in Supplementary Methods.
Total RNA and protein extraction
Total RNA and protein were extracted as described in Supple-
mentary Methods.
Quantitative real-time PCR and RT-qPCR directly applied in
serum
For colorectal cancer tissues and cell lines, the cDNA was
synthesized from 200 ng extracted total RNA using the Prime-
Script RT reagent Kit (Takara Bio Company) and amplified by RT-
qPCR with an SYBR Green kit (Takara Bio Company) on an ABI
PRISM 7500 Sequence Detection System (Applied Biosystems)
with the housekeeping gene GAPDH as an internal control. The
2
DDCt
method was used to determine the relative quantification
of gene expression levels. All the premier sequences were synthe-
sized by RiboBio, and their sequences are shown in Supplemen-
tary Table S1.
For cell-free serum MALAT1 detection, we used our previously
established RT-qPCR-D (RT-qPCR directly applied in serum)
method without RNA extraction (20). Briefly, we prepared the
2preparation buffer that contained 2.5% Tween 20 (EMD
Chemicals), 50 mmol/L Tris (Sigma-Aldrich), and 1 mmol/L
EDTA (Sigma-Aldrich) as Asaga and colleagues described (21).
First, 5 mL of serum were mixed with an equal volume of
2preparation buffer. Subsequently, the above mixture was
reverse transcribed (RT) in triplicates in a 20 mL reaction volume.
Finally, the RT product was diluted 10-fold and centrifuged at
16,000 g for 5 minutes, and a 5-mL supernatant solution was used
as the cDNA template for qPCR. The reagents and reaction
conditions were the same as those for RT-qPCR.
Knockdown of MALAT1 and EZH2 in colorectal cancer cells
Colorectal cancer cells were plated in a 6-well plate in RIPM
1640 supplemented with 10% FBS and cultured until 50% to 70%
confluent. SiRNAs were mixed with Lipofectamine 2000 (Invitro-
gen) in reduced serum medium (Opti-MEM, Gibco) according to
the manufacturer's instructions, and final concentration of siRNAs
was 100 nmol/L. Knockdown effect was examined by RT-qPCR
using RNA extracted 48 hours after transfection. The siRNA
sequences of lncRNA-MALAT1, EZH2, and negative control used
in this study are listed in Supplementary Table S1.
Cell migration and invasion assays
Cell migration and invasion were assessed with Boyden cham-
bers or modified Boyden chambers according to the manufac-
turer's protocol (Becton Dickinson Labware).
Li et al.
Mol Cancer Ther; 16(4) April 2017 Molecular Cancer Therapeutics740
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Cell viability assay
The cell viability assay was performed as described in Supple-
mentary Methods.
RNA immunoprecipitation
An RNA immunoprecipitation (RIP) experiment was per-
formed to investigate whether ribonucleoprotein complex con-
tained lncRNA MALAT1 and its potential binding protein (EZH2)
in colorectal cancer cells. The Magna RIP RNA-Binding Protein
Immunoprecipitation Kit (Millipore) was used according to the
manufacturer's instructions. The RNAs were immunoprecipitated
using anti-EZH2 (Cell Signaling Technology) antibody. Total
RNA and controls were also assayed to demonstrate that the
detected signals were from RNAs specifically binding to EZH2.
The final analysis was performed using RT-qPCR and shown as the
fold enrichment of MALAT1. The RIP RNA fraction Ct value was
normalized to the input RNA fraction Ct value. Primers are listed
in Supplementary Table S1.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using
the EZ ChIP Chromatin Immunoprecipitation Kit (Millipore)
according to the manufacturer's protocol. Briefly, cross-linked
chromatin was sonicated into 200 to 1,000 bp fragments. The
chromatin was immunoprecipitated using anti-EZH2 (Cell Sig-
naling Technology) and anti-H3K27me3 (Millipore) antibodies.
Normal human immunoglobulin G (IgG) was used as a negative
control. RT-qPCR was conducted to detect the relative enrichment
according to the method described above. Primers are listed in
Supplementary Table S1.
Immunofluorescence analysis
HT29 cells were grown to 40% to 50% con fluence and then
transfected with 100 nmol/L of si-MALAT1 or si-EZH2. After 48
hours of incubation, the cells were fixed with 4% paraformalde-
hyde and permeabilized in 0.2% Triton X-100 (Sigma-Aldrich) for
20 minutes. The cells were then blocked with 10% goat serum in
PBS for 1 hour. The cells were incubated with primary anti–E-
cadherin overnight at 4
C and then incubated with the appro-
priate rhodamine-conjugated secondary antibody for 1 hour. The
cells were then washed and incubated with DAPI (Invitrogen) for
nuclear staining. The slides were visualized for immunofluores-
cence with a laser scanning Olympus microscope.
Western blot and antibodies
The primary antibodies used for Western blotting were rabbit
anti-human EZH2 antibody (1:1,000; Cell Signaling Technolo-
gy), rabbit anti-human b-actin antibody (1:1,000; Cell Signaling
Technology), rabbit anti-human Vimentin antibody (1:1,000;
Santa Cruz Biotechnology), and rabbit anti-human E-cadherin
antibody (1:1,000; Santa Cruz Biotechnology). Horseradish per-
oxidase (HRP)–conjugated anti-rabbit antibodies (1:5,000; Santa
Cruz Biotechnology) were used as the secondary antibodies. A
total of 25 mg protein from each sample was separated on 10% Bis-
Tris polyacrylamide gel through electrophoresis and then blotted
onto polyvinylidene fluoride membranes (GE Healthcare). Then,
the membrane was blocked with 5% (5 g/100 mL) nonfat dry milk
(Bio-Rad) in tri-buffered saline plus Tween (TBS-T) buffer for 2
hours. Blots were immunostained with primary antibody at 4
C
overnight and with secondary antibody at room temperature for 1
hour. Immunoblots were visualized using Immobilon Western
Chemiluminescent HRP Substrate (Millipore). Protein levels were
normalized to b-actin.
Statistical analysis
For colorectal cancer versus normal cell lines, colorectal cancer
serum versus healthy control, and colorectal cancer tissue versus
adjacent nontumor tissue, differences were shown in mean
expression and were determined using the Student t test. The
survival curves of colorectal cancer patients were estimated via
the Kaplan–Meier method, and the difference in survival curves
was estimated using the log-rank testing. The correlation analysis
was evaluated by using the Spearman test. The Mann–Whitney
U test or Kruskal–Wallis test was used for evaluating the patho-
logic difference among clinical cohort groups. Count dates were
described as frequency and examined using the Fishe's exact test.
The results were considered statistically significant at P < 0.05.
Error bars in figures represent SD (standard deviation). Statistical
analyses were performed with GraphPad Prism (version 5.01)
software.
Results
MALAT1 is overexpressed in colorectal cancer patients and
correlated with tumor metastasis
RT-qPCR was used to detect MALAT1 expression levels in cell
lines and clinical tissues, normalized to GAPDH. All seven colo-
rectal cancer cell lines expressed higher MALAT1 than the normal
FHC cell line, especially in SW620 cells that originated from
metastasis colorectal cancer (Fig. 1A). The expression of MALAT1
was significantly increased in 68 colorectal cancer tissues com-
pared with paired noncancerous tissues (Fig. 1B). Moreover, the
colorectal cancer tissues in 58.8% (40 of 68) of cases had at least
2-fold higher expression of MALAT1 than paired noncancerous
tissues (Fig. 1C). The samples were also used and divided into
two groups using a median MALAT1 value of 0.398. We then
investigated the association of MALAT1 expression with clinical
parameters, including age, gender, differentiation, pathologic
tumor classification (pT), pathologic lymph node status (pN),
pathologic metastatic status (pM), and stage. The expression of
MALAT1 was significantly higher in pT (pT3þpT4), pN (þ), pM
(þ), and high stage (3þ4) patients compared with lower stage
(1þ2) patients (Supplementary Table S2), which indicated that
MALAT1 may play its oncogenic role primarily in colorectal cancer
with advancing progression, but not in the initial phase. More-
over, OS was significantly shorter in the high MALAT1 expression
group than in the low MALAT1 group (Fig. 1D).
High MALAT1 expression is associated with poor response to
first-line oxaliplatin treatment in colorectal cancer patients
We next sought to explore the association between MALAT1
and response to treatment in an independent set of 53 serum
samples from patients with metastatic colorectal cancer treated
with oxaliplatin by using the RT-qPCR-D method. Patients were
divided into responding (CR þ PR) and nonresponding (SD þ
PD) groups according to RECIST criteria. We found that the
MALAT1 expression level was much higher in patients who did
not respond to treatment than those who experienced response
to chemotherapy (Fig. 1E). This association between MALAT1 and
treatment response was further validated in another set of
48 primary tissue samples from patients receiving oxaliplatin-
based treatment (Fig. 1F).
MALAT1 and Colorectal Cancer Chemotherapy
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We then stratified patients i nto a l ow ( n ¼ 37) and a hi gh
(n ¼ 16) MALAT1 expressi on group wit h an estab lished cut- off
value (0.432) by us ing an ROC cu rve analysis to distinguish the
responding and nonresponding patients (Supplementary Fig.
S1). The di agnosti c sensitivit y and specificit y reached 89.29%
and 52% with the esta blished cut-offs, respectively . In the
validation g roup containing 168 ser um sa mples of colorectal
cancer patients, the p rop ortion of patients not responding to
chemotherapy was significantly higher in the high MALAT1
expression group than in the low expression group (Fig. 1G).
The diagnostic sensitivity and specificity were 75.0% (72/96)
and 61.1% (44/72), and the corresponding positive and neg-
ative predictive values w ere 0.72 (72/100) and 0.65 (4 4/68). In
addition, more patients experienced d istant metastasis in the
high MALAT1 expression group (38 vs. 16). Furthermore, the
Kaplan–Meier survival analysis indicated that high MALAT1
expression was associated with shorter OS and DFS in co lo-
rectal cancer patients (Fig. 1H).
MALAT1 negatively regulates E-cadherin expression in
colorectal cancer cells
Consistent with previo us study (3), our established HT29
OxR cells exhibit morphologic and molecular changes con-
sistent with EMT (shown in Supplementary Results and
Figure 1.
High MALAT1 expression is correlated with metastasis and associated with poor response to oxaliplatin treatment in colorectal cancer (CRC) patients.
A and B, MALAT1 expression was determined by RT-qPCR and normalized against GAPDH in normal colon FHC cells and 7 colorectal cancer cell lines
(A) and 68 paired colorectal cance r tissues and adjacent n oncancerous tissues (B). C, The MALAT1 expression level was analyzed using RT-qPCR
andexpressedaslog
2
fold change (colorectal cancer/normal), and the log
2
fold changes were presented as follows: >1, overexpression (40 cases);
<1, underexpression (12 cases); the remainder were defined as unchanged (16 cases). D, Colorectal cancer primary tissues were divided into a high
and a low MALAT1 expression group by using a median value of 0.398, and the asso ciation of MALAT1 expression with OS was analyzed with a Kaplan–Meier
plot. E and F, The expression of MALAT1 in responding and nonresponding groups was determined in the primary tissues (E)andserum(F)of
colorectal cancer patients (shown in 10%–90%). G, Fifty-three serum samples from colorectal cancer patients were divided into a low and high
MALAT1 expression group by establishing an ROC curve. Then, the proportion of patients that responded or did not respond to chemotherapy in the
high and low MALAT1 expression group was analyzed (P < 0.001, Fisher exact test). H, Kaplan–Meier curves for OS (left) and PFS (right) were drawn
accordingtoMALAT1expressionandwereanalyzedbyusingthelog-ranktest.
Li et al.
Mol Cancer Ther; 16(4) April 2017 Molecular Cancer Therapeutics742
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Supplementary Fig. S2). To define functional links between
MALAT1 and EMT, we examined the effects of MALAT1 knock-
down on E-cadherin expression. Firstly, we assayed the expression
level of MALAT1 and E-cadherin mRNA in colorectal cancer cells.
As shown in Fig. 2A, both MALAT1 and E-Cadherin are highly
expressed in both noninvasive and invasive colorectal cancer
lines, versus normal colon epithelial cells. More importantly, the
MALAT1 levels were relatively higher with concurrent low levels
of E-cadherin in the invasive cell lines (SW620, LoVo, and HT29
OxR cells) compared with those in the noninvasive cell lines
(HCT116, SW480, and HT29 parental cells). It should be pointed
out that significantly higher MALAT1 with lower E-cadherin
expression was found in HT29 OxR cells compared with HT29
parental cells (Fig. 2A, P < 0.05). A significant negative correlation
was also observed between the E-cadherin mRNA levels and
the MALAT1 expression levels in primary colorectal cancer tissues
(r ¼0.4792, P < 0.001; Fig. 2B). We then determined whether
MALAT1 regulates E-cadherin expression in colorectal cancer
cells. As shown in Fig. 2C, the knockdown effect was best using
si-MALAT1 (NO.3) compared with si-MALAT1 (NO.1) and si-
MALAT1 (NO.2). Thus, we choose si-MALAT1 (NO.3) for further
experiments. MALAT1 knockdown significantly increased E-cad-
herin expression at both the transcript and protein levels in
HT29 parental and OxR cells (Fig. 2D and E). More importantly,
MALAT1 knockdown also impaired the migratory and invasive
ability of both cell lines (Fig. 2F).
EZH2 is highly expressed and interacts with the 3
0
end region of
MALAT1 in colorectal cancer
Various reports have indicated that the interaction between
MALAT1 a nd EZH2 might be important for this study (13–14,
22). To verify the potential pathway, we first determined EZH2
expression in co lorectal cancer tissues. Results showed that
EZH2 mRNA expression was significantly higher in colorectal
cancer tiss ues compared with normal colon tissues ( Fig. 3A).
Then, we compared the mRNA expression levels for both
MALAT1 and EZH2 in colorectal cancer tissues and found a
significant positive correlation between the two groups (r ¼
0.6105, Fig. 3B). Furthermore, colorectal cancer patients were
divided into two groups using a median EZH2 val ue of 0.0115,
and the association analysis of EZH2 with clinicopathologic
parameters indicated that the number of patients expressing
high EZH2 was significantly larger in pT (pT3þpT4) and pN (þ)
patients compared with those in pT1þpT2 and pN ()patients
(Supplementary Table S3). OS was also significantly shorter in
the high EZH2 expression group compa red with the low EZH2
group (Fig. 3C).
Subsequently, RIP assay was performed with an antibody
against EZH2 from nuclear extracts of colorectal cancer cells. As
MALAT1 contains more than 8,000 bps, EZH2 protein can only
precipitate the MALAT1 fragments that are responsible for the
interaction. To achieve a more explicit understanding, 6 MALAT1
fragments (M1–M6), covering the whole sequence from 5
0
end to
3
0
end, were separately detected by RT-qPCR with an independent
set of primers (Fig. 3D and Supplementary Table S1). The RIP
results showed a significant enrichment of MALAT1 with EZH2
antibody compared with the nonspecific IgG antibody (Fig. 3E
and Supplementary Fig. S3). Moreover, the region containing the
nucleotides 7501 to 8708 at the 3
0
end of MALAT1 interacted most
strongly with EZH2 (Fig. 3E), but there was no enrichment of
b-actin or lncRNA control (Fig. 3F). These data indicate that
MALAT1 directly interacts with the EZH2, and EZH2 interacts
with the 3
0
end region of MALAT1.
MALAT1 represses E-cadherin expression by associating with
EZH2
To chec k whether E-cad herin express ion is controlle d by
EZH2, we analyzed the E-cadheri n express ion lev el after E ZH2
knockdown. EZH2 mRNA and protein expression levels were
sufficiently suppressed by si-EZH2 transfection i n colorectal
cancer cell s (Suppl ementary Fig. S4). EZH2 knockdown signif-
icantly increased E-ca dherin m RNA and protein le vels in colo-
rectal cancer cells (Fig. 4A and B). si-EZH2 significantly
impaired cell-migratory and -invas ive capacit y in both HT29
parental and OxR cell s (Fig. 4C). Moreover, the percentage of
decreased invasive cells on HT29 OxR cells w as 63.2%, which is
much higher than that of HT29 parental cells (43.9%, P < 0.05).
This may suggest tha t HT29 OxR cells are more sensit ive to
si-EZH2comparedwithHT29parentalcells.
To further address how MALAT1 is involved in EMT through the
enrichment of EZH2, we used ChIP analysis to determine the
effect of si-MALAT1 on histone modification in the E-Cadherin
gene promoter with an anti–H3K27-me3 antibody in SW620
cells. The histone-associated DNAs that were immunoprecipi-
tated with antibody against EZH2 and H3K27-me3 were individ-
ually amplified with primer sets covering the E-Cadherin gene
promoter regions (Fig. 4D). The binding level of EZH2 and
H3K27-me3 was significantly decreased by MALAT1 knockdown
compared with control cells (Fig. 4E and F), whereas binding of
IgG with E-cadherin promoter showed no significant change after
MALAT1 inhibition (Fig. 4G), suggesting that the interaction of
EZH2 and H3K27-me3 in the E-cadherin promoter is specifically
mediated by MALAT1.
MALAT1 knockdown inhibits oxaliplatin-induced EMT and
reverses oxaliplatin resistance through EZH2 in colorectal
cancer
Based on the above results, we attempted to demonstrate the
biological consequences of MALAT1 on oxaliplatin-induced EMT
in a more direct way. Firstly, we evaluated the effect of oxaliplatin
on E-cadherin levels in colorectal cancer cells. SW480 and SW620
cells were incubated without oxaliplatin or with a concentration
of 0.3 mmol/L oxaliplatin for 48 hours. As expected, E-cadherin
mRNA expression was significantly suppressed in colorectal can-
cer cells incubated with oxaliplatin compared with oxaliplatin-
free cells (Fig. 5A). E-cadherin protein levels were also down-
regulated after oxaliplatin treatment (Fig. 5B). At the same time,
the silencing oligonucleotides or negative control were transfected
into another independent group of SW480 and SW620 cells, and
then the cells were incubated with 0.3 mmol/L oxaliplatin for
48 hours. The functional role of the MALAT1-EZH2 pathway in
the oxaliplatin-induced EMT process was then examined. As
shown in Fig. 5C, the suppression of E-cadherin mRNA induced
by oxaliplatin was potently relieved by MALAT1 silencing com-
pared with negative controls. Similar results were also found when
EZH2 was silenced (Fig. 5D). MALAT1 and EZH2 silencing
reversed the oxaliplatin-induced expression change of E-cadherin
and Vimentin at protein levels (Fig. 5E). Moreover, the in situ
expression of E-cadherin was detected by immunofluorescence
in HT29 cells as described in Materials and Methods. Decreased
E-cadherin expression was observed after being treated with
oxaliplatin compared with oxaliplatin-free cells; however, this
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