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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 1, pp. 82–99, January 6, 2017
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

MEG3 Long Noncoding RNA Contributes to the Epigenetic
Regulation of Epithelial-Mesenchymal Transition in Lung
Cancer Cell Lines*□
S

Received for publication, July 28, 2016, and in revised form, November 2, 2016 Published, JBC Papers in Press, November 16, 2016, DOI 10.1074/jbc.M116.750950

Minoru Terashima, Shoichiro Tange, Akihiko Ishimura, and Takeshi Suzuki1
From the Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kanazawa 920-1192, Ishikawa, Japan
Edited by Joel Gottesfeld

The amino-terminal tail of histones can carry post-translational modifications such as methylation, acetylation, ubiquitination, and phosphorylation. These modifications regulate thestructure of chromatin and influence many cellular processes,
including DNA replication, DNA repair, transcription, and cell
cycle progression (1). In particular, the importance of lysine
methylation of histone H3 (Lys-4, Lys-9, Lys-27, and Lys-36) ishighlighted because of its specific dynamics with respect totranscriptional regulation (2, 3). Methylation of H3K4 andH3K36 is closely associated with active transcription, whereas

methylation of H3K27 and H3K9 is linked to transcriptional
repression. These modifications are controlled by two classes of
enzymes with opposing activities: histone lysine methyltransferases (KMTs)2 and lysine demethylases (KDMs). Deregulation of these enzymes has been reported to contribute tohuman diseases such as developmental disorders and many
types of cancer (2– 4).
Using retroviral insertional mutagenesis in mice, we have
tried to identify novel genes involved in cancer development.
As a result, we have isolated hundreds of candidate cancer
genes, which include many genes encoding KMTs and KDMs
(5, 6). We previously reported that KDM5B/PLU1 promoted
cell invasion and epithelial-mesenchymal transition (EMT) ofcancer cells (7, 8) and that DOT1L KMT enhanced cell migration and sphere formation potential of cancer cells (9). Thus our
experiments demonstrated that histone KMTs and KDMs were
implicated not only in the initiation but also in the malignant
progression of cancer.
Aggressive cancer progression is often accompanied with the
EMT program that is activated by extrinsic signals such astransforming growth factor-␤ (TGF-␤) (10, 11). EMT is a paradigm of cell plasticity characterized by the reversible changes
in epithelial and mesenchymal gene expression. Notably, transcriptional repression of CDH1/E-cadherin, a defining marker
of epithelial cells, is important for EMT. Moreover, the plastic
nature of EMT implies that epigenetic regulation such as histone modification, DNA methylation, and microRNA may be
involved (12, 13). Thus the epigenetic analysis of the genes
important for EMT has been carried out, and in particular, thefunctional connection between transcriptional repression of
CDH1/E-cadherin and histone methyl-modifying enzymes has
been investigated (8, 14, 15).
Previously, we showed that EED, a member of the Polycomb
repressive complex-2 (PRC2) that catalyzes H3K27 methylation, was required for TGF-␤-dependent repression of CDH1
and microRNA-200 (miR-200) family genes through histone
H3 methylation, suggesting an essential role of PRC2 in the
EMT of cancer cells (16). PRC2 is a complex of histone H3
methyltransferase essential for specific gene silencing during* This work was supported in part by Grants-in-aid for Scientific Research C
24590346 and 15K08263 (to T. S.) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan. The authors declare that they
have no conflicts of interest with the contents of this article.
□
S
This article contains supplemental Table S1 and Figs. S1 and S2.
1
To whom correspondence should be addressed. Tel.: 81-76-264-6741; Fax:
81-76-234-4502; E-mail: suzuki-t@staff.kanazawa-u.ac.jp.

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2

The abbreviations used are: KMT, lysine methyltransferase; KDM, lysine
demethylase; EMT, epithelial-mesenchymal transition; lncRNA, longnoncoding RNA; QRT-PCR, quantitative reverse transcription-PCR; RIP,
RNA immunoprecipitation; ChIRP, chromatin isolation by RNA purification; TRITC, tetramethylrhodamine isothiocyanate; KD, knockdown; iPS,
induced pluripotent stem.

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Histone methylation is implicated in a number of biologicaland pathological processes, including cancer development. In
this study, we investigated the molecular mechanism for the
recruitment of Polycomb repressive complex-2 (PRC2) and its
accessory component, JARID2, to chromatin, which regulates
methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could
interact with JARID2, was clearly increased during transforming growth factor-␤ (TGF-␤)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-␤-mediatedchanges in cell morphology and cell motility characteristicof EMT and counteracted TGF-␤-dependent changes in theexpression of EMT-related genes such as CDH1, ZEB family,
and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of
TGF-␤ in their expressions. Chromatin immunoprecipitation(ChIP) revealed that MEG3 regulated the recruitment of
JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3
could associate with JARID2 and the regulatory regions of target
genes to recruit the complex. This study demonstrated a crucialrole of MEG3 lncRNA in the epigenetic regulation of the EMT
process in lung cancer cells.

Epigenetic Regulation of EMT by MEG3 lncRNA

Results
Expression of MEG3 Long Noncoding RNA Was Transiently
Induced during TGF-␤-induced EMT—To find the long noncoding RNAs (lncRNAs) involved in TGF-␤-induced EMT of
JANUARY 6, 2017 • VOLUME 292 • NUMBER 1lung cancer cells, we have performed a candidate geneapproach based on the previous studies (32, 33). Because cells
undergoing EMT are thought to acquire stem cell-like properties (31), we picked up the candidate lncRNAs that werereported to be implicated in ES cells or iPS cells (32, 33). Thenwe examined the changes in the expression of these lncRNAs in
the cells after TGF-␤ treatment (Fig. 1, A and B). We used thetwo lung cancer cell lines A549 and LC-2/ad because they aregood model systems showing quick and clear changes for cellmorphology and EMT-related gene expression during EMT
caused by TGF-␤ (see Fig. 2 and supplemental Fig. S1 forthe phenotypes of LC-2/ad cells) (38). Quantitative RT-PCR(QRT-PCR) was carried out to detect the expression of theselncRNAs in A549 and LC-2/ad cells with or without TGF-␤
treatment, and the fold changes were presented. We found that
only MEG3 lncRNA was up-regulated by TGF-␤ in both A549
and LC-2/ad cells (Fig. 1, A and B). Then we examined theexpression changes of MEG3 in TGF-␤-induced EMT processof A549 and LC-2/ad cells (Fig. 1, C and D). The expression of
MEG3 was immediately and transiently induced by TGF-␤,suggesting its potential role in the induction of EMT. Therefore, we decided to focus on MEG3 lncRNA as a good candidatethat might function during TGF-␤-induced EMT.
Knockdown of MEG3 Inhibited the Changes in Cell Morphologies Induced by TGF-␤—To clarify the function of MEG3
lncRNA, we examined the effects of MEG3 knockdown in TGF␤-induced EMT. For knockdown of MEG3, we used two different shRNAs for MEG3 (MEG3 shRNA#1 and shRNA#2).
MEG3 expression was clearly down-regulated in A549 andLC-2/ad cells with the infection of retroviruses expressing each
MEG3 shRNA even in the presence of TGF-␤ (supplemental
Fig. S2, A and B). We showed the data of MEG3 shRNA#1 as arepresentative result (indicated as MEG3 KD) because we confirmed that both MEG3 shRNAs led to the same effects in theexpression of EMT-related genes in our EMT studies (Fig. 3, A
and C). In the presence of TGF-␤, control A549 cells showeddispersed and elongated cell phenotypes characteristic of EMT(Fig. 2A). MEG3 knockdown had little effect on cell shapes byitself, but it inhibited cell morphological changes mediated by
TGF-␤ (Fig. 2A). We next examined the expression of E-cadherin, an epithelial cell marker, by immunofluorescence assay.
Control A549 cells revealed heterogeneous E-cadherin staining, which had almost disappeared in TGF-␤-treated cells (Fig.
2B) (38). MEG3 knockdown increased E-cadherin staining on
the cell membrane compared with the control cells, which was
still observed after TGF-␤ treatment (Fig. 2B). These results
suggested that the epithelial cell characters might be enhanced
by MEG3 knockdown and maintained in the presence of
TGF-␤. Because actin reorganization is associated with EMT,
we observed the status of actin in the cells with TRITC-conjugated phalloidin staining (10). As shown in Fig. 2C, TGF-␤
treatment led to actin stress fiber formation, but in the MEG3
knockdown cells, we never detected actin fiber even after
TGF-␤ treatment.
We further examined the effects of MEG3 knockdown inanother cell line, LC-2/ad. After TGF-␤ treatment, control
LC-2/ad cells became flat and scattered, accompanied by loss of
E-cadherin staining and the appearance of actin fiber (Fig. 2,
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Downloaded from http://www.jbc.org/ at KANAZAWA UNIV on January 10, 2017cancer and development (17–19). PRC2 consists of four core
subunits, SUZ12, EED, RBBP4/7, and the catalytic EZH2.
Proper recruitment of PRC2 to the target genes plays a crucialrole in the epigenetic regulation of gene expression. JARID2, aninteracting component of PRC2, has been reported as one of theessential factors for proper PRC2 recruitment in embryonicstem (ES) cells (20 –23). We also found that JARID2 was indispensable for TGF-␤-induced EMT of lung and colon cancer celllines (24). Our ChIP experiments indicated that JARID2 mightcause TGF-␤-dependent repression of CDH1 and miR-200
family genes through EZH2 recruitment and H3K27 methylation on their regulatory regions. However, in the absence of
TGF-␤, JARID2 showed little effect on the levels of EZH2 occupancies and H3 methylation on these regions. Based on theseresults, we hypothesized that some additional factors and/or
signals induced by TGF-␤ would be required for JARID2 function (24).
Long noncoding RNAs (lncRNAs) have been recognized asimportant regulatory factors in various cellular processes suchas cell proliferation, differentiation, and establishment of cell
identity (25). Expression of lncRNAs reveals highly developmental stage- or cell type-specific patterns and is frequently
deregulated in cancer (26 –28). Expression of lncRNAs reveals
highly developmental stage- or cell type-specific patterns and isfrequently deregulated in cancer (26 –28). Functions oflncRNAs are largely unknown, but some lncRNAs were shownto interact with transcription factors and chromatin regulators
to fine-tune the expression of specific genes (25). PRC2 is one of
the most studied examples of chromatin-modifying factors that
could be recruited and regulated by lncRNAs such as HOTAIRand RepA (29, 30). Thus we hypothesized that lncRNAs might
be involved in the regulation of PRC2 and JARID2 during the
EMT process. Because cells undergoing EMT are proposed toacquire stem cell-like properties (31), we focused on lncRNAs
that were shown to be implicated in ES cells or induced pluripotent stem (iPS) cells (32, 33). Among them, MEG3 lncRNA
was identified as a good candidate that might function in the
TGF-␤-induced EMT process based on its expression pattern(see Fig. 1, A and B). Moreover, during this study, it has beenreported that JARID2 could bind to lncRNAs in mouse ES cells
and that one of the candidates, Meg3, was implicated in PRC2
recruitment at a subset of target genes in pluripotent stem cells(34, 35). Therefore, we tried to examine functional interaction
between MEG3, JARID2, and PRC2 during the EMT process ofcancer cells.
In this study we found that MEG3 lncRNA was essential for
the TGF-␤-induced EMT process in A549 and LC-2/ad lungcancer cell lines. The gene expression program during EMT
was disturbed by MEG3 knockdown and potentiated by MEG3
overexpression. MEG3 was directly involved in the epigenetic
regulation of several EMT-related genes through the recruitment of JARID2 and EZH2 to the chromatin for histone H3
methylation.

Epigenetic Regulation of EMT by MEG3 lncRNA

D–F). MEG3 knockdown strengthened E-cadherin staining
and antagonized the TGF-␤-induced phenotypes observed in
the control cells (Fig. 2, D–F). Taken together, these results
indicated that MEG3 knockdown antagonized TGF-␤-induced
EMT phenotypes of A549 and LC-2/ad lung cancer cells such asmorphological changes and cytoskeletal rearrangements.
To see the effects of MEG3 knockdown in EMT-associatedphenotypes, we performed the transfilter migration assay using
Boyden modified chambers to measure cell migration activities.
As shown in Fig. 2, G and H, the treatment of TGF-␤ significantly increased the number of migrated A549 cells per field.
MEG3 knockdown had little effect on cell migration by itself
but inhibited the TGF-␤-dependent increase of migrated cells(Fig. 2, G and H). These results suggested that MEG3 knockdown inhibited TGF-␤-induced EMT and also EMT-associatedcell migration activity of lung cancer cells.
MEG3 Knockdown Antagonized the Changes in the Expression of EMT-related Genes Caused by TGF-␤—EMT is associated with the changes in the expression of epithelial and mes-

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enchymal marker genes (10). First, we examined the expression
level of an epithelial cell marker, CDH1/E-cadherin, and mesenchymal markers, FN1/fibronectin and vimentin in the cellswith MEG3 knockdown. QRT-PCR revealed that MEG3 knockdown increased CDH1 expression and inhibited transcriptional
repression of CDH1 induced by TGF-␤ in A549 and LC-2/ad
cells (Fig. 3, A and C), which was consistent with the results ofE-cadherin staining (Fig. 2, B and E). In the cases of FN1/fibronectin and vimentin, MEG3 knockdown also counteracted
the effect of TGF-␤ (Fig. 3, A and C). These findings suggested
that MEG3 knockdown might inhibit the EMT-inducing gene
expression program in A549 and LC-2/ad cells.
We further analyzed the expression of ZEB family transcription factors, important regulators of E-cadherin repression(39), and the miR-200 family of microRNAs, specific inhibitorsof the ZEB family (40, 41), in the MEG3 knockdown cells. In
A549 and LC-2/ad cells, TGF-␤ up-regulated the expression of
ZEB1 and ZEB2 and decreased the expression of two representative miRNAs, miR-200a and miR-200c (Fig. 3, A and C).
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FIGURE 1. Expression of long noncoding RNAs during TGF-␤-induced EMT of A549 and LC-2/ad lung cancer cells. A and B, QRT-PCR analysis was
performed to detect the expression of various lncRNAs, which were reported to be implicated in ES cells or iPS cells, in A549 cells (A), and LC-2/ad (B) cells before
and after the treatment of 1 ng/ml TGF-␤ (24 h). Fold changes were calculated and presented. n.d. means not detected (*, p ⬍ 0.01 compared with control; **,p ⬍ 0.05 compared with control). C and D, QRT-PCR was performed to detect the expression of MEG3 lncRNA in A549 cells (C) and LC-2/ad (D) cells before andafter the treatment of 1 ng/ml TGF-␤ (6, 12, 24, 48, and 72 h) (*, p ⬍ 0.01 compared with control).

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FIGURE 2. Knockdown of MEG3 antagonized TGF-␤-induced morphological changes of A549 and LC-2/ad cells and migratory activities of A549 cells.
A and D, cell morphological changes of A549 (A) and LC-2/ad (D) cells after TGF-␤ treatment. A549 or LC-2/ad cells were infected with retroviruses expressing
control shRNA or MEG3 shRNA#1 (described as MEG3 KD) without or with the treatment of 1 ng/ml TGF-␤ for 6 days. Cells were stained with 0.4% crystal violet.
Scale bars, 20 ␮m. B and E, immunofluorescence images of cells showing the localization of E-cadherin. The cells were treated without or with TGF-␤ for 48 h.
The panels of A549 (B) or LC-2/ad (E) cells with the same arrangement with A or D were stained with anti-E-cadherin antibody and DAPI. Scale bars, 10 ␮m. C andF, fluorescence images of A549 (C) and LC-2/ad (F) cells showing reorganization of actin cytoskeleton by staining with TRITC-phalloidin (Actin) and DAPI. The
cells were treated without or with TGF-␤ for 48 h. Scale bars, 10 ␮m. G and H, MEG3 KD inhibited TGF-␤-induced increase of migrated cells through the filter.
Cells that migrated through the filter to the lower surface within 24 h were fixed and stained (G). The number of migrated cells was counted under a lightmicroscope from at least five fields and three experiments, and the averages were calculated (H) (*, p ⬍ 0.01). Scale bars, 60 ␮m.

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FIGURE 3. Knockdown of MEG3 influenced the TGF-␤-dependent changes in the expression of EMT-related genes in A549 or LC-2/ad cells. A and C,
QRT-PCR analysis was performed to detect the expression of CDH1/E-cadherin, FN1/fibronectin, vimentin, ZEB1, ZEB2, miR-200a, miR-200c, JARID2, PMEPA1,
SMAD7, ID1, and ID2 in A549 (A) and LC-2/ad (C) cells infected with retroviruses expressing control shRNA or each MEG3 shRNA (MEG3 sh#1 and sh#2) with orwithout the treatment of 1 ng/ml TGF-␤ for 24 h (*, p ⬍ 0.01 compared with control; **, p ⬍ 0.05 compared with control; ***, p ⬍ 0.01 compared with the sample
without TGF-␤ treatment; ****, p ⬍ 0.05 compared with the sample without TGF-␤ treatment). B and D, immunoblotting was performed to detect theexpression of E-cadherin, fibronectin, vimentin, ZEB1, ZEB2, phosphorylated SMAD3 (p-SMAD3), and GAPDH proteins using the corresponding antibodies in
A549 (B) and LC-2/ad (D) cells. WB, Western blotting.

MEG3 knockdown itself slightly but significantly reduced theexpression of ZEB1 and ZEB2 and increased the expression of
miR-200a and miR-200c (Fig. 3, A and C). In the presence of
TGF-␤, MEG3 knockdown antagonized the TGF-␤-dependentchanges in their expressions (Fig. 3, A and C), confirming its
inhibitory effects. We also examined the expression of JARID2in the MEG3 knockdown cells, because JARID2 was induced by
TGF-␤ and implicated in EMT (24). MEG3 knockdown did nothave a significant effect on JARID2 expression and JARID2

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induction by TGF-␤ in A549 and LC-2/ad cells (Fig. 3, A and C).
JARID2 induction seemed to decrease slightly in the MEG3
knockdown cells compared with the TGF-␤-treated control
cells, but the decrease was not statistically significant (Fig. 3, A
and C). To examine the specificity of MEG3 knockdown effects
in gene regulation, we picked up several of the other known
TGF-␤-responsive genes (42, 43) and analyzed the changes inthe expression of these genes. In A549 and LC-2/ad cells,
TGF-␤ treatment increased the expression of TMEPA1 andVOLUME 292 • NUMBER 1 • JANUARY 6, 2017

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any changes in EZH2 signals and H3K27 and H3K4 methylationon the region of the GAPDH gene with MEG3 knockdown (Fig.
4, D and H). These results revealed that MEG3-mediated epigenetic changes would be specific to the target genes controlled
by MEG3 and TGF-␤, which was consistent with the specificityof transcriptional regulation.
Overexpression of MEG3 Partially Influenced the Gene
Expression Program in EMT—To deepen our understanding for
MEG3 function in EMT regulation, we investigated the effects
of MEG3 overexpression in A549 and LC-2/ad cells. First, weobserved the cell morphologies and the status of E-cadherin
and actin (Fig. 5). The cells with MEG3 overexpression lookedslightly scattered compared with the control cells, but the effectwas not significant (Fig. 5, A and D). More importantly, MEG3
overexpression itself could reduce E-cadherin staining of thecells but not induce actin stress fiber formation (Fig. 5, B, C, E,
and F). In the presence of TGF-␤, we confirmed morphologicalchanges and cytoskeletal rearrangements of the cells associatedwith EMT (Fig. 5). These results suggested that MEG3 overexpression itself could down-regulate the expression of E-cadherin but might not proceed with the EMT process completely.
Next, we analyzed the expression of EMT-related genes in
A549 and LC-2/ad cells with MEG3 overexpression. MEG3
overexpression caused significant changes on the expression of
CDH1/E-cadherin, ZEB1, ZEB2, miR-200a, and miR-200c but
had no effect on the expression of FN1/fibronectin, vimentin,
and JARID2 (Fig. 6, A and C). The expression of CDH1, miR200a, and miR-200c was repressed, and the expression of ZEB1and ZEB2 was significantly up-regulated in the MEG3-overexpressing cells. In the presence of TGF-␤, MEG3 overexpression
potentiated the effects of TGF-␤ in the expression of CDH1/Ecadherin, ZEB1, ZEB2, miR-200a, and miR-200c but not significantly in the expression of FN1/fibronectin, vimentin, and
JARID2 (Fig. 6, A and C). We also confirmed the effects
of MEG3 overexpression in protein expression for several of
the EMT-related genes. MEG3 reduced the expression ofE-cadherin protein, increased ZEB1 and ZEB2 proteins, andenhanced the effects of TGF-␤ in their expressions (Fig. 6, B
and D). These results were consistent with the QRT-PCR
results (Fig. 6, A and C) and indicated that MEG3 overexpression itself partially influenced the gene expression program
during the EMT process. This partial effect in gene expression
might be related to the observed cell phenotypes caused by
MEG3 overexpression (Fig. 5).
We also examined the status of histone H3 methylation and
EZH2 occupancies on the regulatory regions of CDH1, miR200b/200a/429, and miR-200c/141 genes in A549 and LC-2/ad
cells with MEG3 overexpression. In these regions, MEG3 overexpression caused the significant increase of H3K27me3 andEZH2 recruitment and the decrease of H3K4me3 comparedwith the control cells (Fig. 7, A–C and E–G), which was correlated with the decrease of their expressions (Fig. 6). MEG3 overexpression also potentiated the effects of TGF-␤ in the EZH2
occupancies and histone H3 methylation on the target genes(Fig. 7, A–C and E–G). Again, we did not detect any changes in
EZH2 signals and H3 methylation on the regulatory region of
unrelated GAPDH gene by MEG3 overexpression (Fig. 7, D and
H). These results suggested that MEG3 lncRNA could mediate
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SMAD7 and down-regulated the expression of ID1 and ID2(Fig. 3, A and C). MEG3 knockdown did not have a significant
effect in the expression of these genes and their expression
changes induced by TGF-␤. These results suggested that MEG3
was not involved in the expression of all of the TGF-␤-responsive target genes, but might regulate a subset of genes, including
some essential genes for EMT.
Next, we confirmed the changes in protein expression forseveral of the EMT-related gene products in A549 and LC-2/ad
cells. The TGF-␤-dependent decrease of E-cadherin protein
and the increase of fibronectin, vimentin, ZEB1, and ZEB2 proteins were inhibited by MEG3 knockdown (Fig. 3, B and D),
which was consistent with the QRT-PCR results (Fig. 3, A and
C). To see whether MEG3 knockdown would influence the
TGF-␤ signaling pathway in the cells or not, we analyzed thestatus of the phosphorylated SMAD3 protein (11). The phosphorylated SMAD3 proteins were detected after TGF-␤ treatment, and their levels were similar in the control cells and the
MEG3 knockdown cells (Fig. 3B), indicating that MEG3 knockdown would not impair the activation of SMAD3 transcription
factor by TGF-␤. These results suggested that MEG3 might not
influence TGF-␤-induced activation of downstream transcription factors but could contribute to TGF-␤-dependent transcriptional regulation of EMT-related genes in lung cancer
cells.
MEG3 Is Involved in the Recruitment of EZH2 and Histone H3
Methylation on the Regulatory Regions of CDH1 and miR-200
Family Genes—MEG3 lncRNA has been reported to interact
with JARID2 protein in mouse ES cells (34). JARID2 is a cofactor of PRC2 enzyme that controls histone H3K27 methylationand transcriptional repression in ES cells (20 –23). Thus weperformed chromatin immunoprecipitation (ChIP) to analyze
the status of histone H3 methylation on the regulatory regionsof CDH1 and miR-200 family genes, whose expressions wererepressed in TGF-␤-induced EMT. After immunoprecipitation, quantitative PCR was carried out using the primer pairs
located around the transcription start sites of the CDH1 geneand two microRNA clusters as described previously (8, 44).
After TGF-␤ treatment, we could observe the significant
increase of tri-methylated H3K27 (H3K27me3), the enhanced
recruitment of EZH2, a catalytic subunit of PRC2, and the substantial decrease of transcriptionally active H3K4me3 marks on
the regulatory regions of CDH1, miR-200b/200a/429, and miR200c/141 genes in A549 and LC-2/ad cells (Fig. 4, A–C and
E–G). These results strongly suggested that TGF-␤-induced
EZH2 recruitment on these regulatory regions might beresponsible for the transcriptional repression. Knockdown of
MEG3 caused the slight decrease of H3K27me3 and EZH2 signals on these regions compared with the control cells (Fig. 4,
A–C and E–G), which was correlated with the increase of theirexpressions (Fig. 3). In addition, MEG3 knockdown counteracted TGF-␤-mediated changes of histone methylation and
EZH2 recruitment (Fig. 4, A–C and E–G). These findings suggested that endogenous MEG3 lncRNA was involved in EZH2
recruitment and histone H3 methylation on the specific target
genes during the TGF-␤-induced EMT process. Next, the ChIP
assays on the regulatory region of the unrelated GAPDH gene
were performed as a control experiment. We could not observeEpigenetic Regulation of EMT by MEG3 lncRNA

EZH2 recruitment and histone H3 methylation on the specifictarget loci such as CDH1 and miR-200 family genes for transcriptional repression, which might be a critical step for the
EMT process.
Effects of MEG3 Overexpression in EMT Were Cancelled by
JARID2 Knockdown—To understand the mechanism of how
MEG3 regulates the expression of the target EMT-related
genes, we examined the requirement of JARID2 in the processby the JARID2 knockdown experiment. A549 and LC2/ad cellswith or without MEG3 overexpression were infected with thecontrol retrovirus or JARID2 shRNA-expressing retrovirus.
Then we analyzed the expression of EMT-related genes with orwithout TGF-␤ treatment (Fig. 8, A and B). In the case of
CDH1/E-cadherin, JARID2 knockdown itself had no effect on
its expression but inhibited the repression of CDH1 induced by

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TGF-␤ as reported previously (Fig. 8, A and B) (24). Moreover,
JARID2 knockdown inhibited MEG3-induced repression of
CDH1 in the absence or presence of TGF-␤ (Fig. 8, A and B).
Similarly, JARID2 knockdown cancelled the effects of MEG3
overexpression and/or TGF-␤ in the expression of ZEB1, ZEB2,
miR-200a, and miR-200c genes (Fig. 8, A and B). For the mesenchymal markers, FN1/fibronectin and vimentin, JARID2
knockdown inhibited only the effect of TGF-␤, because MEG3
originally had little effect on them (Fig. 6). These results indicated that JARID2 knockdown prevented the ability of overexpressed MEG3 to regulate a subset of EMT-related genes.
Therefore, JARID2 was suggested to be an essential factor for
MEG3 function in EMT-related gene expression.
We also examined the effect of JARID2 knockdown in histone methylation and EZH2 occupancies on the regulatory
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FIGURE 4. Knockdown of MEG3 affected the TGF-␤-induced regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of
CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with retroviruses expressing control shRNA or MEG3shRNA#1 without or with TGF-␤ treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 (A and E), miR-200b/200a/429(B and F), miR-200c/141 (C and G), and GAPDH genes (D and H) in A549 (A–D) or LC-2/ad (E–H) cells are shown. The occupancies of methylated histones or EZH2
protein on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (*, p ⬍ 0.01 compared with
control; **, p ⬍ 0.05 compared with control).

Epigenetic Regulation of EMT by MEG3 lncRNA

regions of CDH1, miR-200b/200a/429, and miR-200c/141
genes in the cells with MEG3 overexpression. On these regions,
JARID2 knockdown counteracted the TGF-␤-mediated increase of H3K27me3 and EZH2 recruitment and the decreaseof H3K4me3 as shown previously (Fig. 9, A and B) (24). In addition, JARID2 knockdown inhibited MEG3-induced changes inthe EZH2 occupancies and histone H3 methylation on the target genes (Fig. 9, A and B). These epigenetic changes caused by
JARID2 knockdown were well correlated with the changes inthe expression of these genes. Again, we did not detect any
changes in EZH2 signals and H3 methylation on the regulatory region of the unrelated GAPDH gene (Fig. 9, D and H).
These results suggested that JARID2 was required for MEG3
lncRNA to induce EZH2 recruitment on the specific target
loci, thereby regulating histone H3 methylation for transcriptional repression.
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Overexpression of MEG3 Enhanced the Recruitment of
JARID2 and EZH2 on the Regulatory Regions of CDH1 and miR200 Family Genes—As the mechanism for the epigenetic regulation of MEG3, it was suggested that MEG3 could bind to
JARID2 and recruit JARID2 and EZH2 to the specific target
regions for histone methylation. However, in the above ChIP
experiments, the recruitment of endogenous JARID2 protein
was not detected on the regulatory regions of CDH1, miR-200b/
200a/429, and miR-200c/141 genes with the anti-JARID2 antibody used in this study, possibly because of its low reactivity for
immunoprecipitation. Thus we decided to examine the recruitment of JARID2 on the target genes when JARID2 was overexpressed in the cells. We first tried to confirm the interaction of
MEG3 and JARID2, which was reported in mouse ES cells (34),by RNA immunoprecipitation (RIP) assay. A549 cells were
infected with the various combinations of the retroviruses
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FIGURE 5. Overexpression of MEG3 reduced the expression of E-cadherin but did not induce the reorganization of actin cytoskeleton of A549 andLC-2/ad cells. A and D, cell morphological changes of A549 (A) or LC-2/ad (D) cells after TGF-␤ treatment. The cells were infected with the control retrovirus or
the retrovirus expressing MEG3 without or with the treatment of 1 ng/ml TGF-␤ for 6 days and were stained with 0.4% crystal violet. Scale bars, 20 ␮m. B and E,
immunofluorescence images of cells showing the localization of E-cadherin. The panels of A549 (B) or LC-2/ad (E) cells were stained with anti-E-cadherin
antibody and DAPI. The cells were treated without or with TGF-␤ for 48 h. Scale bars, 10 ␮m. C and F, fluorescence images of A549 (C) and LC-2/ad (F) cells
showing reorganization of actin cytoskeleton by staining with TRITC-phalloidin (Actin) and DAPI. Scale bars, 10 ␮m.

Epigenetic Regulation of EMT by MEG3 lncRNA

expressing MEG3 and FLAG-tagged JARID2, and the crosslinked cell lysates were prepared. The cell lysates were immunoprecipitated with control antibody (mouse IgG) or antiFLAG antibody, and the co-precipitated RNA was quantified by
QRT-PCR (Fig. 10A). The amplified signal for MEG3 lncRNA
could be detected only when both MEG3 and JARID2 were
overexpressed in the cells, and the cell lysate was immunoprecipitated with anti-FLAG antibody (Fig. 10A), confirming thespecific interaction of overexpressed MEG3 and JARID2. We
did not detect the signals derived from endogenous MEG3
lncRNA possibly due to the sensitivity of this assay (Fig. 10A).
Then we carried out ChIP analysis to see the effect of MEG3
overexpression in the recruitment of FLAG-tagged JARID2 on
the regulatory regions of CDH1, miR-200b/200a/429, and miR200c/141 genes (Fig. 10, B–D). As we reported previously (24),90 JOURNAL OF BIOLOGICAL CHEMISTRY

JARID2 overexpression itself did not alter H3K27 methylation,
EZH2, and FLAG-tagged JARID2 recruitment on these regions
compared with the control cells, but co-treatment of TGF-␤
enhanced the levels of methylation and their recruitment (Fig.
10, B–D). MEG3 overexpression itself caused the significant
increase of H3K27me3 and EZH2 recruitment on these regions
compared with the control cells similarly as shown in Fig. 7 (Fig.
10, B–D). Overexpression of both JARID2 and MEG3 caused
the significant increase of H3K27me3 and the occupancies of
EZH2 and FLAG-tagged JARID2 on these regions comparedwith JARID2-overexpressing cells and MEG3-overexpressing
cells (Fig. 10, B–D). Again, we did not observe any changes in
H3K27me3, EZH2, and JARID2 on the regulatory region of theunrelated GAPDH gene (Fig. 10E). These results indicated that
MEG3 overexpression could increase the recruitment of
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FIGURE 6. Overexpression of MEG3 influenced the expression of part of EMT-related genes in A549 and LC-2/ad cells. QRT-PCR analysis was performed
to detect the expression of CDH1/E-cadherin, FN1/fibronectin, vimentin, ZEB1, ZEB2, miR-200a, miR-200c, and JARID2 in A549 (A) and LC-2/ad (C) cells infectedwith the control retrovirus or the retrovirus expressing MEG3 with or without treatment of TGF-␤ for 24 h (*, p ⬍ 0.01 compared with control; **, p ⬍ 0.05
compared with control; ***, p ⬍ 0.01 compared with the sample without TGF-␤ treatment; ****, p ⬍ 0.05 compared with the sample without TGF-␤ treatment).
B and D, immunoblotting was performed to detect the expression of E-cadherin, fibronectin, vimentin, ZEB1, ZEB2, and GAPDH proteins using the corresponding antibodies in A549 (B) and LC-2/ad (D) cells.

Epigenetic Regulation of EMT by MEG3 lncRNA

JARID2 and EZH2 on the specific target regions for H3K27
methylation and that JARID2 overexpression also could
enhance MEG3-induced EZH2 recruitment and histone
methylation.
We also examined the effects of co-expression of JARID2 andMEG3 in the expression of EMT-related genes in A549 cells.
JARID2 overexpression itself caused no significant changes inthe expression of EMT-related genes but potentiated the effects
of TGF-␤ as reported previously (Fig. 10F) (24). Again, MEG3
overexpression itself influenced the expression of part of the
EMT-related genes such as CDH1/E-cadherin, ZEB1, ZEB2,
miR-200a, and miR-200c. Co-expression of MEG3 and JARID2
induced a significant decrease of CDH1, miR-200a, and miR200c expression and an increase of ZEB1 and ZEB2 expression
JANUARY 6, 2017 • VOLUME 292 • NUMBER 1

compared with JARID2-overexpressing cells and MEG3-overexpressing cells (Fig. 10F). For the expression of FN1/fibronectin and vimentin, MEG3 and/or JARID2 had no effect. These
results indicated that co-expression of JARID2 and MEG3
induced significant expression changes in several of EMT-related genes in the absence of TGF-␤, suggesting the importanceof cooperation of JARID2 and MEG3 in transcriptional regulation during EMT.
Next, we examined whether MEG3 would stimulate theinteraction of JARID2 and EZH2 in the lung cancer cells. A549
cells were infected with the various combinations of the retroviruses expressing MEG3 and FLAG-tagged JARID2, and thecell lysates were prepared. We performed immunoprecipitation analysis with anti-EZH2 antibody followed by immunoJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 7. Overexpression of MEG3 affected the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 geneand miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 withoutor with TGF-␤ treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 (A and E), miR-200b/200a/429 (B and F),
miR-200c/141 (C and G), and GAPDH genes (D and H) in A549 (A–D) or LC-2/ad (E–H) cells are shown. The occupancies of methylated histones or EZH2 proteinon the regions were analyzed by quantitative PCR (*, p ⬍ 0.01 compared with control; **, p ⬍ 0.05 compared with control; ***, p ⬍ 0.01 compared with thesample without TGF-␤ treatment; ****, p ⬍ 0.05 compared with the sample without TGF-␤ treatment). WB, Western blotting.

Epigenetic Regulation of EMT by MEG3 lncRNA

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FIGURE 8. Knockdown of JARID2 cancelled MEG3-mediated changes in the expression of part of EMT-related genes in A549 and LC-2/ad cells. QRT-PCR
analysis was performed to detect the expression of CDH1/E-cadherin, FN1/fibronectin, vimentin, ZEB1, ZEB2, miR-200a, miR-200c, and JARID2 in A549 (A) andLC-2/ad (B) cells infected with the control retrovirus or the retrovirus expressing MEG3 and/or the retrovirus expressing JARID2 shRNA without or withtreatment of TGF-␤ for 24 h (*, p ⬍ 0.01 compared with control; **, p ⬍ 0.05 compared with control; ***, p ⬍ 0.01 compared with the sample without TGF-␤
treatment; ****, p ⬍ 0.05 compared with the sample without TGF-␤ treatment).

blotting with anti-EZH2 and anti-FLAG antibodies. Immunoblot with anti-EZH2 revealed that endogenous EZH2 wasdetected at a similar level with or without expression of MEG3and JARID2 (Fig. 10G, left panels, open arrowhead). In contrast,
immunoblot with anti-FLAG showed that co-precipitated
JARID2 was significantly detected when MEG3 was co-expressed in the cells (Fig. 10G, right upper panel, closed arrowhead). The expression level of FLAG-tagged JARID2 was notaffected by MEG3 co-expression (Fig. 10G, right lower panel,closed arrowhead). Therefore, the complex of EZH2 andJARID2 was found to be increased in the presence of MEG3.

92 JOURNAL OF BIOLOGICAL CHEMISTRY

These results strongly suggested that MEG3 enhanced theinteraction of JARID2 and EZH2 in A549 lung cancer cells.
We also tried to examine whether MEG3 lncRNA could associate with the chromatin of the regulatory regions of CDH1,
miR-200b/200a/429, and miR-200c/141 genes by chromatin
isolation by RNA purification (ChIRP) assay (Fig. 11) (37). A549
cells were infected with the control retrovirus or the retrovirus
expressing MEG3 with or without treatment of TGF-␤. The
cross-linked cell lysates were incubated with biotinylated DNA
probes against MEG3 lncRNA, and the binding complexes wererecovered by streptavidin-conjugated magnet beads. QPCRVOLUME 292 • NUMBER 1 • JANUARY 6, 2017

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FIGURE 9. Knockdown of JARID2 inhibited MEG3-induced changes in the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the
retrovirus expressing MEG3 and/or the retrovirus expressing JARID2 shRNA without or with TGF-␤ treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3
on the regulatory regions of CDH1 (A and E), miR-200b/200a/429 (B and F), miR-200c/141 (C and G), and GAPDH genes (D and H) in A549 (A–D) or LC-2/ad (E–H)
cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p ⬍ 0.01 compared with control;**, p ⬍ 0.05 compared with control; ***, p ⬍ 0.01 compared with the sample without TGF-␤ treatment; ****, p ⬍ 0.05 compared with the sample without TGF-␤
treatment).

was performed to detect the enrichment of the specific regulatory regions that associated with MEG3 lncRNA. TGF-␤ treatment significantly increased the amplified signals for the regulatory regions of CDH1 and miR-200c/141 genes but not theregulatory region of the unrelated GAPDH gene (Fig. 11, A, C,
and D). The signal for the regulatory region of miR-200b/200a/
429 was slightly increased by TGF-␤, but the difference was notstatistically significant (Fig. 11B). These results suggested that
endogenous MEG3 lncRNA might be recruited to the specificregulatory regions in the cells after TGF-␤ treatment. MEG3JANUARY 6, 2017 • VOLUME 292 • NUMBER 1

overexpression caused a clear increase of the amplified signals
for the regulatory regions of CDH1, miR-200b/200a/429, andmiR-200c/141 genes, which was further enhanced by TGF-␤
but not of GAPDH gene (Fig. 11, A–D). These results indicatedthat the TGF-␤ signal was not necessary for the association of
MEG3 lncRNA with the specific target regions but could
enhance the interaction. Furthermore, the levels of MEG3 association with the target sites were well correlated with therecruitment of JARID2 and EZH2 on the chromatin, histone
H3K27 methylation, and transcriptional repression of the tarJOURNAL OF BIOLOGICAL CHEMISTRY93

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Epigenetic Regulation of EMT by MEG3 lncRNA

get genes (Figs. 6, 7 and 10). Altogether, these results suggested
that MEG3 lncRNA could bind to JARID2 and mediated theassembly of the JARID2- and EZH2-containing PRC2 complex
on the regulatory regions of CDH1, miR-200b/200a/429, andmiR-200c/141 genes for histone H3 methylation and transcriptional repression during EMT process of cancer cells.

Discussion
In this study, we found that MEG3 lncRNA was required for
TGF-␤-induced EMT of A549 and LC-2/ad lung cancer celllines. MEG3 knockdown inhibited EMT by antagonizing TGF␤-dependent changes in the expression of EMT-related genes.
In contrast, MEG3 overexpression partially influenced theexpression of EMT-related genes, including CDH1, ZEB family,
and miR-200 family, and contributed to E-cadherin down-regulation. Mechanistic investigations suggested that MEG3 could
associate with the regulatory regions of CDH1 and miR-200
family genes and induce the recruitment of JARID2 and EZH2
and histone H3K27 methylation on these regions for transcriptional repression. This study demonstrated a novel role of

FIGURE 10. MEG3 interacted with JARID2 and stimulated the interaction and recruitment of JARID2 and EZH2 on the regulatory regions of CDH1,
miR-200a, and miR-200c genes for histone H3K27 methylation in A549 cells. A, interaction of MEG3 and JARID2 detected by RIP. A549 cells were infectedwith the various combinations (as indicated) of the retroviruses expressing MEG3 and FLAG-tagged JARID2. The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG) or anti-FLAG antibody, and the co-precipitated RNA was transcribed to cDNA. QPCR was performed to detect theenrichment of MEG3 in the precipitates. n.d. means not detected. B–E, A549 cells were infected with the various combinations (as indicated) of the retroviruses
without or with TGF-␤ treatment. ChIP analyses of H3K27me3, EZH2, and FLAG-tagged JARID2 on the regulatory regions of CDH1 (B), miR-200b/200a/429 (C),miR-200c/141 (D), and GAPDH genes (E) are shown (*, p ⬍ 0.01 compared with control; ***, p ⬍ 0.01 compared with the sample without TGF-␤ treatment; #, p ⬍0.01 compared with the sample of JARID2 overexpressed cells; ##, p ⬍ 0.05 compared with the sample of MEG3 overexpressed cells). F, QRT-PCR analysis was
performed to detect the expression of CDH1/E-cadherin, FN1/fibronectin, vimentin, ZEB1, ZEB2, miR-200a, and miR-200c in A549 cells infected with the various
combinations of the retroviruses (*, p ⬍ 0.01 compared with control; ***, p ⬍ 0.01 compared with the sample without TGF-␤ treatment; #, p ⬍ 0.01 comparedwith the sample of JARID2 overexpressed cells; ##, p ⬍ 0.05 compared with the sample of MEG3 overexpressed cells). G, interaction of JARID2 and EZH2 wasenhanced by MEG3. A549 cell lysates were immunoprecipitated (IP) with anti-EZH2 antibody and then subjected to immunoblotting with anti-EZH2 (left upper
panel) and anti-FLAG antibodies (right upper panel). The cell lysates were also directly subjected to immunoblotting with anti-EZH2 and anti-FLAG antibodies
to see the expression level of endogenous EZH2 and FLAG-tagged JARID2 proteins (lower panels). Open and closed arrowheads indicate endogenous EZH2
protein and FLAG-tagged JARID2 protein, respectively. WB, Western blotting.

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FIGURE 11. MEG3 could associate with the specific regulatory regions of CDH1, miR-200a, and miR-200c genes in A549 cells. ChIRP analyses of MEG3 on
the regulatory regions of CDH1 (A), miR-200b/200a/429 (B), miR-200c/141 (C), and GAPDH genes (D) in A549 cells are shown. The cells were infected with thecontrol retrovirus or the retrovirus expressing MEG3 with or without treatment of TGF-␤. The cross-linked cell lysates were incubated with biotinylated DNA
probes against MEG3 lncRNA, and the binding complexes were recovered by streptavidin-conjugated magnet beads. QPCR was performed to detect theenrichment of the specific regulatory regions that associated with MEG3 lncRNA. (*, p ⬍ 0.01 compared with control; **, p ⬍ 0.05 compared with control; ***,
p ⬍ 0.01 comparing to the sample without TGF-␤ treatment).

Epigenetic Regulation of EMT by MEG3 lncRNA

96 JOURNAL OF BIOLOGICAL CHEMISTRY

LC-2/ad lung cancer cells, was highly dependent on the function of MEG3, JARID2, and PRC2.
The experiments of MEG3 overexpression provide us
another interesting clue for understanding the epigenetic regulation of EMT. MEG3 overexpression could induce the interaction and recruitment of JARID2 and EZH2, H3K27 methylation, and transcriptional repression on CDH1 and miR-200
family genes in the absence of TGF-␤ (Figs. 6, 7, and 10). Thiswas in contrast to the previous observation that JARID2 overexpression itself had no effect (24). However, MEG3 itself could
not induce the EMT phenotype completely because it couldchange the expression of only a subset of the EMT-related
genes (Figs. 5 and 6). For example, the expression of mesenchymal markers, FN1/fibronectin and vimentin was not altered by
MEG3 overexpression. These results suggested that the MEG3dependent function of JARID2 and PRC2 on the specific target
genes was required but not sufficient for the EMT-inducing
program in lung cancer cells.
The presence of repressive H3K27me3 and active H3K4me3
marks on the gene promoter was not restricted to multipotent
cells but was detected in more differentiated cells (47). For thegenes that possessed these opposing methylation marks, it wasreported that relative intensities of H3K27me3 and H3K4me3
determined their transcription levels (48). Our ChIP experiments were correlated with these previous studies; TGF-␤
resulted in the increase of EZH2 recruitment and K27me3 andthe substantial decrease of K4me3 on the CDH1, miR-200b/
200a/429, and miR-200c/141 genes, thereby causing transcriptional repression, but MEG3 knockdown inhibited TGF-␤dependent epigenetic changes, consequently relieving transcriptional repression (Figs. 3 and 4). In mammalian cells,
H3K27 methylation is catalyzed by Polycomb family, and H3K4
methylation is mostly regulated by COMPASS family proteins(49). These families are well known for their opposing roles inbalancing gene expression. In this study, the function of PRC2
in H3K27 methylation and transcriptional repression was
mainly investigated, but the regulation of H3K4 methylation
remained unknown. However, we and another group (8, 15)previously proposed a critical role of H3K4-methyl-modifying
enzymes, including KDM5B and LSD1 in the promotion of
EMT. Furthermore, it has been reported that HOTAIR lncRNA
could mediate interaction of the PRC2- and LSD1-containing
complex and coordinate the regulation of H3K27 and H3K4
methylation on the target genes (45). To understand the epigenetic regulation of EMT in detail, further studies will be
required for the functional interaction of the H3K4-methylmodifying complex and JARID2-containing PRC2 complex.
Rapidly accumulating evidence reveals that lncRNA isinvolved in the initiation and progression of human cancer(26 –28). In addition to the observed deregulation of lncRNA
expression in cancer, lncRNAs have been demonstrated to act
as oncogenes or tumor suppressor genes. Although lncRNAs
have been scarcely functionally characterized, some of themcan serve as decoys to bind microRNAs, scaffolds to regulate
protein-protein interaction, or guides to recruit epigenetic regulators on chromatin (25). The MEG3 gene encodes one of thelncRNAs and is expressed in many normal tissues. In many
types of human tumors and cancer cell lines, loss of MEG3
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MEG3 lncRNA in the epigenetic regulation of EMT process ofcancer cells.
PRC2 plays an essential role in development, stem cell identity, and disease pathogenesis, including cancer (17, 18). The
catalytic subunit, EZH2, regulates histone H3K27 methylationand establishes a chromatin structure for transcriptional
repression. EZH2 overexpression is frequently observed inhuman solid tumors and is associated with a poor prognosis inseveral tumor types (19). Thus increased activity of EZH2 or
PRC2 is proposed to be responsible for malignant phenotypesof cancer cells. It has been demonstrated that EZH2 could
down-regulate the expression of CDH1 and miR-200 family
genes through histone H3K27 methylation in cancer cells (14).
We also found that EED, a core component of PRC2, and
JARID2, an accessory factor of PRC2, were required for transcriptional repression of these genes through H3K27 methylation, which was essential for TGF-␤-induced EMT (16, 24).
Here we showed that MEG3 knockdown counteracted TGF-␤induced EMT of lung cancer cells by inhibiting EZH2 recruitment, H3K27 methylation, and transcriptional repression ofthese genes. Therefore, our studies validated the importance of
PRC2 recruitment and activity during the EMT-inducing transcriptional program.
The core components of the mammalian PRC2 complex do
not have sequence-specific DNA binding activities. Thus it
would be an important issue how PRC2 is recruited to its specific target genes for transcriptional repression. So far, chromatin targeting mechanisms of PRC2 have been proposed, which
include the interactions with various DNA-binding factors
and long noncoding RNAs. During embryonic development,
JARID2 is one of the essential factors for PRC2 recruitment(20 –23). We also proposed that JARID2 was essential for PRC2
recruitment in TGF-␤-induced EMT of lung cancer cells (24).
Recently, a growing body of evidence has indicated that longnoncoding RNAs play a crucial role in the recruitment of PRC2
and JARID2 to the target loci (34, 35, 45, 46). Although JARID2
and PRC2 could associate with many lncRNAs, Meg3 wasshown to be one of the important regulators of JARID2 andPRC2 recruitment in mouse ES cells (34, 35). Meg3 bound to
JARID2, stimulated the assembly of JARID2 and PRC2, andrecruited them to the specific target genes in pluripotent stem
cells. It was also emphasized that Meg3 affected JARID2 andPRC2 recruitment on only a subset of genes but not all of PRC2regulated loci (34). Our previous studies revealed that transcriptional regulation of CDH1 and miR-200 family genes wasdependent on JARID2 and PRC2, because knockdown of
JARID2 or EED completely inhibited TGF-␤-induced repression of these genes. In this study, we showed that MEG3 knockdown also inhibited EZH2 recruitment, H3K27 methylation,and transcriptional repression of these genes, and eventually
EMT was induced by TGF-␤ (Figs. 2– 4). In the MEG3 knockdown cells, elevated expression of endogenous JARID2 wasobserved after TGF-␤ treatment (Fig. 3, A and C), but JARID2
might not contribute to EZH2 recruitment and transcriptional
repression of these genes without MEG3 expression (Figs. 3 and
4). These results suggested that transcriptional regulation of
the critical target genes such as the CDH1 and miR-200 families, which were responsible for EMT progression in A549 andEpigenetic Regulation of EMT by MEG3 lncRNA

Experimental Procedures
Plasmids—The retrovirus vectors expressing small hairpin
RNAs (shRNAs) were constructed as described previously (7).
The oligonucleotide sequences for shRNAs are listed in supplemental Table S1. The sequences of control and JARID2 shRNAs
were described previously (7, 24). For human MEG3 cDNA, theprimer set described in supplemental Table S1 was designed
based on the previous report (34) and was used for cloning. The
amplified cDNA was cloned into pDON-5 Neo plasmid(Takara, Ohtsu, Japan) to produce retrovirus-expressing
MEG3. pMXs-puro plasmid was kindly provided by Dr. C.
Takahashi (Kanazawa University, Japan), and the plasmid that
produces retrovirus expressing FLAG-tagged JARID2 was constructed as described previously (24).
Cell Culture and Transfections—LC-2/ad human lung cancer
cell line was purchased from RIKEN BRC, Japan, and was maintained in the mixture of RPMI 1640 and Ham’s F-12 medium
with 10% FBS, 2 mM glutamine, and penicillin/streptomycin(Sigma) at 37 °C in 5% CO2. A549 human lung cancer cell line
was kindly provided by Dr. Y. Endo (Kanazawa University,
Japan) and maintained as described previously (16). For EMT
induction, A549 and LC-2/ad cells were treated with 1 ng/ml
TGF-␤ (R&D Systems, Minneapolis, MN) for 24 h to 6 days.
The protocols for the production and infection of shRNA or
cDNA-expressing retroviruses were essentially the same as
described previously (7).
Quantitative PCR—Preparation of RNA and quantitative
RT-PCR was performed as described previously (7). QuantitaJANUARY 6, 2017 • VOLUME 292 • NUMBER 1

tive PCR data were normalized with control human GAPDH
expression. The averages from at least three independent
experiments are shown with the standard deviations. p values
were calculated between control and the samples using
Student’s t test. Primers used for the quantitative PCR were
described previously (7, 8, 24) and are listed in supplemental
Table S1. For the quantification of microRNAs, we used
TaqMan microRNA assays (Applied Biosystems, Waltham,MA) for miR-200a (catalog no. 000502) and miR-200c (Applied
Biosystems). All data were normalized with RNU6B (Applied
Biosystems) expression.
Cell Staining, Immunofluorescence, and Immunoblotting—
To observe the changes of cell morphologies, A549 or LC-2/ad
cells were stained with 0.4% crystal violet. To visualize the actin
cytoskeleton, the cells were stained with 0.25 ␮M TRITC-conjugated phalloidin (Sigma). For indirect immunofluorescence,the specimens were treated with anti-E-cadherin antibody (catalog no. 610181, BD Biosciences) and incubated with Alexa546conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were
stained with 4⬘,6-diamidino-2-phenylindole (DAPI). Immunoblotting was done as described previously (8). We used anti-Ecadherin, anti-fibronectin (SAB4500974, Sigma), anti-vimentin(ab8069, Abcam, Cambridge, MA), anti-ZEB1 (catalog no.
3396, Cell Signaling, Danvers, MA), anti-ZEB2 (catalog no.
61096, Active Motif, Carlsbad, CA), anti-phosphorylated
SMAD3 (ab51451, Abcam), and anti-GAPDH (6C5, Millipore)
antibodies.
Cell Migration Assay—Cell migration abilities were measured in modified Boyden chambers consisting of Transwell
membrane filter inserts (catalog no. 3422, Corning Costar).
Serum-starved cells (2 ⫻ 105) suspended in 100 ␮l of DMEM
containing 1 mg/ml BSA and 0.5% FBS were cultured in eachTranswell chamber and allowed to migrate toward the underside of the membrane for 24 h for A549 cells. The lower chamber contained DMEM with 10% FBS. For TGF-␤-treated cells,
cells were pre-treated with 1 ng/ml TGF-␤ for 4 days and thenallowed to migrate. Cells that had not penetrated the filter were
wiped out, and cells on the lower surface of the filter were
stained with 0.4% crystal violet. The number of migrated cells
was counted under a light microscope from at least five fields
and three experiments.
ChIP Assays—ChIP assays were carried out essentially as
described previously (7, 36). The cross-linked cell lysates were
immunoprecipitated with the following antibodies: antiH3K27me3 and anti-H3K4me3 (36); anti-EZH2 (catalog no.
17-662, Millipore); anti-JARID2 (catalog no. NB100-2214,
Novus Biologicals, Littleton, CO); and anti-FLAG (M2, catalog
no. F1804, Sigma). Quantitative PCR was performed to detect
the enrichment of a specific amplified region. Percentage
enrichment over input chromatin was presented. Primers used
for the QPCR correspond to region a of CDH1 gene, region b of
miR-200b/a/429 gene, region b of miR-200c/141 gene; andregion a of GAPDH gene, as described previously (8).
RIP—The preparation of cross-linked cell lysates was essentially the same with our ChIP experiments except that cells
were lysed with RIP buffer (50 mM Tris-Cl, pH 7.5, 150 mM
NaCl, 10 mM EDTA, 0.5% Nonidet P-40, protease inhibitors(Nakalai, Kyoto, Japan) and SUPERase-In (Thermo Fisher SciJOURNAL OF BIOLOGICAL CHEMISTRY

97

Downloaded from http://www.jbc.org/ at KANAZAWA UNIV on January 10, 2017expression has been detected (50). Re-introduction of MEG3
inhibited cell proliferation and colony formation of cancer celllines, partly by MEG3-mediated induction of apoptosis (51, 52).
MEG3 could also stabilize p53 protein and stimulated p53-mediated transcription of the genes (51, 53). Thus it is hypothesized that MEG3 is a tumor suppressor gene (50). In contrast tothese results, our study indicated an important function of
MEG3 in EMT, a hallmark of malignant tumor progression. In
general, the roles of epigenetic enzymes and cofactors in cancer
are sometimes controversial. Overexpression of EZH2 wasdetected in many types of tumors and correlated with poor
prognosis (19), but its mutation was also detected in various
myeloid and lymphoid neoplasms (54). JARID2 gene deletion
was reported in leukemic transformation of chronic myeloid
diseases (55), but a higher level expression of JARID2 was associated with metastasis in rhabdomyosarcomas (56). Thus thecontribution of these epigenetic regulators to cancer development is highly dependent on many factors and cellular context.
Therefore, careful analyses and discussions should be performed to understand the roles of the epigenetic regulatory
factors.
We have found a novel function of MEG3 lncRNA during
TGF-␤-induced EMT of A549 and LC-2/ad lung cancer celllines. MEG3 played an important role in the EMT process byregulating EMT-related gene expression through the modulation of PRC2 recruitment and histone H3 methylation.
Our study provides novel insights into the involvement oflong noncoding RNAs for epigenetic mechanism of cancer
malignancies.

Epigenetic Regulation of EMT by MEG3 lncRNA

Author Contributions—T. S. and M. T. designed the experiments.
M. T. performed most of the experiments. S. T. and A. I. performed
part of the experiments. T. S. wrote the manuscript with the help of
M. T. and A. I.
Acknowledgments—We thank Drs. C. Takahashi and Y. Endo
(Kanazawa University, Japan) for providing the plasmid and the cellline. We also thank Dr. H. Kimura (Tokyo Institute of Technology,
Japan) and Dr. N. Nozaki (Monoclonal Antibody Institute, Japan) for
providing the antibodies that specifically recognize methylated H3K
residues.
Note Added in Proof—In the version that was published as a Paper in
Press on November 16, 2016, the incorrect E-cadherin immunoblot
was used in Fig. 3D. This error has now been corrected and does notaffect the results or conclusions of this work.

98 JOURNAL OF BIOLOGICAL CHEMISTRY

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input DNA was presented.

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MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation ofEpithelial-Mesenchymal Transition in Lung Cancer Cell Lines
Minoru Terashima, Shoichiro Tange, Akihiko Ishimura and Takeshi Suzuki
J. Biol. Chem. 2017, 292:82-99.
doi: 10.1074/jbc.M116.750950 originally published online November 16, 2016

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