Involvement of aberrantly activated HOTAIR/EZH2/miR-193a feedback loop in progression of prostate cancer
Ling et al. Journal of Experimental & Clinical Cancer Research
Involvement of aberrantly activated HOTAIR/EZH2/miR-193a feedback loop in progression of prostate cancer
Zhixin Ling 0 1 3
Xiaoyan Wang 0 2
Tao Tao 0 6
Lei Zhang 0 1 3
Han Guan 5
Zonghao You 1 3
Kai Lu 1 3
Guangyuan Zhang 1 3
Shuqiu Chen 1 3
Jianping Wu 1 3
Jinke Qian 4
Hui Liu 4
Bin Xu 1 3
Ming Chen 1 3
0 Equal contributors
1 Department of Urology, Affiliated Zhongda Hospital of Southeast University , Nanjing, Jiangsu 210009 , China
2 Department of Nursing, Affiliated Zhongda Hospital of Southeast University , Nanjing, Jiangsu 210009 , China
3 Surgical Research Center, Institute of Urology, Medical School of Southeast University , Nanjing, Jiangsu 210009 , China
4 Department of Urology, Binhai People's Hospital , Yancheng, Jiangsu 224500 , China
5 Department of Urology, the First Affiliated Hospital of Bengbu Medical College , Bengbu 233004 , China
6 Department of Urology, Anhui Provincial Hospital, Anhui Medical University , Hefei 230001 , China
Background: Though androgen deprivation therapy is the standard treatment for prostate cancer (PCa), most patients would inevitably progress to castration-resistant prostate cancer (CRPC) which is the main cause of PCa death. Therefore, the identification of novel molecular mechanism regulating cancer progression and achievement of new insight into target therapy would be necessary for improving the benefits of PCa patients. This study aims to study the function and regulatory mechanism of HOTAIR/EZH2/miR-193a feedback loop in PCa progression. Methods: MSKCC and TCGA datasets were used to identify miR-193a expression profile in PCa. Cell Counting Kit-8 (CCK-8) assays, colony formation, invasion, migration, flow cytometry, a xenograft model and Gene Set Enrichment Analysis were used to detect and analyze the biological function of miR-193a. Then, we assessed the role of HOTAIR and EZH2 in regulation of miR-193a expression by using plasmid, lentivirus and small interfering RNA (siRNA). Luciferase reporter assays and chromatin immunoprecipitation assays were performed to detect the transcriptional activation of miR-193a by EZH2 and HOTAIR. Further, qRT-PCR and luciferase reporter assays were conducted to examine the regulatory role of miR-193a controlling the HOTAIR expression in PCa. Finally, the correlation between HOTAIR, EZH2 and miR-193a expression were analyzed using In situ hybridization and immunohistochemistry. Results: We found that miR-193a was significantly downregulated in metastatic PCa through mining MSKCC and TCGA datasets. In vitro studies revealed that miR-193a inhibited PCa cell growth, suppressed migration and invasion, and promoted apoptosis; in vivo results demonstrated that overexpression of miR-193a mediated by lentivirus dramatically reduced PCa xenograft tumor growth. Importantly, we found EZH2 coupled with HOTAIR to repress miR-193a expression through trimethylation of H3K27 at miR-193a promoter in PC3 and DU145 cells. Interestingly, further evidence illustrated that miR-193a directly targets HOTAIR showing as significantly reduced HOTAIR level in miR-193a overexpressed cells and tissues. The expression level of miR-193a was inversely associated with that of HOTAIR and EZH2 in PCa. Conclusion: This study firstly demonstrated that miR-193a acted as tumor suppressor in CRPC and the autoregulatory feedback loop of HOTAIR/EZH2/miR-193a served an important mechanism in PCa development. Targeting this aberrantly activated feedback loop may provide a potential therapeutic strategy.
Prostate cancer; MiR-193a; HOTAIR; EZH2; Progression
Prostate cancer is the most commonly diagnosed
malignancy in American men and is one of leading causes of
cancer-related death among aging population [
biggest challenge for PCa treatment is that most patients
would inevitably progress to castration-resistant prostate
cancer within 2 years’ androgen deprivation therapy,
which is considered as main cause of prostate cancer
patient death. A wide spectrum of genetic aberrations
was associated with PCa development, however the
exact molecular mechanism underlying PCa progression
Enhancer of zeste homolog 2 (EZH2) has been widely
studied in the area of cancer epigenetics in recent years
]. EZH2 was first observed in cancer when it was
identified as one of the most elevated genes in metastatic
PCa and closely correlated with poor prognosis .
Given its dysregulation in PCa and its oncogenic role in
PCa cells proliferation and metastasis, substantial efforts
have been dedicated to identify its underlying molecular
regulatory mechanisms and its potential therapy
application in PCa. EZH2 is one of the core enzymatic subunit
of histone methyltransferase polycomb repressor
complex 2 (PRC2) which methylates lysine27 of histone H3
(H3K27) to promote transcriptional silencing of many
tumor suppressive genes [
]. In addition, several
studies also demonstrated the PRC2-independent function of
EZH2 in transcriptional activator rather than repression
]. A wide spectrum of genetic as well as epigenetic
aberrations are associated with cancer development,
many of which are associated with histone
methyltransferase EZH2 dysregulation. However, the exact
functional role of EZH2-mediated epigenetic silencing of
tumor suppressive miRNAs in prostate cancer
progression has not been systematically studied.
The action of microRNAs (miRNAs) has been
studied in detail as destabilizers and repressors of
translation of protein-coding transcripts (mRNAs) to
regulate diverse biological function including proliferation,
differentiation, apoptosis, metabolic progression in various
cancers including PCa. MicroRNA-193a (miR-193a) was
initially discovered in 2008, and it was identified as a
tumor suppressive miRNA in oral carcinoma, lung cancer,
colorectal cancer, and malignant pleural mesothelioma
]. It has been reported that DNA methylation is
associated with miR-193a downregulation in acute myeloid
leukemia, hepatocellular carcinoma, non-small cell lung
cancer, and oral cancer [
]. In a previous miRNA
microarray analysis, we detected a panel of miRNAs,
including miR-193a, whose expression are downregulated in
CRPC clinical samples [
]. However, the exact biological
function of miR-193a in tumorigenesis of PCa remains
largely unknown. Moreover, the molecular mechanism of
miR-193a silencing in PCa is still unclear.
Long non-coding RNAs (lncRNAs) are defined as
nonprotein coding transcripts longer than 200 nucleotides.
They were initially argued to be spurious transcriptional
noise. However, increasing discoveries of dysregulated
lncRNA expression among various cancer types
implicated that aberrant lncRNA expression may be major
contributor to tumorigenesis [
]. For last decade,
lncRNA HOX transcript antisense RNA (HOTAIR) has
attracted considerable scientific attention in numerous
cancers. Accumulating evidence indicate that HOTAIR
may function as an oncogene in the malignant progression
of various cancers including prostate cancer [
indicating its potential role of effective therapeutic target
in cancers. HOTAIR could directly binds to the AR
protein to prevent its ubiquitination and protein
degradation, thereby promoting PCa cell growth and invasion
]. And HOTAIR is well known for its interacting with
key epigenetic regulators such as PRC2 and histone
demethylase LSD1 to induce gene silencing and
chromatin dynamics, which appears to be misregulated in a
variety of cancers [
], whereas it’s regulatory role of
genes expression in prostate cancer remains limited.
Furthermore, numerous studies during last decade
have illustrated that lncRNAs can be targeted by
miRNAs to reduce lncRNA stability. LncRNA
MALAT1 can be targeted by microRNA-9 in the
human primary glioblastoma cell line U87MG and the
human Hodgkin cell line L428 [
cancerrelated lncRNA PTCSC3 is targeted and repressed by
miR-574-5p in thyroid cancer cell lines [
]. It is
reported that recruitment of let-7 by HuR reduced
the HOTAIR stability and decreased its expression
]. So far, few studies have revealed the potential
miRNAs that can directly negatively regulate HOTAIR
expression in PCa.
In this study, we systemically investigated the biological
functions of miR-193a and results suggested that
miR193a functions as a tumor suppressor in PCa. And
HOTAIR could interact with EZH2 to repress miR-193a
expression by epigenetic modification. On other hand,
miR-193a directly targets HOTAIR and reduces HOTAIR
expression in PCa. This study demonstrated that the
autoregulatory feedback loop of HOTAIR/EZH2/miR-193a
plays a key role in PCa development.
Cell culture and tissue collection
The human PCa cell line DU145 and PC3 were
purchased from ATCC (Manassas, VA, USA). Cells
were cultured in RPMI-1640 (Gibco, Grand Island,
New York State, USA) supplemented with 10% fetal
bovine serum (Gibco), 100 U/ml penicillin, and
100μg/ml streptomycin at 37 °C in a 5% CO2
incubator. PCa (n = 31) samples were collected from
Zhongda Hospital Affiliated with Southeast University.
PCa tissues were obtained from early-stage patients
who underwent radical prostatectomy and never
received previous treatment. Clinicopatholigical
features of these specimens were examined by senior
pathologist and diagnosed according to pathological
evidence. All the patients whose samples enrolled in
this study have been informed and signed consents
for their tissues used in scientific research, and this
research also obtained approval from ethics of
Zhongda Hospital Affiliated with Southeast University.
Oligonucleotides, plasmid, lentivirus, and cell transfection
Oligonucleotides were chemically synthesized by
GenePharma (Shanghai, China) according to the
sequences of has-miR-193a mimic (miR-193a), 5′-A
ACUGGCCUACAAAGUCCCAGU-3′ (sense), 5′-UGG
hsamiR-193a inhibitor (anti-miR-193a), 5′-ACUGGGACU
UUGUAGGCCAGUU-3′; scrambled miRNA mimic
(antisense); scrambled miRNA inhibitor (anti-NC), 5′-CA
GUACUUUUGUGUAGUACAA-3′; EZH2 siRNA
(sense), 5′-CU GAAACAGCUGCCUUAGCTT-3′
(antisense); HOTAIR siRNA 1 (si-HOTAIR1), 5′-GCACA
GAGCAACUCUAUAATT-3′ (sense), 5′-UUAUAGAG
UUGCUCUGUGCTT-3′ (antisense); HOTAIR siRNA 2
5′-GCCUUUGGAAGCUCUUGAATT3′ (sense), 5′-UUCAAGAGCUUCCAAAGGCTT-3′
(antisense); siRNA negative control (si-NC), 5′-UUCUC
CGAACGUGUCACGUTT-3′ (sense), 5′-ACGUGACACG
UUCGGAGAATT-3′ (antisense). Human EZH2 cDNA
was chemically synthesized and subcloned into Nhel and
XhoI restriction site of pcDNA3.1 (+) plasmid (Invitrogen,
Carlsbad, CA, USA) to generate pcDNA3.1-EZH2 plasmid
which was purchased from Sangon Biotech Co., Ltd.
(Shanghai, China). The oligonucleotides and plasmids were
transfected using Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA) following the manufacturer’s protocol. Lentivirus
carrying overexpressing Hsa-miR-193a lentiviral vectors
(GV309) were from GeneChem (Shanghai, China) and
named with LV-miR-193a. LV-shEZH2 is EZH2 knocking
down expressing lentivirus, of which double-stranded
EZH2-shRNA oligonucleotide was cloned into GV248
vector by GeneChem (Shanghai, China). LV-NC, the GFP
vector, was used as control. And the lentiviruses were used
to infect cells in the presence of Polybrene. After 48 h,
puromycin was added in fresh medium to select of stable
clones. Western blot analysis and real-time quantitative
reverse transcriptionpolymerase chain reaction (qRT-PCR)
were used to determine the efficiency of knockdown and
Data mining and bioinformatics analysis
As previously described [
], we retrieved and
reanalyzed the original miRNAs expression and clinical
data from the Memorial Sloan Kettering Cancer Center
(MSKCC) (www.mskcc.org) and The Cancer Genome
Atlas (TCGA) (http://cancergenome.nih.gov) databases
to investigate clinical relevance of miR-193a with the
pathological traits of patients.
RNA isolation and real-time quantitative reverse transcription and polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells or tissues using
TRIzol (Takara, Shiga, Japan) according to the
manufacturer’s instruction. For miR-193a, qRT-PCR reaction was
performed with a HiScript RT kit (VazymeBiotech,
Nanjing, China) and AceQ SYBR Green PCR Master
Mix (VazymeBiotech) following the manufacturer’s
protocols. Primers were chemically synthesized by SprinGen
Biotech (Nanjing, China). The miR-193a primers for
amplification were as follows: stem–loop reverse
transcription primer: 5′-CTCAACTGGTGTCGTGGAGTC
GGCAATTCAGTTGAGACTGGGAC -3′; specific
forward primer: 5′-ACACTCCAGCTGGGAACT
GGCCTACAAAGTCCC -3′; specific reverse primer:
5′TGGTGTCGTGGAGTCG-3′. The U6 snRNA primers
were as follows: forward: 5′-CTCGCTTCGGCAGC
5′-AACGCTTCACGAATTTGCGT3′. The HOTAIR primers were: forward: 5′-CAGTGGG
GAACTCTGACTCG-3′; reverse: 5′-GTGCCTGGTG
CTCTCTTACC-3′; The GAPDH primers were: forward:
5′-TGCACCACCAACTGCTTAGC-3′; reverse: 5′-GG
CATGGACTGTGGTCATGAG-3′. PCR was conducted
with a 20 μl reaction volume that contained 2 μl of RT
products, 1 μl of 10 μM miR-193a or U6 primer set,
10 μl of 2× SYBR Green PCR Master Mix, and 7 μl of
ddH2O. Cycling conditions were as follows: 95 °C for
3 min, 95 °C for 15 s, and 60 °C for 60 s. The last two
steps were conducted with 40 cycles. qRT-PCR was
performed using 7500 Real-Time PCR System (Applied
Biosystems, Foster City, CA, USA). The gene expression
threshold cycle (CT) values of miR-193a were calculated
by normalizing to the internal control U6, and HOTAIR
expression was normalized to GAPDH. Relative
quantification values were calculated via the 2−ΔΔCt method.
Cell proliferation assay
PC3 and DU145 cells were seeded at 3 × 105 cells per
well in six-well plates and then cultured overnight. At
48 h post-transfection with oligonucleotides or plasmid,
cells were trypsinized and seeded at 3000 cells per well
in 96-well plates. Cell proliferation was measured by
CCK-8 kit (KeyGene Biotech, Nanjing, China) every
24 h according to the manufacturer’s instruction.
Absorbance was detected at wavelength of 450 nm. Five
wells were measured for cell viability in each treatment
group. Three independent experiments were carried out
with each 96-well plate.
Cell colony formation assay
After 48 h transfection, cells were trypsinized and
seeded in 6-well plate at a density of 800 cells/well
and cultured for 10 to 14 days until colony appeared.
Cells were washed with 0.01 M phosphate-buffered
saline (PBS, 137 mM NaCl, 10 mM Na2HPO4,
1.8 mM KH2PO4, 2.7 mM KCl, and pH 7.4), fixed
with methanol for 20 min, and finally stained with
0.5% crystal violet (make a solution of 0.5% crystal
violet in 20% methanol) for 20 min at room
temperature. The number of colonies was counted
only when they contained more than 50 cells.
Cell apoptosis assay
Apoptotic cells were stained by using Annexin
VFITC/Propidium Iodide (PI) contained in Apoptosis
Kit (KeyGene Biotech, Nanjing, China). After 48 h of
transfection, PCa cells were stained with 5 μl
Annexin V-FITC and 5 μl PI and cultured in dark at
room temperature for 15 min. These cells were
analyzed by flow cytometry, and results were processed
with Cell Quest ProSoftware (BD Biosciences, San
Jose, CA, USA).
In order to label nuclei of apoptotic cells, PC3 and
DU145 cells were plated on glass coverslips in 6-well
plates, transfected, and fixed in 4% paraformaldehyde 48 h
post-transfection. We performed the terminal
deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)
staining using an In Situ Cell Death Detection Kit (Roche,
Mannheim, Germany) according to the manufacturer’s
protocol. The number of TUNEL-positive nuclei was
counted and divided by the number of DAPI-stained
nuclei to calculate the percentage of TUNEL-positive cells.
Three coverslips were analyzed per condition.
Transwell migration and invasion assay
Migration and invasion assays were performed by
using a Transwell chamber with or without Matrigel
(BD Biosciences) according to the manufacturer’s
instructions. After 48 h of transfection, cells were
trypsinized and seeded in top chamber with a density
of 1 × 105 cells per well with 200 ul serum-free
medium. 600ul of 10% FBS RPMI-1640 medium was
added to the lower chamber. Migration and invasion
assays were assessed at 8 and 24 h, respectively. Cells
on the top chamber were removed, and cells on lower
chamber were then fixed with methanol and stained
with 0.5% crystal violet and scored.
In situ hybridization (ISH) and immunohistochemical staining (IHC)
ISH and IHC were performed and subsequent results
were analyzed as previously described [
]. In brief, the
triple digoxigenin-labeled antisense locked nucleic acid
(LNA)-modified probes for HOTAIR and miR-193a were
synthesized by Boster Biotech (Wuhan, China). ISH was
conducted according to the manufacturer’s instruction
of the HOTAIR and microRNA ISH Optimization Kits
(Boster, Wuhan, China). IHC was performed using
antiEZH2 antibody (1:250, Abcam, Cambrige, MA, USA)
according to the manufacturer’s instructions. The
original magnification: ×200.The specific evaluation of gene
expression via ISH or IHC was calculated as previously
]. In brief, sections with no labeling or less
than 5% labeled cells were scored as 0, 5%–30% of cells
as 1, 31%–70% of cells as 2, and ≥71% as 3. The staining
intensity was scored similarly using 0 for negative
staining, 1 for weakly positive, 2 for moderately positive, and
3 for strongly positive. The scores of percentage of
positive tumor cells and staining grade were calculated to
generate the immune-reactive score for each specimen.
A combined score of 0–1 indicates negative expression
(−), 2–3 indicates weak expression (+), 4–5 indicated
moderate expression (++), and 6 indicates strong
expression (+++). Each sample was examined separately and
scored by two pathologists [
Xenograft tumor assay
BALB/C nude male mice (six-week old) were purchased
from Model Animal Research Center of Nanjing University.
All animal experiments were conducted according to the
National Institute of Health Guide for the Care and Use of
Laboratory Animals and approved by the ethics committee
of the Affiliated Zhongda Hospital of Southeast University.
PC3 (5 × 106) cells which have been stably infected with
LV-miR-193a or LV-NC were subcutaneously injected into
each flank of the mice. Tumor size was measured every
week with the following formula: (length × width2)/2. At
the end of experiments, all mice were sacrificed and tumors
were dissected and weighed. Partial xenograft tumor tissues
were used for RNA extraction and detection of HOTAIR
and miR-193a expression. And the rest tissues were
formalin fixed immediately and paraffin embedded. Thereafter
tissues were stained with antibody against Ki-67, CD31 and
CD34 (all 1:100, Boster, Wuhan, China) according to the
RNA sequencing and bioinformatics analysis
PC3 and DU145 cell lines stably overexpressing miR-193a
were established via LV-miR-193a infection and
puromycin selection. RNA sequencing was assigned to and
performed by KangChen Bio-tech Ltd., Co. (Shanghai,
China). A total of 2 μg RNA were extracted from
LV-miR193a or LV-NC expressing cells, respectively, and assigned
to RNA-sequencing using Illumina-HiSeq4000 system.
Gene Enrichment Set Analysis (GSEA) was used to
identify gene sets or pathways which were relevant to
miR-193a expression profile in PCa cells (http://
www.broadinstitute.org/gsea/index.jsp). Hallmark of gene
sets were obtained from the Molecular Signatures
Database on that website. Normalized enrichment score
(NES) and false discovery rate (FDR) were used to analyze
across the gene sets.
Western blot analysis
PC3/DU145 Cells were lysed in RIPA buffer (50 mM
TrisCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1%
NP-40) supplemented with 1% proteinase inhibitors
(Sigma, St. Louis, MO, USA) and 1 mM PMSF (Beyotime,
Hangzhou, China) for total protein preparation. Briefly, 30
μg protein samples were analyzed with 10% SDS-PAGE
and then transferred into a polyvinylidene fluoride
membrane (Millipore, Billerica, MA, USA). Blots were blocked
with 5% skim milk for 1 h at room temperature, and
incubated with specific primary antibodies at 4°C overnight.
The membranes were washed three times with TBST
(TBS-1% Tween 20) buffer and incubated with secondary
antibody at room temperature for 1 h, and detected with
enhanced chemiluminescence (ECL, Beyotime, Hangzhou,
China). Primary antibodies used were rabbit anti-EZH2
(1:1000, Abcam, Cambrige, MA, USA), rabbit
antiH3K27me3 (1:1000, Abcam, Cambrige, MA, USA), rabbit
anti-H3(1:1000, Proteintech, Rosemont, IL, USA),
HRPconjugated monoclonal mouse anti-GAPDH (1:1000;
KangChen, China;) and HRP-labeled goat anti-rabbit
secondary antibody (1:3000; Zhongshan Goldenbridge
Biotechnology, Beijing, China).
Luciferase reporter assay
The exact transcription start site (TSS) of miR-193a
has been reported previously [
], 2Kb upstream of
TSS were retrieved from NCBI Genome
Bioinformatics (http://www.ncbi.nlm.nih.gov/). The promoter
region of has-miR-193a was chemically synthesised and
cloned into the KpnI and HindIII restriction sites by
GenScript (Nanjing, China), which is downstream the
open reading frame of luciferase in the
pGL3-basicVector (Promega, Madison, WI, USA) to generate the
pGL3-miR-193a promoter reporter. For promoter
luciferase reporter assay, cells were co-transfected with
pGL3-miR-193a promoter and pRL-TK. The 2.3 kb
full sequences of wild-type HOTAIR cDNA or mutant
HOTAIR cDNA were synthesised by Generay Biotech
(Shanghai, China). We then subcloned full cDNA
sequence of wild type and mutant HOTAIR into the
XhoI and NotI restriction sites of psi-CHECK™-2
vector (Promega, Madison, WI, USA) to generate
psi-CHECK-HOTAIRMut reporters. Luciferase assay activity was measured 48 h
after transfection using the Dual-Luciferase Reporter Assay
System (Promega, Madison, WI, USA).
Chromatin immuoprecipitation (ChIP)
PC3 and DU145 cells which were transfected with si-NC or
si-HOTAIR2 for 72 h and processed with ChIP assay kit
according to the manufacturer’s instructions (Beyotime,
China). Antibodies used includes EZH2 antibody (1:50,
Abcam, Cambridge, MA, USA), H3K27me3 antibody (1:50,
Abcam, Cambridge, MA, USA) and IgG (1:50, Millipore,
Billerica, MA, USA). Gene Expression Omnibus (GEO)
datasets (DU145 H3K27me3:
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1138596; PC3 H3K27me3:
1383872) were retrieved and analyzed for the H3K27me3
peak loci at miR-193a promoter region. ChIP qPCR
Primers were specifically designed according to the
H3K27me3 loci in PCa cells. The precipitated DNA
quantitated by qRT-PCR and normalized by respective 1% ChIP
input. ChIP primer sequences used are as follows: sense,
Each value in this study was obtained from at least
three independent experiments and presented as
mean ± SD. The significance of difference among the
means was calculated using Student’s t-tests for
twogroup comparisons with Statistical Package of the
Social Sciences software version 19.0 (SPSS, Inc.
Chicago, IL, USA). A two-sided P value of <0.05 was
considered statistically significant.
Experimental scheme and miR-193a expression correlates with prostate cancer clinical features
In our previous microarray analysis [
], we have
detected a total of 452 miRNAs that were differentially
expressed between CRPC and ADPC (androgen
dependent prostate cancer). Among them, 275 miRNAs
were significantly downregulated in CRPC with fold
change >2, compared with that in ADPC. EZH2
enhances tumorigenesis and is commonly upregulated in
various types of cancers including PCa. Its oncogenic
activity in cancer mainly depends on EZH2-mediatd
epigenetic modification, such as DNA methylation and
histone methylation, resulting in aberrantly silencing of
tumor suppressive genes in cancer. In order to
investigate these potential underexpressed miRNAs which were
possibly regulated by epigenetic modification, we
reanalyzed the GEO dataset (GSE26996) to identify EZH2
negatively-regulated miRNAs in DU145 [
to illustrate the potential miRNAs that can regulate
HOTAIR in prostate cancer, we searched miRcode
algorithm (http://www.mircode.org) and DIANA Tools
.php?r=lncBase/indexbio). Disciplinary overlaps was
showed by intersecting these three groups of miRNAs.
16 miRNAs were shared (Fig. 1a). By aligning these
miRNAs to MSKCC prostate cancer dataset (GSE21032)
], miR-193a was found to be one of the most
significantly downregulated miRNAs in metastatic PCa
tissues compared with primary cancer (Additional file 1:
Table S1). However, few studies have focused on the role
of miR-193a in PCa, thus prompting us to focus on it
for the present study.
MSKCC dataset includes 28 normal adjacent tissues
(NAT), 99 primary prostate cancer tissues (primary
PCA), and 14 metastatic prostate cancer tissues (MET).
As is shown in Fig. 1b, miR-193a was not significantly
underexpressed in primary prostate cancer tissues
(P = 0.0858). However, its expression in metastatic
cancer was significantly decreased compared to primary
cancer (P < 0.05). ROC analysis demonstrated that the
expression of miR-193a can be used to discriminate
between metastatic and primary prostate cancers (Fig. 1c).
To further validate its expression profile in PCa, the
largest TCGA-prostate adenocarcinoma database was
investigated and we found that miR-193a expression was
significantly downregulated in higher-T stage tumors
(Fig. 1d, P < 0.05). ROC analysis also showed that the
level of miR-193a could be used to discriminate between
pT3–4 and pT1–2 PCa (Fig. 1e). All results from
MSKCC and TCGA databases imply that miR-193a
represents a poor prognostic factor of prostate cancer.
To strengthen above findings, we evaluated the
miR193a expression in our clinical specimens via ISH and
found that lower level of miR-193a might be correlated
with tumor progression as miR-193a was aberrantly
downregulated in high-Gleason score tumors (Gleason
score: 8–10) (Fig. 1f, P < 0.05).
Taken together, all these results suggested that
aberrantly expressed miR-193a involves in the progression of
prostate cancer and may act as a tumor suppressor.
Biological effects of EZH2 on PCa cell growth and colony forming in a miR-193a-dependent manner
EZH2 was downregulated with si-EZH2 or
overexpressed with pcDNA3.1-EZH2 to evaluate its biological
effects on cell proliferation in PC3 and DU145 cell lines.
The knock-down or overexpression efficiency was
verified through western blot analysis (Fig. 2a-c). qRT-PCR
was used to assess the inhibitory efficiency for miR-193a
after EZH2 depletion and overexpression efficiency for
miR-193a after upregulation of EZH2 (Fig. 2d and e).
The CCK-8 assay was used to detect the importance of
miR-193a in EZH2-associated PCa cell growth and
proliferation. Ectopic expression of EZH2 significantly
promoted cell viability and proliferation at 48, 72 and
96 h (P < 0.05) (Fig. 2f and g). Functional experiments
were performed on the EZH2-overexpressing PCa cells
supplemented with miR-193a mimics to confirm
whether downregulation of miR-193a was required for
the EZH2 mediated increase in cell proliferation and
viability. Results indicated that miR-193a significantly
alleviated the promoting effect of EZH2 on cell growth
(Fig. 2f and g). Conversely, anti-miR-193a (miR-193a
inhibitor) could also partially abrogate the inhibition
effect of proliferation caused by knock-down of EZH2 in
PCa cells at 48, 72 and 96 h (P < 0.05) (Fig. 2h and i).
Similarly, EZH2 overexperssion significantly enhance cell
viability of PCa cells based on colony formation assay
and miR-193a mimics supplementary partially
eliminated the promoting effect. EZH2 depletion significantly
inhibited the proliferation of PCa cells while
anti-miR193a alleviated the inhibitory effect (Fig. 2j-l). GSEA
showed that a negatively enriched expression of gene
sets was involved in mitotic spindle assembly
(NES = −1.64, FDR = 0.02, P = 0.069) (Fig. 2m) and G2/
M cell-cycle checkpoint (NES = −2.0, FDR < 0.05,
P < 0.05) (Fig. 2n) in miR-193a-overexpressing cells.
Collectively, these results indicated that ectopic
expression of miR-193a inhibited cell viability and proliferation
of PCa cells and EZH2 could modulate cell growth at
least in a miR-193a-dependent manner.
EZH2 modulates apoptosis of PCa cells in a miR-193adependent manner and miR-193a suppresses PCa cell migration and invasion in vitro
EZH2 was downregulated by si-EZH2 and upregulated
by transfection with pcDNA3.1-EZH2 to assess the role
of EZH2 in PCa cell apoptosis. Meanwhile, the role of
miR-193a in PCa cell apoptosis was also explored via
transfection with miR-193a mimics after EZH2 depletion
or miR-193a inhibitor after overexpression of EZH2. No
significant difference was observed between cells
transfected with either pcDNA3.1-EZH2 or pcDNA3.1
(P > 0.05). EZH2 depletion increased the apoptosis rate
in both PC3 and DU145 cells which could be partially
rescued by supplementing the cells with anti-miR-193a
(apoptosis rate: 22.0 ± 0.6% vs.15.2 ± 0.5% in PC3 and
15.5 ± 0.4% vs. 9.6 ± 0.4% in DU145, P < 0.05). On the
other hand, upregulation of miR-193a in
EZH2overexpressing cells could significantly induce apoptosis
in PCa cells (apoptosis rate: 10.0 ± 0.6% vs.1.1 ± 0.2% in
PC3 and 9.7 ± 0.5% vs. 4.6 ± 0.3% in DU145, P < 0.05)
(Fig. 3a-c). To further elucidate the role of miR-193a on
PCa cell apoptosis, we conducted the TUNEL assay
(Additional file 2: Figure S3). TUNEL assay also showed
similar results as Annexin V-FITC/PI assay did. These
results suggested that EZH2 modulated the cell
apoptosis of PCa at least partially in a miR-193a-dependent
The aforementioned data linked the downregulation of
miR-193a to prostate cancer, especially metastatic
cancer, indicating that miR-193a may play a critical role
in PCa metastasis. To test this hypothesis, we stably
infected human metastatic prostate cancer cell lines (PC3
and DU145) with LV-miR-193a to upregulate the
miR-193a expression thereby assessing its impact on cell
migration and invasion by performing migration and
invasion assays. The results indicated that the
overexpression of miR-193a in DU145 and PC3 cell lines
strongly suppressed cell migration and invasion abilities
(Fig. 3d-g, P < 0.05). GSEA showed that a negatively
enriched expression of genes sets was involved in
hallmarks of transforming growth factor beta (TGF-β)
signaling (NES = −1.79, FDR = 0.01, P = 0.015) (Fig. 3h),
tumor necrosis factor alpha (TNF-α) via nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-kB)
signaling (NES = −1.77, FDR = 0.005, P = 0.017) (Fig. 3i) and
KRAS prostate up signaling (NES = −1.69, FDR = 0.08,
P = 0.203) (Fig. 3j) in miR-193a-overexpressing PCa cells. It
is well known that TGF-β signaling plays a significant role in
regulation of epithelial mesenchymal transition in PCa
by promoting migration and invasion abilities [
Moreover, NF-kB signaling could also be activated by
TNF-α and enhance the invasion ability of CRPC cells
in vitro [
]. Stable knock-down of KRAS signaling
has also been reported to suppress PCa cell migration
and invasion [
]. In summary, miR-193a could
markedly restrain the invasiveness of PC3 and DU145 cells
probably by negatively regulating of several
Ectopic expression of miR-193a suppresses the formation of prostate xenograft tumors in vivo
To investigate whether miR-193a possesses tumor
suppressive ability in vivo. We performed xenograft
tumor experiments in nude mice by monitoring tumor
incidence, latency and endpoint weight. Stably
overexpressing of miR-193a PC3 cells was generated by
infecting with lentivirus LV-miR-193a (Fig. 4a) and these cells
are then subsequently implanted into nude mice. Results
revealed that ectopic expression of miR-193a remarkedly
suppressed PCa tumor growth as manifested by reduced
tumor size and tumor weight (Fig. 4b-e).
We then carried out immunohistochemical staining of
Ki67, CD31 and CD34 in the xenograft tumors (Fig. 4f ).
The results illustrated that reduced Ki67-positive cells,
CD31-positive cells and CD34-positive cells were found
in miR-193a-overexpressed xenograft tumor cells. These
data suggested that miR-193a suppressed prostate tumor
regeneration and growth by inhibition of proliferation,
angiogenesis and invasion.
All together, these experiments further confirmed that
miR-193a serves as a suppressor in tumorigenesis of
EZH2 coupled with HOTAIR to silence miR-193a through introducing trimethylation of H3K27 at miR-193a promoter region
The in vitro studies have suggested that the oncogenic
activities of EZH2 could be contributed by loss of
miR193a in CRPC cell lines. In order to prove that EZH2
had a direct role in repressing miR-193a in PC3 and
DU145, we depleted EZH2 by stably infection of
lentiviral particles of LV-shEZH2 in PCa cells, and measured
the level of miR-193a. Upon inhibition of EZH2,
there were upregulation of miR-193a expression in
PC3 and DU145 cells (Fig. 5a1-a2, P < 0.05).
Conversely, we observed that ectopic expression of
EZH2 could significantly reduce the level of
miR193a expression (Fig. 5b1-b2, P < 0.05) when
overexpressing EZH2 by transfecting pcDNA3.1-EZH2 into
PC3 and DU145 cells.
It is reported that EZH2 as a component of PRC2
coupled with lncRNA HOTAIR during the gene
silencing of tumor suppressor, and the HOTAIR-dependent
recruitment of PRC2 was often observed in region
enriched with CpG islands [
]. HOTAIR is upregulated
in CRPC cell lines and tissues [
], and it is
specifically highly expressed in metastatic PCa, we hypothesized
that HOTAIR played a role in guiding EZH2 to
miR193a promoter region via recognizing the CpG island
(Fig. 5g). To prove that HOTAIR was important in
EZH2 mediated silencing of miR-193a, we inhibited the
HOTAIR expression by siRNAs in EZH2-overexpressing
PCa cells. qRT-PCR indicated that HOTAIR expression
was significantly reduced by HOTAIR siRNAs, and
inducing higher expression of miR-193a even when
EZH2 was overexpressed in PC3 and DU145 cells
(Fig. 5c1-c4, P < 0.05). These results suggested that
EZH2 might couple with HOTAIR to suppress miR-193a
in PCa cells.
As we had shown that HOTAIR and EZH2 were
critical in repressing miR-193a and silencing of
miR193a contributed to PCa tumorigenesis, we tried to
reveal the underlying suppression mechanism of
EZH2 on miR-193a. We hypothesized that HOTAIR
regulates transcription by directing PRC2 complex via
controlling the epigenetic state and downstream gene
expression. Western blot assay showed that the
enrichment of H3K27me3 significantly decreased when
EZH2 was knocked down in LV-shEZH2 infected PCa
cells and vice versa (Fig. 5d and e; P < 0.05). To test
that HOTAIR played a vital role in EZH2-mediated
induction of trimethylation of H3K27, we examined
expression of EZH2 and H3K27me3 in PCa cells
under HOTAIR expression interference. Results
showed that both expression of EZH2 and H3K27me3
largely declined in the HOTAIR knocking down PCa
cells (Fig. 5f1-f2; P < 0.05).
Moreover, we cloned 2.0 kb fragments upstream of TSS
of human pri-miR-193a stem-loop into pGL3-basic-vector
to generate luciferase construct pGL3-193a-promoter
reporter. Then we carried out luciferase reporter assay by
cotransfecting pGL3-193a-promoter and si-EZH2 or
pcDNA3.1-EZH2 into PC3 and DU145 cells. As expected,
co-transfection of the two cell lines with
pGL3-193apromoter and si-EZH2 led to increased luciferase activity
significantly. In contrast, co-transfection with
pGL3-193apromoterand pcDNA3.1-EZH2 markedly reduced the
luciferase activity in the same cells (Fig. 5h1-h2, P < 0.05).These
results suggested that EZH2 directly regulates miR-193a
activity through its promoter. To address whether HOTAIR
regulates miR-193a via EZH2, ChIP assays were conducted
to measure the enrichment levels of EZH2 and H3K27me3
at the promoter region of miR-193a. GEO datasets
(GSM1383872 and GSM1138596) were reanalyzed to
determine the binding site of H3K27me3 at miR-193a
promoter region in PC3 and DU145 cells (Fig. 5g). We
observed that silencing of HOTAIR led to reduction of
EZH2 occupancy and H3K27me3 level at the miR-193a
promoter region (Fig. 5i1-i2; P < 0.05). It suggests that
HOTAIR was required for gene repression effect of EZH2
to miR-193a. Taken together, we showed that EZH2
coupled with HOTAIR to induce H3K27 trimethylation at
miR-193a promoter, which reduced miR-193a expression
in PC3 and DU145 cells (Fig. 5j).
Mir-193a directly targets HOTAIR and negatively modulates its expression in PCa
As miRcode algorithm and DIANA Tools showed
HOTAIR has miR-193a binding site (Fig. 6e), we
wondered whether miR-193a could directly modulate
HOTAIR expression. First, we observed ectopic
miR193a expression reduced HOTAIR level in PCa cells
either via transfecting with miR-193a mimics or
infecting with LV-miR-193a (Fig. 6a and b; P < 0.05).
Second, HOTAIR expression level was significantly
decreased in xenograft tumor tissues which were
generated from PC3 cells stably overexpressing miR-193a
(Fig. 6c and d; P < 0.05). Third, we subcloned full
length cDNA sequence of HOTAIR (WT) or HOTAIR
(Mut) into psi-CHECK™-2 vector (Fig. 6e), and
cotransfected with miR-193a mimics or scramble mimics
into PC3 and DU145 cells. The luciferase reporter
assay demonstrated that miR-193a significantly
reduced luciferase activity (wild type). Mutation of
miR-193a binding site in HOTAIR abrogated the
inhibitory effects (Fig. 6f, P < 0.05). Altogether, above
results clearly indicate that miR-193a directly targets
HOTAIR and negatively modulates HOTAIR
expression in prostate cancer.
Taken all together, we found
HOTAIR/EZH2/miR193a feedback loop in PCa, that HOTAIR is required for
EZH2 mediated miR-193a silencing, which in return
upregulates HOTAIR expression. In order to further
investigate their relationship, we evaluated EZH2,
miR-193a and HOTAIR expression through IHC and
ISH staining in 31 PCa specimens (Fig. 6g). Spearman
correlation analysis showed significant inverse
correlation between HOTAIR and miR-193a (r = −0.67,
P < 0.001) as well as between EZH2 and miR-193a
(r = −0.59, P < 0.001) (Fig. 6i). These clinical expression
data further supported the HOTAIR/EZH2/miR-193a
negative feedback loop.
Here, we identified a HOTAIR-triggered feedback
loop that involves EZH2-mediated repression of
miR193a and controls tumorigenesis and prostate cancer
progression. This study also provides what we believe
is the first in vitro and in vivo proof for a
tumorsuppressive function of miR-193a in prostate cancer.
We also illustrated a interesting cross-regulation
between miR-193a and oncogene HOTAIR that
miR193a directly targets HOTAIR and modulates its
expression in prostate cancer. These findings
demonstrated the vital role of HOTAIR/EZH2/miR-193a
feedback loop in progression of prostate cancer.
The dysregulation of miR-193a has been frequently
reported in various types of cancers. Previous studies
indicated that miR-193a was markedly downregulated
in oral carcinoma, lung cancer, colorectal cancer, and
malignant pleural mesothelioma and served as a
tumor suppressor in these cancers [
miR-193a also exhibited the promoting effects of
chemoresistance in bladder cancer and oesophageal
]. These findings implied that miR-193a
could function either as tumor suppressor or multiple
drug resistance gene in a cellular context-dependent
manner. Though recently Jia L et al. reported that
androgen receptor-regulated miR-193a in C4-2B cells
targets AJUBA and promotes migration ability in
AR-positive prostate cancer cell lines [
the expression profile of miR-193a in clinical PCa
specimens and the exact biological function of
miR-193a in prostate cancer have not been
systematically studied. In our current study, we discovered
that miR-193a levels were significantly
downregulated in metastatic prostate cancer via reanalysis of
MSKCC and TCGA datasets, and its expression was
negatively correlated with the advanced stages and
high gleason scores of PCa patients. Further, from
the in vitro and in vivo studies, we showed that
miR-193a exhibited its abilities to inhibit cell
proliferation, induce apoptosis, suppress migration,
invasion and xenograft tumor growth. These results
suggested that miR-193a behaved as a tumor
suppressor in tumorigenesis of CRPC.
The molecular mechanism that underlie aberrant
expression of miRNAs may result from DNA
hypermethylation, histone modification, induction of
heterochromatin, and alteration of Dicer abundance [
It has been reported that miR-193a downregulation is
associated with DNA methylation in acute myeloid
leukemia, hepatocellular carcinoma, non-small cell
lung cancer, and oral cancer [
]. To date, little
is known about the reason underlying miR-193a
silencing in CRPC cells. Cao Q et al. demonstrated
that a group of miRNAs was upregulated by EZH2
knock-down in DU145 cell including miR-193a ,
implicating its downregulation in PCa may result
from epigenetic modification. Through qRT-PCR and
western blot analysis, we showed that H3K27
methyltransferase EZH2 played a vital role in suppression of
miR-193a expression in CRPC cells. Luciferase
reporter assay revealed that EZH2 could directly bind
to the promoter of miR-193a and induce
trimethylation of H3K27 thereby silencing miR-193a expression.
The interaction between HOTAIR and PRC2 was
critical for the regulation of target gene silencing. Tsai
et al. demonstrated that PRC2 directly binds to the
5′-end of HOTAIR (1-300 nt). HOTAIR functioned as
modular scaffold, guided EZH2 and recruited them to
the target gene loci and silence the transcription
through trimethylation of H3K27 [
]. Li et al.
investigated the binding interactions between PCR2 and
HOTAIR, and showed that a loss of miR-34a
suppression was observed in HPDE cells expressing HOTAIR
lack of EZH2-interacting region, indicating the crucial role
of HOTAIR-EZH2 interaction in gene silencing [
this study, we observed that knock-down of HOTAIR led
to reduced enrichment of EZH2 and H3K27me3 at the
miR-193a promoter region. Interestingly, depletion of
HOTAIR could still increase the miR-193a expression
even in EZH2-overexpressing PCa cells, implying
EZH2mediated miR-193a suppression was dependent on
HOTAIR. Previous study has illustrated that enrichment
of the CG-rich motifs in CpG islands was critical for
HOTAIR-dependent recruitment of PRC2 in target gene
]. A stretch of CpG island could be observed at
the promoter region of miR-193a which is important for
HOTAIR recognition and subsequently guiding
EZH2associated gene repression. Collectively, EZH2 coupled
with HOTAIR to suppress miR-193a expression by
epigenetic modification in CRPC cells.
MiRNAs are known as critical modulators of gene
expression via post-transcriptional gene silencing. The
abundance of numerous lncRNAs could also be
regulated by miRNAs [
]. Chiyomaru et al. revealed
that miR-141 could directly bind to HOTAIR in a
sequence-specific manner and suppress HOTAIR
expression, thus inhibiting proliferation and invasion of
cancer cells [
]. Tao et al. demonstrated that HOTAIR
was a direct target of miR-148 and was inhibited by
overexpression of miR-148 in breast cancer [
addition, miR-34a was also shown to lower the stability
of HOTAIR in PC3 and DU145 cells [
]. In present
study, we found that HOTAIR has the target sequences
of miR-193a. Our luciferase reporter assay and real-time
PCR results showed that miR-193a binds to the
HOTAIR and downregulates HOTAIR expression in
PCa cell lines. This study is the first to demonstrate that
miR-193a directly targets HOTAIR in both PC3 and
DU145 PCa cells.
As miR-193a was epigenetic silenced by EZH2 and
HOTAIR, in return the suppression effect of HOTAIR
by miR-193a was largely abrogated. Thus, the tumor
suppressor miR-193a was continuously inhibited and
oncogene HOTAIR remained highly expressed in
prostate cancer, which promoting initial prostate cancer
developed into highly aggressive cancer type. The
clinical expression of these genes in tumor tissues further
verified the regulatory mechanism of HOTAIR/EZH2/
miR-193a feedback loop in prostate cancer. Therefore,
targeting this novel feedback loop would be a promising
treatment strategy to prostate cancer.
In summary, we found that miR-193a is downregulated
in metastatic prostate cancer and plays tumor
suppressive function in prostate cancer, which was attributed to
HOTAIR mediated EZH2 targeting to the promoter of
miR-193a. Moreover, HOTAIR is a direct negative target
of miR-193a. Thus, a HOTAIR/EZH2/miR-193a
feedback loop is formed, which plays a vital role in the
progress of CRPC and may be a promising therapy
target for PCa treatment.
Additional file 1: Table S1. expression profile of miRNAs with a
statistically significant (P < 0.05) change in MET and PCA via reanalysis of
MSKCC dataset. (PDF 206 kb)
Additional file 2: Figure S3. TUNEL apoptosis assays were applied to
show the modulation of apoptosis by EZH2 and miR-193a in PC3 and
DU145 cells. (TIFF 1131 kb)
ADPC: Androgen dependent prostate cancer; ChIP: Chromatin
immuoprecipitation; CRPC: Castration-resistant prostate cancer;
EZH2: Enhancer of zeste homolog 2; GEO: Gene Expression Omnibus;
GSEA: Gene Set Enrichment Analysis; H3K27me3: Trimethylation of histone
H3 lysine 27; HOTAIR: HOX transcript antisense RNA; lncRNA: Long
noncoding RNA; MSKCC: Memorial Sloan Kettering Cancer Center; NF-kB: Nuclear
factor kappa-light-chain-enhancer of activated B cells; PRC2: Histone
methyltransferase polycomb repressor complex 2; TCGA: The Cancer
Genome Atlas; TGF-β: Transforming growth factor beta; TNF-α: Tumor
necrosis factor alpha; TSS: Transcription start site
We thank Dr. Liu Xiufang from MD Anderson Cancer Center for his
significant editorial assistance and input.
This work was supported by grants from National Natural Science
Foundation of China (NO. 81672551, 81,572,517, 81,370,849, 81,300,472),
Natural Science Foundation of Jiangsu Province (BK20161434, BL2013032,
BK20150642 and BK2012336), Six talent peaks project in Jiangsu Province,
Jiangsu Provincial Medical Innovation Team (CXTDA2017025), Jiangsu
Provincial Medical Talent (ZDRCA2016080), Jiangsu Provincial Medical Youth
Talent (QNRC2016821, QRNC2016820), Fundamental Research Funds for the
Central Universities and the Scientific Research Innovation Program for
College and University Graduates of Jiangsu Province (KYZZ16_0135).
Availability of data and materials
The datasets generated or analyzed during this current study are
available in MSKCC, TCGA and GEO databases with the accession
number cited in the article.
BX and MC supervised the whole project, designed the experiments, and
analyzed the data. ZL performed the experiments, analyzed the data, wrote
the manuscript, and prepared the figure. XW, TT and LZ contributed to the
experiments and analyzed the data. HG, ZY, KL, GZ, SC, JW, JQ and HL
contributed to the experiments and provided technical support. All authors
read and approved the final manuscript.
Ethics approval and consent to participate
All animal experiments were approved by the Animal Center of Southeast
University and performed following International Guidelines and Protocols.
The patients were informed about the study and were asked to sign
consents for their tissues used in the scientific research, and this research
also obtained approval from ethics of Zhongda Hospital Affiliated with
Consent for publication
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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