FEBS Letters 589 (2015) 645–650

journal homepage: www.FEBSLetters.org

MiR-204, down-regulated in retinoblastoma, regulates proliferation

and invasion of human retinoblastoma cells by targeting CyclinD2
and MMP-9
XianJin Wu a,1, Yong Zeng b,1, ShaoKe Wu b, JiXin Zhong c, YuZhou Wang c, JunFa Xu d,⇑
a
Department of Clinical Laboratory, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, People’s Republic of China
b
Orthopedic Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, People’s Republic of China
c
Oncology Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, People’s Republic of China
d
Institute of Laboratory Medicine, Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan, Guangdong, People’s Republic
of China

a r t i c l e i n f o a b s t r a c t

Article history: Aberrant expression of miR-204 had been frequently reported in cancer studies; however, the mech-
Received 17 August 2014 anism of its function in retinoblastoma remained unknown. Here, we reported that miR-204 was
Revised 14 January 2015 frequently downregulated in retinoblastoma tissues and cell lines. Enforced expression of miR-
Accepted 22 January 2015
204 inhibited retinoblastoma cells’ proliferation and invasion. In vivo study indicated that restora-
Available online 31 January 2015
tion of miR-204 inhibited tumor growth. CyclinD2 and MMP-9 were identified as potential targets of
Edited by Tamas Dalmay miR-204. In addition, a reverse correlation between miR-204 and CyclinD2 or MMP-9 expression was
noted in retinoblastoma tissues. Taken together, our results identified a crucial tumor suppressive
Keywords:
role of miR-204 in the progression of retinoblastoma.
miR-204 Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
CyclinD2
MMP-9
Retinoblastoma

1. Introduction oncogenes or tumor suppressor genes [5]. Interestingly, recent

Retinoblastoma (Rb), a deadly pediatric eye cancer, is the most
common intraocular tumor in children [1]. The mortality rate
among children diagnosed with Rb is 50–70% in the underdevel-
oped countries [2]. Children with Rb are at risk for three life-
threatening problems, including metastasis of Rb, intracranial
neuroblastic malignancy (trilateral Rb), and second primary
tumors. In order to improve the therapeutic outcome of patients
with retinoblastoma, there is an urgent need to further study the
biology and molecular mechanisms of retinoblastoma and identify
the specific biomarkers that cause tumor progression.
MicroRNAs (miRNAs), which are single-stranded 19–25 nucleo-
tide short RNAs, function as negative posttranscriptional regulators
of target genes [3]. MiRNAs play significant role in lots of physio-
logical and pathological process, such as differentiation, prolifera-
tion, metabolism, and death [4]. In addition, accumulating data
suggests that miRNAs are frequently dysregulated and play critical
roles in various types of cancers through acting as potent
⇑ Corresponding author. Fax: +86 76922896377.
E-mail address: junfaxu001@126.com (J. Xu).
1
XianJin Wu and Yong Zeng contributed equally to this work.
studies show that miRNAs were involved in the development of
retinoblastoma. For example, Mu et al. report reduced expression
of the tumor suppressor microRNA let-7 in retinoblastoma [6].
Conkrite et al. showed that overexpression of miR-17–92 is impor-
tant in the formation of a cohort of retinoblastomas [7]. The miR-
34 family, which expression is usually downregulated in many
cancer cell lines, is variably expressed in retinoblastomas. Restora-
tion of its expression can inhibit the proliferation of retinoblas-
toma cells [8]. In addition, some plasma miRNA levels are found
to be of value in RB diagnosis [9]. These findings suggest that tar-
geting the miRNAs may potentially lead to a novel strategy of diag-
nose and therapy to retinoblastomas.
A number of studies have demonstrated that deregulation of
miR-204 expression is involved in the initiation and progression
of cancer by affecting tumor cell function, including growth, inva-
sion and metastasis [10,11]. However, the expression of miR-204 in
human retinoblastoma patients, and its functions in human retino-
blastoma cells, as well as the molecular mechanisms by which
miR-204 exerts its functions, has not been fully understood. In
the present study, we sought to investigate the potential role of
miR-204 in the development and progression of retinoblastoma.
We identified that miR-204 could regulate the proliferation and

http://dx.doi.org/10.1016/j.febslet.2015.01.030
0014-5793/Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
646 X. Wu et al. / FEBS Letters 589 (2015) 645–650
invasion of retinoblastoma cells by directly targeting the CyclinD2
and MMP-9 genes.
2. Materials and methods
2.1. Cell lines and patient samples
Human retinoblastoma cell lines were purchased from the Insti-
tute of Biochemistry and Cell Biology of the Chinese Academy of
Sciences (Shanghai, China). The identities of the cell lines were val-
idated by STR profiling. The STR profiling of SO-RB50 cell line was
presented in Supplement Table 2. All cell lines were maintained in
a humid wet atmosphere containing 5% CO2 at 37 °C in RPMI-1640
medium supplemented with 10% newborn bovine serum, 100 U/
mL penicillin, and 100 mg/mL streptomycin. Retinoblastoma tis-
sues and normal retina tissues were obtained from the Center of
Ophthalmology, Affiliated Hospital of Guangdong Medical College,
Zhanjiang, Guangdong, China. This study was approved by the
institute research ethics committee of Affiliated Hospital of
Guangdong Medical College.
2.2. RNA isolation and quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, Carls-
bad, California, USA). For qPCR, RNA was reverse transcribed to
cDNA from 1 lg of total RNA using a Reverse Transcription Kit
(Takara). Real-time PCR analyses were conducted with Power SYBR
Green (Life Technologies). Real-time PCR was performed on an ABI
Prism 7000 Sequence Detection System with SYBR Premix Ex Taq
(TaKaRa). Results were normalized to the expression of U6 or GAP-
DH. Melt curve analysis was used to assess qPCR amplicon length
with intercalating dye qPCR assays. The primers used for miR-204
were 50 -GGGCTTCCCTTT GTCATCCT-30 (forward) and 50 -GTGCAGGG
TCCGAGGT-30 (reverse). The primers for U6 50 -CTCGCTTCGGCAGC
ACA-30 (forward) and 50 -AACGCTTCACGAATTTGCGT-30 (reverse).
The primers for GAPDH were 50 -AACGTGTCAGTGGTGGACCTG-30
(forward) and 50 -AGTGGGTGTCGCTGTTGAAGT-30 (reverse). The
primers for CyclinD2 were 50 -ACCTTCCGCAGTGCTCCTA-30 (for-
ward) and 50 -CCCAGCCAAGAAACGGTCC-30 (reverse). The primers
for MMP9 were 50 - TGTACCGCTATGGTTACACTCG-30 (forward) and
50 -GGCAGGGACAGTTGCTTCT-30 (reverse). The TaqMan PCR assay
was used to measure the mature miR-204 and the primers were
used as previously described [12].The relative expression of each
gene was calculated and normalized using the 244Ct method
relative to U6 snRNA or GAPDH.
2.3. Plasmid, microRNA mimic, lentivirus production and infection
Full-length CyclinD2 and MMP-9 cDNA entirely lacking the
30 -UTR was subcloned into the eukaryotic expression vector
pcDNA3.1(+) (Invitrogen). The CyclinD2 and MMP-9 30 UTR target
site for miR-204 were amplified by PCR cloned into the XbaI site
of pGL3 control (Promega, Madison, USA). This vector was
sequenced and named WT 30 UTR. Site-directed mutagenesis of
the miR-204 target-site in the CyclinD2 and MMP-9 30 UTR was
carried out using the Quick-change mutagenesis kit (Strata-gene,
Heidelberg, Germany) and named MT 30 UTR. The sequences
of miR-204 mimic 50 -UUCCCUUUGUCAUCCUAUGCCU-30 (forward)
and 50 -GCAUAGGAUGACAAAGGGAAUU-30 (reverse). The
sequences of miR-204 mimic control were 50 -UUCUCCGAACGUGU
CACGUTT-30 (forward) and 50 -ACGUGACACGUUCGGAGAATT-30
(reverse). They were chemically synthesized by Shanghai
GenePharma Company (Shanghai, China). The sequences targeting
human CyclinD2 mRNA 50 -UGCUCCUCAAUAGCCUGCAGCAGUA-30
and the si-negative control (si-NC) were designed by RiboBio
(RiboBioInc, China). Oligonucleotide transfection was performed
with Lipofectamine 2000 reagent (Invitrogen).
The pLV-has-miR-204 plasmid and the negative control pLV-
miRNA-vector were purchased from Biosettia Inc. (Biosettia, San
Diego, USA). Viral packaging and infection were performed accord-
ing to standard protocols as recommended by the manufacturer.
The packaged lentiviruses were named LV-miR-204. The empty
lentiviral vector LV-ctrl was used as a control.
2.4. Luciferase reporter assay
For the reporter assays, WT or MT 30 UTR vector and the control
vector pRL-CMV ((cytomegalovirus) coding for Renilla luciferase,
Promega) were cotransfected. We performed the luciferase assays
using Y79 cells transiently transfected with Renilla constructs (as
an internal control) or plasmids pMIRGLO-CylinD2-30 UTR-WT or
pMIRGLO-CylinD2-30 UTR-MT with or without miR-204 mimic or
miR-204 mimic control using the Dual Luciferase Assay system fol-
lowing the manufacturer’s instructions (Promega, Madison, WI).
Luciferase activity was measured 36 h after transfection. All lucif-
erase activity readings were normalized relative to the activity of
the Renilla luciferase control and the results were expressed as rel-
ative luciferase activity (Firefly LUC/Renilla LUC). All experiments
were performed in triplicate at least 3 times independently.
2.5. MTT, colony formation, migration and invasion assays
The MTT assay and colony formation assays were carried out as
described previously [13,14]. Cell migration and invasion assays
were carried out according to previous description [15]. For cell
migration assays, 1  104 cells in 100 ml PRMI-1640 medium with-
out FBS were seeded on a fibronectincoated polycarbonate mem-
brane insert in a transwell apparatus (Costar, MA). In the lower
chamber, 600 ml PRMI-1640 medium with 10% FBS was added as
a chemoattractant. After the cells were incubated for 8 h at 37 °C
in a 5% CO2 atmosphere, the insert was washed with PBS, and cells
on the top surface of the insert were removed with a cotton swab.
Cells adhering to the lower surface were fixed with methanol,
stained with Giemsa solution and counted under a microscope in
five predetermined fields (200). For cell invasion assays, the pro-
cedure was similar to the cell migration assay, except transwell
membranes were precoated with 24 lg/ll Matrigel (R&D Systems,
USA). Cells adhering to the lower surface were counted the same
way as the cell migration assay.
2.6. Western blot assay
Protein samples were resolved by 10% SDS–PAGE and then
transferred to PVDF membranes. The membranes were blocked
and probed with antibodies against CyclinD2 (C-17, sc-81, Santa
Cruz Biotechnology, Inc.), MMP-9 (2c3, sc-21733, Santa Cruz Bio-
technology, Inc.) and GAPDH (G-9, sc-365062, Santa Cruz Biotech-
nology, Inc.), followed by probed with the secondary antibodies
accordingly. The dilution of the primary antibodies were 1:500,
while the dilution of the secondary antibodies were 1:1000. Band
detection via enzyme-linked chemiluminescence was performed
according to the manufacturer’s protocol (ECL; Pierce Biotechnol-
ogy Inc., Rockford, USA).
2.7. In vivo growth assay
Five animals for each group were used for these experiments.
For the in vivo tumor growth studies, 1  106 Y79 cells, stably
expressing miR-204, or the control vector, were injected subcuta-
neously in the upper back of BALB/C-nu/nu athymic nude mice.
 X. Wu et al. / FEBS Letters 589 (2015) 645–650
After 21 days, tumor samples were carefully removed and
weighed. All animal procedures were performed in accordance
with institutional guidelines.
2.8. Statistical analysis
Statistical analysis was performed using a SPSS software pack-
age (SPSS Standard version 16.0, SPSS Inc). Data were expressed
as the mean ± S.D. from at least three independent experiments.
Student’s t-test was used for comparison between two groups,
and one-way ANOVA was used for comparison among multiple
groups. Correlation was analyzed by Spearman test. p Values < 0.05
were considered significant.
3. Results
3.1. miR-204 expression is down-regulated in primary retinoblastoma
tissues and cell lines
Real-time PCR was performed to examine the expression levels
of miR-204 in a subset of 26 primary human retinoblastoma
tissues as compared to 8 normal pediatric retinas (patient
demographics can be found in Supplementary Table 1). As shown
in Fig. 1A, the expression levels of miR-204 were significantly
decreased in retinoblastoma tissues compared to normal pediatric
retinas (p < 0.05). Next, we evaluated the expression of miR-204 in
three human retinoblastoma cell lines (Y79, WERI-RB-1, and
SO-RB50). In comparison to 8 normal pediatric retina controls,
miR-204 was down-regulated in these cell lines (Fig. 1B). We also
detected the mature miR-204 by TaqMan PCR in the human retino-
blastoma tissues. We found that there was a positive correlation
between pre-miR-204 and mature miR-204 (Supplement Fig. 3).
3.2. miR-204 regulates cell proliferation, migration and invasion
in vitro and in vivo
First, miR-204 mimic and miR control were transiently transfec-
ted into Y79 cells, qRT-PCR was used to confirm miR-204 overex-
pression (Fig. 2A). The MTT and colony formation assays were
performed to investigate the effect of miR-204 in retinoblastoma
cell proliferation. The results showed that restoration of miR-204
significantly inhibited the proliferation of Y79 cells (Fig. 2B). Inter-
estingly, silencing of CyclinD2 mimicked the effect of miR-204 on
cell growth in vitro (Supplementary Fig. 1A–C). To examine the
effect of miR-204 on cell migration, Y79 cells transfected with
miI-204 mimic or miR control were cultured on transwell
apparatus. We found that the percentage of migrated cells was
Fig. 1. miR-204 expression is down-regulated in primary retinoblastoma tissues and cell lines. (A) The expression levels of miR-204 were significantly decreased in
retinoblastoma tissues compared to normal pediatric retinas (student’s t-test was used and p-values less than 0.05 indicated statistically significant difference). (B) miR-204
was down-regulated in retinoblastoma cell lines, when compared with normal pediatric retinas (one-way AVOVA was used for comparison among these groups).
647
significantly less in miR-204-mimics transfected group, when com-
pared with the control group (Fig. 2C). Using a boyden chamber
coated with matrigel, we determined changes in cell invasiveness.
When compared with the control group, Y79 cells transfected with
miI-204 mimic exhibited significantly decreased invasiveness
(Fig. 2D). Interestingly, SO-RB50 cells transfected with miR-204
also exhibited significantly decreased proliferation and invasive-
ness (Supplementary Fig. 2A–C).
Moreover, we investigated the effects of miR-204 on growth
and invasion of Y79 cells in vivo. We initially infected Y79 cells
with lentiviral vectors expression miR-204 stablely (LV-miR-204)
or the negative control (LV-Ctrl). Stable transfection of miR-204
into Y79 cells resulted in decreased growth and tumor weight of
subcutaneous xenograft tumors in nude mice, when compared
with those stably transfected with empty vector (Fig. 2E). These
results suggested that miR-204 could inhibit the growth of retino-
blastoma cells in vivo.
3.3. CyclinD2 and MMP-9 are potential direct targets of miR-204 in
retinoblastoma cells
It is generally accepted that miRNAs exert their function by reg-
ulating expression of their downstream target genes. Thus, puta-
tive miR-204 targets were predicted by using target prediction
programs, miRanda and TargetScan. Our analysis revealed that
CyclinD2 and MMP-9 were two potential targets of miR-204. The
30 -UTR of CyclinD2 and MMP-9 mRNA contains a complementary
site for the seed region of miR-204 (Fig. 3A).
To verify whether or not CyclinD2 and MMP-9 are direct targets
of miR-204, CyclinD2 and MMP-9 wild-type 30 -UTRs and mutants
containing the miR-204 binding sites were cloned downstream of
the luciferase open reading frame. The results showed that miR-
204 significantly inhibited the luciferase activity of the CyclinD2
and MMP-9 WT 30 -UTR but not that of the mutant in Y79 cells
(Fig. 3B). To directly assessed the effect of miR-204 on CyclinD2
and MMP-9 expression, we transfected miR-204 into Y79 cells
and found that overexpression of miR-204 reduced the CyclinD2
and MMP-9 mRNA and protein levels (Fig. 3C and D). Taken
together, these results indicated that CyclinD2 and MMP-9 were
direct targets of miR-204 in retinoblastoma cells.
3.4. miR-204 levels are inversely correlated with mRNA expression of
CyclinD2 and MMP-9 in retinoblastoma tissues
We further examined the mRNA expression of CyclinD2 and
MMP-9 in the 26 cases of retinoblastoma tissues used before. As
shown in Fig. 4A and B, when CyclinD2 or MMP-9 mRNA levels
648 X. Wu et al. / FEBS Letters 589 (2015) 645–650

Fig. 2. miR-204 regulates cell proliferation, migration and invasion in vitro and in vivo. (A) qRT-PCR assay confirmed that miR-204 was overexpressed in miR-204 mimic-
transfected cells, compared with those transfected with miR control. (B) miR-204 inhibited Y79 cell proliferation, as determined by MTT assay (left panel) and colony
formation (right panel). (C) miR-204 decreased migration ability of Y79 cells, as determined by transwell assay. (D) The boyden assay demonstrated that miR-204 impaired
Y79 cells’ invasion ability. (E) Stable transfection of miR-204 into Y79 cells resulted in decreased growth and tumor weight of subcutaneous xenograft tumors in nude mice,
when compared with those stably transfected with empty vector. (Student’s t-test was used and p-values less than 0.05 indicated statistically significant difference.)

were plotted against miR-204 expression, a significant inverse cor-
relation was observed (2-tailed Spearman’s correlation, p = 0.000
and 0.002, respectively).
4. Discussion
Despite the advancements in treatment options, improvements
in retinoblastoma patient survival have been limited owing to lack
of early detection. Biomarkers to improve retinoblastoma diagno-
sis, prognosis and prediction of treatment response therefore rep-
resent opportunities to improve patient outcome. In recent years,
investigation of epigenetic biomarkers such as miRNA expression,
has implicated that these alterations may be enticing translational
biomarker candidates in retinoblastoma [16,17]. It is well
documented that microRNAs could contribute to cancer
development by acting as oncogenes or tumor suppressor genes.
Recently, we and other researchers have found that miR-204 func-
tioned as a tumor suppressor in a subset of cancer [11,18–21].A
previous study had demonstrated the significant role of miR-204
in controlling vertebrate eye development. The ablation of miR-
204 expression could result in an eye phenotype characterized by
microphthalmia, abnormal lens formation, and altered dorsoven-
tral (D-V) patterning of the retina [22]. However, the underlying
mechanism of miR-204 in retinoblastoma has not been explored.
In this study, we found that the expression of miR-204 in reti-
noblastoma specimens was significantly lower than that in normal
pediatric retinas. Interestingly, the expression of miR-204 in
human retinoblastoma cell lines was also down-regulated. Subse-
quently, we found that restoration of miR-204 in retinoblastoma
cells inhibited cell growth in vitro. In addition, restoration of
miR-204 significantly decreased the migration and invasion capac-
ity of retinoblastoma cells. Furthermore, we observed that miR-204
decreased cell growth in vivo. Our results were analogous to an
emerging body of literature, which suggested that miR-204 func-
tioned as a tumor suppressor [23–25].
Subsequently, CyclinD2 and MMP-9 were identified as potential
functional targets of miR-204. Our results showed that miR-204
bound the complementary sites of 30 -UTR of both CyclinD2 and
 X. Wu et al. / FEBS Letters 589 (2015) 645–650 649

Fig. 3. CyclinD2 and MMP-9 are potential direct targets of miR-204. (A) The 30 -UTR of CyclinD2 and MMP-9 mRNA contains a complementary site for the seed region of miR-
204. (The MT sequence, which were indicated by blue color, represent mutants containing the miR-204 binding sites.) (B) miR-204 significantly inhibited the luciferase
activity of the CyclinD2 (left panel) and MMP-9 (right panel) WT 30 -UTR but not that of the mutant in Y79 cells. (One-way AVOVA was used for comparison among these
groups.) (C) Restoration of miR-204 reduced the CyclinD2 (left panel) and MMP-9 (right panel) mRNA levels (student’s t-test was used and p-values less than 0.05 indicated
statistically significant difference). (D) Overexpression of miR-204 reduced the CyclinD2 (left panel) and MMP-9 (right panel) protein levels.

Fig. 4. miR-204 levels are inversely correlated with mRNA expression of CyclinD2 and MMP-9 in retinoblastoma tissues (correlation was analyzed by Spearman test).

MMP-9, and dramatically decreased their expression. In addition, a
significant inverse correlation between the levels of miR-204 and
mRNA expression of CyclinD2 and MMP-9 was found in human
retinoblastoma tissues. These observations provide the first line
of evidence, to our knowledge, that miR-204 mechanistically acts
via the regulation of CyclinD2 and MMP-9. Elevated CyclinD2 has
been observed in various cancers and aberrant expression of Cyc-
linD2 often leads to proliferation of cancer cells [26,27]. In addi-
tion, our data suggested that silencing of CyclinD2 inhibited the
growth of retinoblastoma cells in vitro. Metastasis is a complex
process of tumor cell invasion to new tissues, involving the coordi-
nation of several signaling pathways that allow changes in cell
morphology, changes in adhesion and migration capabilities
between the cells and the extracellular matrix (ECM) and changes
in cell–cell interaction [28,29]. Degradation of the ECM by cancer
cells is mediated via protease, such as matrix metalloproteinases
(MMPs), serine proteinase, and cathepsins [30,31]. MMP-9 is one
of the most vital enzymes for the degradation of the main constit-
uent of the basement membrane, and is therefore involved in can-
cer invasion and metastasis [32]. A previous report found that
MMP-9 was significantly higher in retinoblastoma tissue than in
normal retina and suggested that MMP-9 could be connected to
the invasion and development of retinoblastoma cells [33]. In the
present study, we speculated that miR-204 may modulate prolifer-
ation and invasion, at least partly, by regulating CyclinD2 and
MMP-9 expression in retinoblastoma cells. Interestingly, CyclinD2
and MMP-9 expression may be regulated by other mechanism in
retinoblastoma. For instance, CyclinD2 was negatively regulated
by let-7 [34], which was often down-regulated in retinoblastoma
[6]. Further research is needed for deeper understanding of the
650 X. Wu et al. / FEBS Letters 589 (2015) 645–650
biological behavior of miR-204 and other microRNAs in the pro-
gression of retinoblastoma.
As the limit on the number of retinoblastoma samples and cell
types, more elaborate studies will be necessary for further explora-
tion of the potential role of miR-204 in development of retinoblas-
toma. In addition, the eyes used for comparisons of miR-204
expression were postnatal. To better compare the expression of
miR-204 between retinoblastoma and normal eyes, fetal retina
should be enrolled in the study. However, our findings on miR-
204 are encouraging and suggest that this miRNA could be a poten-
tial target for the treatment of retinoblastoma in the future.
Acknowledgement
The work was supported by National Natural Science Founda-
tion of China (No.: 81101553).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.febslet.2015.01.
030.
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