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ARTICLE # 14735-AJ
Regulation of MicroRNA 145 on the apoptosis of Human
retinoblastoma Y79 cells
Wang Xiaoqin, Chen Zhen, Xng Yiqiao
Department of Ophthalmology of Renmin Hopital of Wuhan University, Wuhan,
430060 Hubei, China
Corresponding Author: Xng Yiqiao, Department of Ophthalmology of Renmin Hopital,
Wuhan University, Wuhan, 430060 Hubei, China
Abstract: This study explored the effect of MicroRNA 145（miR-145）
on the the proliferation and apoptosis of human retinoblastoma Y79 cells.
Cultured human retinoblastoma cells Y79 cells were divided into four
groups：miR-145 intervention group, negative miRNA control group,
empty liposome group and blank control group. miR-145 was transfected
into Y79 cells by lipofection. Real-time PCR confirmed the expression of
miR-145. CCK-8 was used to test the cells inhibitor. Flow cytometry was
used to detect cell cycle and apoptosis. Annexin V-affinity assay was used
to detetect apoptosis. The data were analyzed by SPSS11.5. miR-145 was
effectively trancfected into Y79 cells, using lipofection. Real-time PCR
displayed increased expression of mature miR-145 of miR-145
intervention group (79.06 ± 3.45), statistically different from blank
control group (1.00 ± 0.02), empty liposome group (0.93 ± 0.02) and
negative miRNA control group (1.06 ± 0.03). The results of CCK-8
showed the proliferation inhibition rate (21.64%) of miR-145 intervention
group was significantly higher than negative miRNA control group
(2.57%), empty liposomes group (3.97%) and blank control group (0%),
and the difference was statistically significant (F = 34.13 , P <0.05). Flow
cytometry demonstrated miR-145 inhibited the cell cycle G1 phase of
Y79 cell. The results of Annexin V / PI double immunofluorescent
staining demonstrated that Annexin V positive staining of miR-145
transfected cells showed significantly more green fluorescence. The
apoptosis-positive rate of miR-145 intervention Group (Annexin
V-positive) was 11.10%, which was significantly higher than empty
liposomes group (6.17%), negative miRNA control group (6.45%) and
blank control group (6.11%). This studies indicated that miR-145 can
restrain the proliferation and induced the apoptosis in Y79 during early
stage of apoptosis.
Key words: miR-145, Human, retinoblastoma, proliferation, apoptosis
INTRODUCTION
Retinoblastoma is a malignant tumor on the retina which occurs
in children. Once diagnosed, most patient receive eyeball enucleating,
which is grieved for patients and parents. Though retinoblastoma
radiotherapy is a effective treatment, it can conduce adverse reactions as
radioactive optic neuropathy and secondary malignant tumor. Therefore,
exploring new treatment for retinoblastoma is significant. MicroRNA is a
short non-coding RNA with very few nucleotides (21-23), involved in
ontogenesis, apoptosis, proliferation and differentiation(Esquela-Kerscher
and Slack 2006; Cannell, Kong et al. 2008; Stefani and Slack 2008), and is
closely related to tumorigenesis, metastasis, differentiation(Lu, Getz et al.
2005; Jannot and Simard 2006). MiR-145 is located on chromosome 5
(5q32-33) reported as tumor suppressor gene(Boultwood, Fidler et al.
2002). MiR-145 was liposome-mediated transferred into cultured human
retinoblastoma cells Y79 cells. We observe the effect of MiR-145 on the
proliferation and apoptosis of retinoblastoma with the attempt to explore
the molecular mechanism of new treatment for retinoblastoma.
MARERIALS AND METHODS
Cell Grouping and Treatment: An human retinoblastoma cells Y79 cells
was obtained from the American Type Culture Collection (ATCC) and
cultured in RPMI 1640 containing 10% FBS in an atmosphere with 5%
CO and 90% humidity at 37°C. The medium was replenished every 2–3
days. Before experiment, cells were maintained in serum-free medium for
24h. We searched for the target genes of miR-145 by using miRBase.
miR-145mimics（miR09122105714）and negative miRNA was
synthetized by Guangzhou RiboBio Co., Ltd. Y79 cells was transfected
with Lipofectamine 2000 reagent （Invitrogen）.Conventional cultured
Y79 cell was centrifuged and the supernate was discarded. After
resuspended with RPMI 1640 containing 10% FBS, the cell density is
4× 105 /ml.Cells were seeded in 96-well plates. The trancfection reagent
were added in wells. The concentration of miRNA was 50nmol/ml, the
concentration of liposome was 5μg/ml. 24h after transfection, cells were
tested. Y79 cells were divided into four groups, including miR-145
intervention group, negative miRNA contrl group, empty liposome group
and blank control group.
Real-time PCR: Cells were collected by centrifugation at 1200r/min for
5min.Total RNA was extracted with Trizol(Invitrogen) and stored at
-20℃.RT-PCR was performed according to the manufacturer’s
instruction(Fermentas).Primer of miRNA retroviral (cat.4366596),
miR-145 primer(cat.4373133) and was synthetized by Guangzhou
RiboBio Co., Ltd.) SYBR Green PCR Master Mix kit (Biosystems,USA)
according to manufacturer’s instructions. The reaction mixture included 1
μL of cDNA, 10μL of 2×POWER SYBRGreen PCR Master Mix, 4uL
of each primer and 1μL of DEPC-treated water.The PCR cycling
protocol was as follows: 40 cycles of 50℃ for 2min, 95℃ for 10min, 95℃
for 30s, 60℃ for 1min, 72℃ for 15s. Fluorescence signals were
collected at 60℃. The melt curve was delineated with data collected at
60-95℃.The experiment was repeated three times and the 2－ΔΔCt method
was employed to calculate the eapression of taeget gene. U6 served as an
internal reference. The formulas for calculation were as
ΔCt=Ct－iR-145－CtU6, ΔΔCt=ΔCt(intervention group)-Δ
Ct(control group). According to the formulas, the fold change miR-145
mRNA expression was calculated as
2－ΔΔCt.
Cell Counting Kit-8 (CCK-8): Cells inhibitor(CI) was tested using
CCK-8 cell viability assay.48h after transfection,10μL of CCK-8 was
added in every well, followed by incubated for 60min. Absorbance(A
value) of intervention group(IG) and control group(CG) was measured
at 450 nm using a microplate reader. Cells inhibitor(CI) was measured
according to the formulas as CI=1-(IG/CG)×100%
Flow cytometry: Cell cycle was detected by flow cytometry. 24h after
transfection, cells were harvested and resuspended with PBS, and the
density of cells were 1×106/ml. Cells were fixed in 70% alcohol for
30min, centrifuged for 5min at 500r/min, then the alcohol was discarded.
Add 40μL of Phosphorous Acid sodium citrate, the cells were placed in
room temperature for 30min. Cells were centrifuged for 5min at 500r/min
again. Cells were washed with PBS. Add RNase A and incubated at 37°C
for 1h. At that time, 50μL of propidium iodide was added, and keep in
dark place at 4°C for 30min. The cell cycle was analyzed by flow
cytometry.
Flow cytometry was also used to detect apoptosis. Cells were harvested
and resuspended with PBS, and the density of cells were 3×105/ml. The
mixture was concluded with 500μL of cell suspension, 5μL of Annexin
V-FITC and 10μL of Propidium iodide (PI), and kept in dark place at
room temperature for 15min. The cell apoptosis was analyzed by flow
cytometry(Becton Dickinson FACS Calibur).
Annexin V-affinity assay: Annexin V-affinity assay detected the
apoptosis of cells. Annexin V-FITC Apoptosis Detection Kit was used.
24h after transfection, cells were harvested and resuspended with PBS,
and the density of cells were 3×105/ml. The mixture included 500μL of
cell suspension, 5μL of Annexin V-FITC and 10μL of Propidium
iodide (PI), and kept in dark place at room temperature for 15min. The
mixture was dropwise added on glass slide, then observed using Inverted
Microscope (Olympus IX51)
Statistic analysis :The data were analyzed by SPSS11.5. Each
experiment was repeated three times, and average data are expressed.
each group of data with the Levene test confirmed the homogeneity of
variance using one-way ANOVA. Pairwise comparisons using LSD t test.
P <0.05 was considered statistically significant.
RESULTS
The expression of miR-145 of four groups: Compared with the blank
control group (1.00 ± 0.02), empty liposome group (0.93 ± 0.02) and
negative miRNA control group (1.06 ± 0.03), expression of mature
miR-145 of miR-145 intervention group (79.06 ± 3.45)was increased, and
the difference was statistically significant (F = 229.853, P <0.05). The
difference between the empty liposomes group, negative control group
and the control group was not statistically significant (P> 0.05).
The effection of miR-145 on cell proliferation of Y79 cells: Compared
with the blank control group, empty liposomes group and negative
miRNA control group, miR-145 intervention group show significantly
inhibition of proliferation of miR-145 Y79 cell. miR-145 group
proliferation inhibition rate (21.64%) was significantly higher than the
negative miRNA control group (2.57%), the empty liposomes group
(3.97%) and the control group (0%), and with a statistically significant
difference (F = 34.13 , P <0.05). The difference between the empty
liposome group, negative miRNA control group and blank control group
was not statistically significant (P> 0.05).
The effection of miR-145 on cell cycle of Y79 cells: Distribution of the
cell cycle phase of miR-145 intervention group was changed. The G0/G1
phase cells decreased. The S phase cells increased. The G2 / M phase
cells increased. miR-145 inhibited the G1 phase of cell cycle of Y79 cell
(P <0.05) (table 1).
The effection of miR-145 on cell apoptosis of Y79 cells: The results of
Annexin V / PI double immunofluorescent staining demonstrated that
miR-145 cells Annexin V positive staining showed significantly more
green fluorescence, while the Annexin V-positive rate of other three
groups was very low. Four groups of PI-stained cells were rarely ,which
demonstrated that miR-145 induced the Y79 cells occured during early
stage of apoptosis (Figure 1 ). The results of flow cytometry quantitative
analysis of miR-145 on apoptosis demonstrated coincident trends with
Annexin V / PI double staining immunofluorescence. The
apoptosis-positive rate of miR-145 Group (Annexin V-positive) was
11.10%, which was significantly higher than the empty liposomes group
(6.17%), negative miRNA control group (6.45%) and blank control group
(6.11%).The difference was statistically significance (F = 35.434, P
<0.05). The difference between the empty liposomes group, negative
miRNA control group and blank control group was not statistically
significant (P> 0.05, Figure 2).
DISCUSSION
Since Iin-4 was first reported by Iee et al at 1993, the role of small RNA
molecules in the regulation of gene expression get more attention.
miRNA is a class of 20 to 24 nucleotides belong to non-coding
single-stranded RNA, which is widely distributed in tissues and cells. The
majority of miRNA is highly conserved, time-ordered, tissue-specifically
regulate development and participate in cell differentiation, proliferation
and death. The the miRNA is known contribute to the regulation of gene
expression primarily in the post-transcriptional stage, working as a
negative regulator of gene expression (Bartel 2004). Tumorigenesis is the
result of the imbalance between cell proliferation, differentiation and
apoptosis. A growing number of studies have confirmed miRNA is
closely related to a variety of tumor occurrence and development. miRNA
can target on tumor suppressor gene of the amplified region. It
upregulated tumor suppressor gene and play the role of suppressing
tumorigenesis. miRNA can also target on Oncogenes. It down-regulate
the expression levels of oncogene, and prevent malignant
transformation(Jannot and Simard 2006).
Those two types of miRNA oncogenes and tumor suppressor genes play
an important role in Tumorigenesis and tumor development. Although
those endogenous miRNA account for only 2% of the total number of
human genes, they regulate more than 30% of expression of the gene in
the human genome. And the mechanisms of microRNA-mediated gene
regulation is completely new (McManus 2003).
Studies have shown that expression of miR-145 in breast cancer, colon
cancer and lymphoma cells are downward(Akao, Nakagawa et al. 2007).
The function of MiR-145 in retinoblastoma not been reported. Our
experiment found that miR-145 expressed at a very low
level in retinoblastoma Y79 in vitro,coinciding with the findings of
miR-145 expression on other types of tumors.We using
liposome-mediated transient transfection technology for further
understanding of its function. Y79 cells cells was transfected with
miR-145 mimetics. Y79 cells were divided into four groups,including
miR-145 intervention group, negative miRNA intervention group, empty
liposome group and blank control group. 48h after miR-145 was
transfected into retinoblastoma cells, the proliferation of retinoblastoma
cells was significantly suppressed. Cell cycle is suppressed in G1 phase,
suggesting that miR-145 inhibit cell growth of retinoblastoma. This
miR-145 inhibits colon cancer cell growth also been demonstrated(Xu,
Liu et al. 2012). In order to observe the change of retinoblastoma tumor
Y79 apoptosis after transfection of miR-145 before , we using Annexin V
/ PI double immunofluorescence staining and flow cytometry. The results
demonstrated that Annexin V positive cells of miR-145 intervention
group increased significantly, higher than the negative control group,
empty liposomes groups and blank groups, the difference is statistically
significant. Zhang et al found that miR-145 inhibited expression of
DEF-45, induced apoptosis in colon cancer cells(Zhang, Guo et al. 2010).
The mechanisms of miR-145 inducing Y79 cell proliferation and
apoptosis need further research.
The gene involved in signal transduction,chromatin regulation, protein
transcription, protein metabolic, protein modification may be a potential
target of the miR-145 gene (Michael, SM et al. 2003). The study found
that miR-145 by combining insulin receptor substrate -1 (IRS-1) and the
3-untranslated region (3'UTR) to downregulate the expression of insulin
receptor, then inhibiting the growth of colon cancer cells (Shi,
Sepp-Lorenzino et al. 2007). There are research confirmed that miR-145
can downregulate the expression of the N-ras oncogene,and play an
inhibitory effect on colorectal cancer. Our study demonstrated that
miR-145 play an important role in the proliferation and apoptosis of
retinoblastoma.We expected miR-145 become a effective treatment for
retinoblastoma, and the specific target of miR-145 in the retinoblastoma
gene regulation mechanism needs further research.
tein expression. Compared to in vitro environment, in vivo environment
is more complex and the role of miR-145 in Rb cells in vivo need more
research.
CONCLUSION
In this studies, researchers transfeceted miR-145 into cultured human
retinoblastoma cells Y79 cells, and indicated that increased expression
was related with restrained proliferation of retinoblastoma Y79 cells.
Furthermore, we observed that miR-145 induced apoptosis of Y79 cells
was during G1 phase of cell cycle, consistent with our hypothesis that
upregulated miR-145 may profitable for treatment for retinoblastoma.
ACKNOWLEDGEMENTS
This study was supported by the Fundamental Research Funds for the Central
Universities
of
China
(No.
201130202020007)
(http://www.gs.whu.edu.cn/newscenter/ readnews.asp? News ID=5728)
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Figure legends
Figure 1 The results of Annexin V / PI double immunofluorescent staining of Y78(48
h after transfection). a, b, c, d were the photos observed using inverted bright-field
microscope (×100) cells of each group.e, f, g, h were the photos of Annexin V
staining ,which showed green fluorescence. i, j, k, l were the photos of PI staining,
which showed red fluorescence.
Figure 2 The effection of miR-145 on cell apoptosis of Y79 cells
table 1 The effection of miR-145 on Y79 cell cycle
Percentage of the number of cells in different cycles
groups
（%）
G0/G1
S
G2/M
blank control group
80.03±2.31
17.45±1.76
2.51±0.76
miR-145 intervention group
64.20±1.93*
31.00±1.87*
4.80±0.54*
negative miRNA control group
80.40±2.56
17.48±1.55
2.12±0.32
empty liposome group
80.48±3.73
17.14±2.06
2.37±0.43
F value
71.842
152.867
26.536
P value
0.000
0.000
0.002
miR-145 intervention group compared with blank control group,*P<0.01