MiR-29a Inhibits Cell Proliferation and Induces Cell Cycle Arrest through the Downregulation of p42.3 in Human Gastric Cancer
et al. (2011) MiR-29a Inhibits Cell Proliferation and Induces Cell Cycle Arrest through the Downregulation of
p42.3 in Human Gastric Cancer. PLoS ONE 6(10): e25872. doi:10.1371/journal.pone.0025872
MiR-29a Inhibits Cell Proliferation and Induces Cell Cycle Arrest through the Downregulation of p42.3 in Human Gastric Cancer
Yun Cui 0
Wen-Yu Su 0
Jing Xing 0
Ying-Chao Wang 0
Ping Wang 0
Xiao-Yu Chen 0
Zhi-Yong Shen 0
Hui Cao 0
You-Yong Lu 0
Jing-Yuan Fang 0
Alfons Navarro, University of Barcelona, Spain
0 1 Division of Gastroenterology and Hepatology, Shanghai Jiao-Tong University School of Medicine Renji Hospital, Shanghai Institute of Digestive Disease , Shanghai , China , 2 GI Division, No.9 People's Hospital, Shanghai Jiaotong University School of Medicine , Shanghai , China , 3 GI Surgical Division, Shanghai Jiaotong University School of Medicine Renji Hospital , Shanghai, China, 4 Laboratory of Molecular Oncology , Beijing Institute for Cancer Research, School of Oncology, Peking University , Hai-Dian District, Beijing , China
As a newly identified and characterized gene, p42.3 is associated with cell proliferation and tumorigenicity. The expression of p42.3 is upregulated in human gastric cancer (GC), but its underlying mechanisms of action are not well understood. MicroRNAs (miRNAs) are known to play vital regulatory roles in many cellular processes. Here we utilized bioinformatics and experimental approaches to investigate the regulatory relationship between miRNAs and the p42.3 gene. We showed that miR-29a could repress p42.3 expression at both the mRNA and protein levels via directly binding to its 3'UTR. Furthermore, an inverse relationship was observed between miR-29a and p42.3 expression in gastric cancer cell lines and GC tissue samples, especially in cases where p42.3 was downregulated. Taken together, we have elucidated previously unrecognized roles of miR-29a and indicated that miR-29a may function, at least partially, by targeting the p42.3 gene in human GC.
Funding: This work was supported by grants from the National Basic Research Program of China 973 program (2010CB5293, the National High Technology
Research and Development Program of China (863 Program)(2006AA02A402, the National Natural Science Foundation of Key Program (No. 30830055) and the
Ministry of Public Health, China (No. 200802094) to FJY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
p42.3 is a novel gene that has been recently isolated and
identified by the mRNA differential display (mRNADD)
technique. The full-length cDNA of p42.3 is approximately 4.0 kb,
and the gene encodes a 389 amino acid (aa) protein that is
estimated to have a molecular mass of 42.3 kDa. Further research
has revealed that its expression is cell cycle-dependent in gastric
cancer (GC) cell lines. Its protein expression peaks during the M
phase of the cell cycle, before gradually decreasing after cell
division; this indicates that p42.3 may be involved in cell cycle
regulation. Furthermore, silencing of p42.3 by small interfering
RNA (siRNA) results in the upregulation of CHK2 and the
downregulation of cyclin B1, which are two key proteins involved
in cell cycle regulation [1,2]. While RT-PCR and
immunohistochemical analyses have shown that p42.3 is upregulated in GC
compared with normal tissue samples, functional research has
suggested that the depletion of p42.3 may not only result in the
inhibition of GC cell proliferation and colony formation in vitro,
but may also significantly reduce tumorigenicity in nude mice .
Although previous studies have suggested a critical role for the
p42.3 gene in the pathology of GC, the specific underlying
mechanisms of its action remain to be clarified.
MicroRNAs (miRNAs) consist of a class of small (,22
nucleotides), endogenous, non-coding RNAs that are known to
play important regulatory roles in gene expression . The primary
miRNA transcript is called pri-miRNA , which is transcribed by
RNA polymerase II or III [6,7]. The pri-miRNA is then cleaved by
the Drosha-DGCR8 microprocessor complex to produce the
precursor hairpin molecule (pre-miRNA) which is then exported
from the nucleus to the cytoplasm by exportin25/Ran-GTP. With
the assistance of a complex that contains the RNase Dicer and the
double-stranded RNA-binding protein, TRBP, the ,70-nucleotide
pre-miRNA is processed into mature miRNA . The functional
strand of the mature miRNA is loaded into the RNA-induced
silencing complex (RISC), which contains the proteins, argonaute
(Ago) and Tnrc6, while the other strand is usually degraded . The
mature miRNA guides the RISC to the imperfect complementary
sequences in target mRNAs to repress the cognate mRNA
translation, promote transcript decay, or both . It is estimated
that most coding genes are probably regulated by miRNAs and,
whilst a miRNA may regulate more than one target genes, certain
genes can be regulated by multiple miRNAs .
Growing evidence suggests that miRNAs are involved in a wide
range of physiological and pathological processes, including
development, differentiation, proliferation and apoptosis [12.13,14,15].
Although abnormalities of miRNA expression have been determined
in many human tumors, including colorectal, gastric and breast
cancers [16,17], the number of such tumors is still expanding.
However, the detailed functions of miRNA in tumors remain to be
Recent studies have suggested that miR-29 has complex
functions in various diseases. MiR-29a may behave as a tumor
suppressor in both lung and pancreatic cancer cell lines, and thus
the exogenous overexpression of miR-29a results in a significant
reduction in the invasive potential and proliferation of these cell
lines . The tumor suppressor role of miR-29a is also supported
by its observed downregulation in a broad spectrum of solid
tumors, including neuroblastoma, sarcomas and brain tumors
. In contrast, miR-29a is upregulated in indolent human B-cell
chronic lymphocytic leukemia (B-CLL)  and acute myeloid
leukemia (AML) , which suggests a possible tumor promoter
role. In addition, the aberrant expression of miR-29a can be found
in many non-malignant diseases, including liver fibrosis ,
diabetes  and Alzheimers disease . Although many genes
have already been confirmed to be the direct targets of miR-29a,
such as PPM1D , PI3K  and neuron navigator 3 ,
they represent a very small fraction of the total genes that miR-29a
In the present report, we demonstrate that p42.3 expression was
controlled at the levels of both mRNA and protein by miR-29a via
direct targeting of the 3UTR of p42.3. MiR-29a could suppress
cell proliferation and induce cell cycle arrest, at least in part, via
the downregulation of p42.3 expression. Moreover, we found that
the expression of p42.3 protein was inversely correlated with
miR29a expression in human GC tissues.
p42.33UTR is putatively targeted by miR-29a
Putative miRNAs that were predicted to target the p42.3 gene
by more than one database were analyzed. The top two miRNAs
which were predicted for three times were selected for further
confirmation and the other three miRNAs, including miR-29a,
whose putative binding sites were close to those of the top two
were also selected as candidates for validation. MiR-29a was
predicted by the TargetScan and miRGEN databases, which
indicated that its putative binding site was at positions 213219 of
the p42.33UTR (Figure 1A). The other candidates are not listed
Directly targeting the p42.33UTR by miR-29a
The reporter gene assay was employed to validate whether
p42.3 was a direct target of miR-29a. Wild-type and mutant
p42.33UTR containing the putative binding site of miR-29a
were cloned into individual plasmids and fused with the reporter
gene. The fluorescent intensity of the reporter gene was
significantly decreased in the group that was co-transfected with
WTpcDNA3/EGFP/p42.3 and pcDNA3.1/pri-miR-29a
compared to the control. In addition, there was no significant decline
of the fluorescent intensity in the group that was co-transfected
with MUTpcDNA3/EGFP/p42.3 (Figure 1B), further confirming
that miR-29a induced the downregulation of p42.3 gene
expression via the specific binding of the putative site of the
Expression of miR-29a and p42.3 are inversely related in
GC cell lines
To ascertain the link between p42.3 and miR-29a, we studied
the endogenous expression of miR-29a and p42.3 in six human
GC cell lines (SGC-7901, MKN-28, MKN-45, MGC-803, SNU-1
and AGS) and one normal gastric epithelium cell line (GES-1;
Figure 2AC). We found that the level of p42.3 mRNA was
significantly higher than the normal control in all GC cell lines
with the exception of SGC-7901, where the difference was not
statistically significant. The expression level of p42.3 was variable
at the protein level, but was significantly higher in all of the GC
cell lines when compared with GES-1.We also showed that
miR29a expression was low in four of the GC cell lines (SGC-7901,
MKN-45, MGC-803 and AGS), which revealed an inverse
relationship with p42.3 expression. Although the expression of
miR-29a was high in SNU-1, this was not statistically significant. It
is worth mentioning that the high expression of miR-29a in
MKN28 was an exception.
MiR-29a regulates p42.3 expression
To evaluate whether p42.3 was repressed by miR-29a, we
treated MKN-45 cells with the mimics and inhibitors of miR-29a
for 48 h in order to exogenously up- and downregulate the
expression of miR-29a specifically; the expression of p42.3 was
then determined. After the transfection of MKN-45 cells with the
mimics, the miR-29a expression increased by approximately
10fold, while the treatment with the inhibitors decreased the
miR29a level by more than 50% (Figure 3AB). This suggested that
both the mimics and inhibitors of miR-29a worked efficiently in
our experiments. Overexpression of miR-29a could significantly
repress the expression of p42.3 at both the mRNA and protein
levels, which was a similar effect to the silencing of p42.3 by
p42.3 siRNA (si-p42.3). We also detected that CHK2 was
upregulated and cyclinB1 was downregulated after the silencing
of p42.3 by p42.3 siRNA. Interestingly, similar effects were
exerted on CHK2 and cyclinB1 by the overexpression of
miR29a in MKN-45 cells (Figure 3CD). In addition, transfection of
miR-29a inhibitors dramatically decreased CHK2 expression and
increased p42.3 and cyclinB1 expression (Figure 3EF). This
suggested that miR-29a could regulate the expression of the p42.3
gene in MKN-45 cells.
MiR-29a inhibits cell proliferation in vitro
According to the data of the cell proliferation assay, we drew the
absorbency curves at the wavelength of 450 nm after transfection
for different durations. We found that cell proliferation was
significantly inhibited after the transfection of MKN-45 cells with
p42.3 siRNA for 48 h and 72 h, and a similar pattern was noted
after cells were transfected with mimics of miR-29a. However, the
degree of repression achieved by miR-29a mimics was greater
than p42.3 siRNA-induced downregulation (Figure 4A). On the
contrary, cell growth was promoted by approximately 10%,
compared to the negative control when transfected with inhibitors
of miR-29a for 48 h, but there was a decline at 72 h (Figure 4B).
This indicated that miR-29a could inhibit cell proliferation via
repressing the expression of p42.3.
MiR-29a blocks cell cycle progression
Silencing of p42.3 by p42.3 siRNA can result in cell cycle arrest;
therefore, we investigated whether miR-29a could affect the cell
cycle progression via targeting the p42.3 gene. After MKN-45 cells
were transfected with p42.3 siRNA for 48 h, we found that the cell
cycle was blocked at the G1 phase (75.93%, P,0.05), compared
with the negative control (66.18%). We found that mimics of
miR29a could also induce G1 phase arrest in MKN-45 cells when
treated for 48 h (75.56%, P,0.05; Figure 5).
Figure 1. MiR-29a targets a putative binding site in p42.33UTR. A. The sequence of miR-29a with the putative binding site in the human
p42.3 gene. The putative binding site with the mutant is shown in the lower panel. B. Regulation of reporter gene expression by miR-29a in MKN-45
cells co-transfected with the pri-miR-29a and reporter gene containing the putative binding site. **: P,0.01.
Expression of miR-29a and p42.3 protein in GC and their
correlation with clinicopathological characteristics
Using a quantitative real-time PCR technique, miR-29a was
detected in 60 pairs of GC tissues and their matched non-cancer
adjacent tissues, while p42.3 protein level was also evaluated in
these tissues by Western blotting. Out of 60 GC tissues samples,
p42.3 expression was high in 35 cases (35/60, 58.33%) relative to
their matched non-cancer adjacent tissues. In the 25 cases in
which p42.3 expression was downregulated, miR-29a expression
was high in 21 cases. MiR-29a expression was low in 27 cases (27/
60, 45%). The above data suggests an inverse relationship between
miR-29a and p42.3 protein expression in tissue samples (P = 0.000,
Table 1 and Figure 6), but the correlation coefficient was not good
(r = 20.316).
Furthermore, miR-29a and p42.3 protein expression were
evaluated with regards to the clinicopathological characteristics of
the 60 patients from whom tissue samples were taken. Our
findings suggested that there were no obvious correlations between
p42.3 protein and miR-29a expression, respectively, with
clinicopathological features (Table 2).
The p42.3 gene is highly conserved in mammals and, as an
oncogene, it may play an important role in the progressive
transformation of normal gastric epithelium cells to cancer cells. Its
differential expression during the cell cycle stages reveals that
p42.3 may be involved in cell cycle regulation. This was further
confirmed by our results, which showed that p42.3 silencing could
alter the expression of two key proteins, CHK2 and cyclin B1, that
are involved in cell cycle regulation. Using loss-of-function
experiments, we demonstrated that p42.3 may stimulate cellular
proliferation and as reported, our results showed that p42.3 was
overexpressed in GC tissues when compared with the adjacent
non-cancer mucosa . However, the molecular mechanisms
resulting in this aberrant expression of p42.3 gene in GC is poorly
Figure 5. Effect of miR-29a on cell cycle in the MKN-45 cell line. Flow cytometric analysis confirmed that both p42.3 siRNA and miR-29a
mimics induced G1 phase arrest in the MKN-45 cell line.
understood, as evident from the lack of available literature.
MiRNAs can regulate gene expression by targeting the binding
sites in the target mRNAs  and, in human cancers, many
miRNAs have already been implicated. However, the function of
only a few has been understood to date, especially in GC .
In this report, we selected miR-29a for further investigation by a
reporter gene system and ultimately identified that p42.3 was a
direct target gene of miR-29a. Our data suggested that the four
databases (TargetScan, microRNA.org, MicroCosm Targets
Version 5 and miRGen) used were efficient, but not perfect, tools
for the prediction of miRNA targets and provided experimental
evidence for improvements to the underlying algorithm.
We showed that p42.3 expression was inversely related with
miR-29a expression in four GC cell lines. In three of these,
MKN45, MGC-803 and AGS, this was evident at both the mRNA and
protein level, but this was not the case in the SGC-7901 cell line.
In addition, miR-29a expression was not low in the MKN-28 and
SNU-1 cell lines. Taken together, these observations allowed us to
hypothesize that the regulatory role of miR-29a is complicated in
GC cell lines and that miR-29a may play different roles in
different cellular backgrounds. However, the detailed mechanisms
of miR-29a functions in GC cell lines require further investigation.
In our study, miR-29a overexpression could induce similar
effects to those achieved by the silencing of p42.3 by p42.3 siRNA.
Both of p42.3 mRNA and protein were downregulated after cells
were transfected with p42.3 siRNA or miR-29a mimics and the
cell proliferation and cell cycle were suppressed when cells
underwent the same interference. It suggested that miR-29a may
be involved in the pathogenesis of GC via the repression of p42.3
gene expression. However, we found from the growth curves that
the degree of repression by miR-29a mimics was greater than by
p42.3 siRNA. In our opinion, the main reason for this may be that
specific miRNAs could regulate hundreds of genes to control
cellular function. In the present study, miR-29a may inhibit cell
proliferation, at least in part, by targeting p42.3.
Our data showed that although the rate of high expression of
miR-29a was 55% (33/60) in GC, p42.3 expression was low in 21
of these cases. This suggested that miR-29a may have a vital role
in GC tissues with low expression levels of p42.3.
aMedian of relative expression, with 25 th75 th percentile in parenthesis.
In the present study, we validated that p42.3 is a direct target of
miR-29a. We have also demonstrated that miR-29a can inhibit
cell proliferation and block the cell cycle, at least in part, via the
repression of p42.3 expression in GC. We conclude that miR-29a
may play a critical role in regulating the expression of p42.3 in
GC. Interestingly we found that p42.3 gene could also regulate the
expression of miR-29a (the data are not shown here), but the
underlying regulatory mechanism will be explored in the future.
Materials and Methods
Four efficient computational approaches that utilize different
evaluating systems  were used for the prediction of the
regulatory miRNAs that target the p42.3 gene, including
TargetScan (http://www.targetscan.org/), microRNA.org (http://www.
microrna.org/microrna/getGeneForm.do/), MicroCosm Targets
Version 5 (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/
targets/v5/) and miRGen
(http://www.diana.pcbi.upenn.edu/cgibin/miRGen/v3/Targets.cgi#Results). Targets were selected for
further confirmation from the group of miRNAs that were common
to the results generated from more than one search.
Sixty pairs of histopathologically confirmed GC and adjacent
non-cancer tissue samples were obtained from patients who
underwent surgical resection at the Renji Hospital affiliated to the
Shanghai Jiaotong University School of Medicine, China between
July 2007 and January 2009. The matched non-cancer adjacent
tissues were obtained at least 5 cm away from the tumor site. The
study was approved by the Research Ethics Committee of
Shanghai Jiaotong University and informed consent was obtained
from all patients, while written consent were obtained from each
Cell lines and culture conditions
Human GC cell lines, SGC-7901, MKN-28, MKN-45,
MGC803, SNU-1 and AGS, and the GES-1 normal gastric epithelium
cell line, which were purchased from ATCC (USA), were
maintained in RPMI 1640 medium (Gibco, Gaithersburg, MD,
USA) supplemented with 10% fetal bovine serum (Invitrogen,
Carlsbad, CA, USA). Cells were cultured at 37uC in a 5% CO2
incubator. A solution of trypsin (0.25%) was used to detach the
cells from the culture flask.
To construct the expression vectors pri-miR-29a was first
amplified with primers designed by Primer Premier 5.0 software
(Table 3) and then cloned into pcDNA3.1 (Invitrogen). To
produce the plasmids that contained the putative binding site of
miR-29a, both wild-type and mutant sequences from position 111
to 380 of the p42.33UTR were chemically synthesized
(Figure 1A) and then were cloned to the downstream of the
EGFP gene at BamHI and EcoRI sites in the pcDNA3/EGFP
vector (Saierbio, Tianjin, China).
Reporter gene assay
MKN-45 cells were seeded in triplicate wells of a 24-well plate on
the day before transfection. PcDNA3.1 (+)/ primary-miR-29a were
co-transfected with wild-type and mut-pcDNA3/EGFP/p42.3
3UTR respectively. Then, 0.5 mg of pcDNA3.1 (+)/
primary-miR29a and 0.5 mg of pcDNA3/EGFP/p42.33UTR were added into
each of the wells, while 0.1 mg of vector pDsRed-C1 (Clontech),
which expressed the RFP, were added to every well as an endogenous
reference. Cells that were only transfected with pcDNA3/EGFP/
p42.33UTR or pcDNA3/EGFP/p42.33UTR+ pcDNA3.1 (+)
were used as the controls. Cells were collected 48 h after transfection
and analyzed based on the intensity of EGFP and RFP fluorescence
Transfection of MKN-45 cells was performed using the
Lipofectamine 2000 Reagent (Invitrogen) following the protocol
of the manufacturer. MKN-45 cells were seeded into 6-well plates
or 96-well plates at 30% confluence the day prior to the
transfection. Mimics (100 nM, GenePharma) and inhibitors
(200 nM, RIBOBIO) of miR-29a were used to exogenously
upand downregulate the expression of miR-29a. To silence p42.3
gene expression, the MKN-45 cells were transfected with siRNA
against p42.3 (si-p42.3, 100 nM, GenePharma). The control RNA
(named as NC) was non-homologous to any human genome
sequence. The sequences of the oligo nucleotides are shown in
Total RNA was isolated using Trizol reagent (Invitrogen) and
subsequently treated with RNase-free DNase I (Fermentas, San
Diego, CA, USA). Total RNA (500 ng) was reverse transcribed
using the PrimeScript RT reagent Kit (TaKaRa, Dalian, Japan).
The primer sequences used to amplify p42.3 and GAPDH are
shown in Table 3. To analyze the expression of the mature
miR29a, 2 mg of the total RNA was subjected to reverse transcription
using the All-in-One MiRNA Q-PCR Detection Kit
(GeneCopoeia, Guangzhou, China). Quantitative real-time PCR for
mature miR-29a and U6 was performed according to the
manufacturers instructions using the ABI 7300 real-time PCR
system and specific primers designed by GeneCopoeia, China.
The relative expression of p42.3 and miR-29a was normalized to
an endogenous reference (GAPDH and U6 small nuclear RNA
respectively) and relative to the control. The results were presented
as fold change, calculated using the 2(2DDCT) method ; a
relative expression ratio of ,1.0 was considered as low, while a
ratio of .1.0 was considered as high expression .
Total protein from cultured cells and tissues were extracted by
RIPA Lysis Buffer containing PMSF, according to the
manufacturers instruction (Beyotime, Shanghai, China). Protein
concentration was measured using the Bradford method . Overall,
40 mg of protein were electrophoresed through 10% SDS
polyacrylamide gels and were then transferred to a PVDF
membrane (Millipore). The membrane was incubated with p42.3
antibody (1:500, Abmart, China), CHK2 antibody (1:1,000, CST),
cyclinB1 antibody (1:1,000, CST) or GAPDH (1:5,000, Kang
Chen, China) at 4uC overnight. Secondary antibodies were labeled
with HRP (Kang Chen, China) and the signals were detected using
ECL kit (Pierce Biotech., Rockford, IL, USA). Subsequently, the
images were analyzed by ImageJ 1.43 software. The protein
expression was normalized to an endogenous reference (GAPDH)
and relative to the control. A relative expression ratio of ,1.0 was
considered as low expression, while a ratio of .1.0 was considered
as high expression .
Cell proliferation assay
Harvested MKN-45 cells (approximately 5 x 104 cells) were
seeded into 96-well culture plates. Cellular proliferation was
measured at 24 h, 48 h and 72 h post-transfection, respectively,
using the Cell Counting Kit-8 (DOJINDO, Japan) according to
the manufacturers protocol. The absorbance at a wavelength of
450 nm, which shows positive relation to the capacity of cellular
proliferation, was determined by a spectrophotometer (E-LIZA
Cell cycle assay
Forty-eight hours after transfection, the cells were lifted using
0.25% trypsin and washed in DPBS (Gibco); they were then fixed
in 70% ethanol at 220uC for 24 h. For flow cytometric analysis
(EPICS XL Beckman Coulter), cells were incubated in RNAse
(Fermentas) at 37uC for 30 min, treated with PI (Sigma) and
suspended in 300 ml DPBS.
Data were shown as mean 6 SD from at least three separate
experiments. The significance was analyzed with the Students t-test
and non-parametric tests (Mann-Whitney U test and Kruskal-Wallis
H test). The statistical significance of correlation between the
expression of miR-29a and p42.3 protein was calculated by the
chisquare test and Spearman9s rank correlation. Statistical analysis was
performed using SPSS 13.0 software (IBM, USA) and differences
were considered statistically significant at P , 0.05.
Conceived and designed the experiments: YC WYS JX YYL JYF.
Performed the experiments: YC WYS. Analyzed the data: YC WYS JX
YCW PW. Contributed reagents/materials/analysis tools: PW XYC ZYS
HC. Wrote the paper: YC WYS JX JYF.
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