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Acta Biochim |
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doi: 10.1093/abbs/gmp006. |
MicroRNA
expression profiling during neural differentiation of mouse embryonic carcinoma
P19 cells
Huang Bing1, Wei Li1,
Botao Zhao1, Caihong
Xia2, Ruqiang Liang1, Kangcheng Ruan1, Naihe
Jing2, and Youxin Jin1*
1 State Key Laboratory of Molecular Biology，，
2 Key Laboratory of Molecular Cell Biology, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
*Corresponding authors: Tel, 86-21-54921222; Fax, 86-21-54921011;
E-mail, [email protected]
Abstract MicroRNAs (or miRNAs) are small
non-coding RNAs (21 to 25 nucleotides) that are
involved in a wide range of activities related to the development and
differentiation of cells. Comparison of the miRNA
expression profiles of mouse P19 embryonic carcinoma cells with those of
differentiated neural stem cells showed that the expression level of 65 miRNAs changed (2.0-fold) after differentiation. MiR
Keywords microRNA; embryonic carcinoma P19 cell; neural stem cells; neural
differentiation
Received: October 7, 2008 Accepted: November 28, 2008
Introduction
The study of neuronal differentiation of embryonic stem
cells has generated considerable interest in the last few years. These studies
facilitated a better understanding of the fundamental aspects of neurogenesis. Since the first report on derivation of human
embryonic stem (ES) cells, a number of studies have explored the possibility of
directing the differentiation of ES cells towards neuronal development [1].
However, a number of challenges still need to be overcome, including gaining a
better understanding of the mechanisms involved and developing techniques that
allow the generation of homogeneous neuronal and glial
subtypes.
The mouse embryonic carcinoma (EC) P19 cell line is a teratocarcinoma cell line derived from transplanted epiblast mouse embryonic cells. Studies on P19 cells have
provided a significant amount of information on the mechanisms of neural and
skeletal muscle differentiation. Aggregated P19 EC cells can be induced to
differentiate into neurons and glial cells in the
presence of retinoic acid (RA) [2]. This method is commonly used for the
molecular analysis of neural induction and differentiation [3]. However, only a
small percentage of neural stem cells can be induced from P19 cells. Moreover,
many different cell types exist in the induced cell population, and these
non-neural cells may interfere with the detailed analysis of neural
differentiation [4,5]. RA treatment also disturbs
neural patterning and the neuronal identities of the ES cell aggregates [6].
Neural precursors produced by RA induction appear to be developmentally
restricted, and can only generate a limited range of neural cell types [7]. In
order to obtain a high percentage (about 95%) of neural stem cells from the P19
EC cells, we employed a method established by Xia et al [8].
MicroRNAs (miRNAs) are a class of small, non-coding, regulatory RNA
molecules. MiRNAs have been used as regulators of
differentiation processes in different experimental models for neuronal
development research [9]. Many miRNAs involved in the
developmental processes exhibit a highly tissue-specific expression, e.g., some
miRNAs are specifically expressed in the neural
system [10–13]. Certain miRNAs appear to be
correlated with the maintenance of pluripotency in
cells during early mammalian development [14].
In this study, we aimed to understand the functions of miRNAs in neural development and neuron function rather
than in the neuronal differentiation of embryonic stem cells. We used the microarray
to monitor the expression profiles of mouse miRNAs
during the course of P19 neuronal differentiation. The results indicated that
there were significant differences between the miRNA
expression in P19 EC cells and the resultant differentiated neural stem cells.
Cell culture and induction of differentiation
P
Construction of small RNA cDNA
library
The total RNA was extracted with the RNArose
reagent (Sangon,
MiRNA microarray
Each microarray contained 406 capture probes. The probes
were perfectly matched for all miRNAs registered and
annotated in the miRBase [16] at the Wellcome Trust Sanger Institute from human, mouse, and rat.
There were 233 mouse miRNAs and 14 negative control
probes (complementary to mRNAs, tRNAs,
rRNAs, random sequences, etc.). All the microarray
methods were performed as described previously [15], except that the
hybridization temperature was adjusted to
Microarray data analysis
The analysis of microarray data was performed as
described previously [9]. All hybridizations were normalized by the total
intensity and the Student’s t test was performed for the values. For each
probe, we calculated the arithmetic mean of at least 2 replicates from 2
independent hybridizations at each time point. The probes with mean values less
than 1000 at all time points were considered undetectable and were filtered out
of the analysis. The probes that had P values less than 0.001 and changed more
than 2-fold were selected for Northern blot validation.
Northern blot analysis
Total RNA was extracted with the RNArose
reagent. Total RNA (10 g) was resolved on a 12% acrylamide/
Analysis of promoters of the miRNA
gene cluster and target miRNA
The preliminary information of the selected miRNAs was obtained from the website http://microrna.sanger.ac.uk/
[16]. Gene sequences about 1 kb upstream of the gene clusters were extracted
for promoter prediction. The BDGP Neural Network Promoter Prediction program (http://www.fruitfly.org/)
was used for promoter prediction [17]. We then extracted sequences about 2 kb
upstream of the predicted promoters and used TRANSFAC software [18] to predict
the transcript factor binding sites. The candidate target genes for the 15 miRNAs that showed significant changes were predicted using
the miRanda [19,20]
and TargetScan algorithms [21].
Results
MiRNA expression
changed significantly during neuronal differentiation of P19 cells
We used microarray analysis to examine the differential
expression of miRNAs in the P19 EC cells and the
neural stem cells that were derived from the EC cells. An example of the scan
images of non-induced p19 cells (labeled as p19) and neural stem cells (labeled
as p19N) is shown in supplementary Fig. S1. The data indicated that there were
significant changes in the miRNA expression during
the differentiation of P19 cells. In comparison with the expression levels in
the P19 cells, 34 miRNAs in the P19N cells exhibited
at least 2-fold up-regulation, while 31 miRNAs in the
P19N cells exhibited at least 2-fold down-regulation (Table 1).
We also observed that many miRNAs
were expressed at low levels in the induced stem cells, a finding consistent
with a previous study [22]. However, the number of up-regulated miRNAs was more than that of the down-regulated miRNAs. While 77 miRNAs exhibited
at least 1.5-fold up-regulation, 49 miRNAs exhibited
at least 1.0-fold down-regulation. The up-regulation of miRNAs
appeared to be common during differentiation. However, while the ratios of up-regulation
were not very high, those of down-regulation were much more significant.
Although various kinds of miRNAs participated in this
process, the most dramatic changes were observed in the miRNAs
whose expression was reduced, which indicated that they were probably the key
regulators in maintaining the multipotentiality of
stem cells.
Confirmation of miRNA
microarray data
In order to confirm the results from the miRNA microarray experiments, we chose 13 miRNAs for Northern blot analysis. The results are shown in
Fig. 1. Seven of the 13 miRNAs, i.e., miR-9, miR-16,
miR
and account for 95% of the cell population on the fourth day of the induction
period, the number of pluripotent cells (Oct4+Sox+)
reduced gradually [8]. To study the elaborate changes in miRNAs
during this process, the P19 cells were collected, the total RNA was isolated,
and Northern blot analysis was carried out to analyze the miRNA
expression patterns on the 1st, 2nd, 3rd, and 4th day after N2B27 induction.
With a decrease in the percentage of pluripotent
cells and an increase in the number of neural stem cells, the miRNAs miR-16, miR-106b, miR
Discussion
Similar expression tendency of the miRNA
cluster
The tendency of miRNA genes to
occur in clusters [25] prompted us to analyze the chromosomal distribution of
the differentially expressing miRNAs. We found that
except for some miRNAs that were expressed alone,
most of these miRNAs showed cluster distribution and
they were distributed either in the intron of the
protein gene or in the gene-spacer region. On the basis of the microarray data,
9 of the 10 miRNA clusters were found to exhibit increased
expression levels after their differentiation (Table 2S), while 7 other
clusters exhibited decreased expression level (Table 3S). It appeared that miRNAs in the same gene cluster usually changed their expression
in the same way (i.e., either as an increase or as a decrease in expression).
Previous studies have shown that there are 50 pre-miRNA sequences located in the distal region of chromosome
Potential binding sites for transcript factors and target
genes that may participate in regulation
It was reported that miRNAs usually formed a reciprocal
negative feedback loop in the neural system [26]. Using a computer-assisted approach,
many potential binding sites for transcript factors were predicted (data not
shown). MiRNAs suppress the mRNA translation or
affect its stability by pairing with the 3′-UTR of the target mRNA.
Further, we predicted a total of 248 potential target genes for the 15
significantly changed miRNAs using the miRanda [19,20] and TargetScan algorithms [21]. Most of these target genes were
signal transducers, transporters, kinases,
transcription regulators, and enzyme regulators (Table 3). Interestingly, most
of the predicted target genes encoded for the predicted transcript factors. The
mechanisms behind the regulation of miRNAs by these
factors in neural development processes still need to be elucidated. In
conclusion, these miRNAs, with significantly changed
expression levels, could play important roles in the maintenance of stem-cell multipotency and neuronal differentiation in early embryonic
development.
Funding
This work was supported by the grants from the National
Key Basic Research and Development Program (2005CB724602), the National Natural
Science Foundation of China (No. 30430210), the
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