In our study, the function of miR124a in neurogene-sis was analyzed using biomarker genes of stem cells and neurons, and the expression level of miR124a investigated by qRT-PCR during th
Trang 1repression of the chromosome 14 ORF 24 gene during
neurogenesis
Hae Young Ko1,2,3, Dong Soo Lee1,4and Soonhag Kim5
1 Department of Nuclear Medicine, Seoul National University College of Medicine, Korea
2 Interdisciplinary Course of Radiation Applied Life Science, Seoul National University College of Medicine, Korea
3 Institute of Radiation Medicine, Medical Research Center, Seoul, Korea
4 Department of Molecular Medicine and Biopharmaceutical Science, Seoul National University College of Medicine, Korea
5 Laboratory of Molecular Imaging, CHA Stem Cell Institute, CHA University, Seoul, Korea
Introduction
MicroRNAs (miRNAs), a class of small noncoding
RNAs, are 22-nucleotide single-strand RNA
mole-cules that are expressed in both plants and animals
[1,2] In general, miRNAs are incorporated into the RNA-induced silencing complex, and perfectly or imperfectly bind to the 3¢-UTR of its target mRNA to
Keywords
c14orf24; imaging; microRNA124a;
neurogenesis; target gene
Correspondence
S Kim, Laboratory of Molecular Imaging,
CHA Stem Cell Institute, CHA University,
605-21 Yoeksam 1-dong, Gangnam-gu,
Seoul, 135-081, Korea
Fax: +82 2 3468 3373
Tel: +82 2 3468 2830
E-mail: kimsoonhag@empal.com
D S Lee, Department of Nuclear Medicine,
Seoul National University, College of
Medicine, 28 Yongon-dong, Chongno-gu,
Seoul, 110-744, Korea
Fax: +82 2 3668 7090
Tel: +82 2 2072 2501
E-mail: dsl@plaza.snu.ac.kr
(Received 16 April 2009, revised 15 June
2009, accepted 29 June 2009)
doi:10.1111/j.1742-4658.2009.07185.x
The function of microRNAs (miRNAs) is translational repression or mRNA cleavage of target genes by binding to 3¢-UTRs of target mRNA
In this study, we investigated the functions and the target genes of micro-RNA124a (miR124a), and imaged the miR124a-mediated repression of chromosome 14 open reading frame24 (c14orf24, unknown function) during neurogenesis, using noninvasive luciferase systems The expression and functions of miR124a were investigated in neuronal differentiation of P19 cells (P19 is a mouse embryonic carcinoma cell line) by qPCR and RT-PCR The predicted target genes of miR124a were found by searching a bioinformatics database and confirmed by RT-PCR analysis Remarkable repression of c14orf24 by miR124a was detected during neurogenesis, and was imaged using in vitro and in vivo luciferase systems The expression of miR124a was highly upregulated during neuronal differentiation Overex-pression of miR124a in P19 cells resulted in a preneuronal gene exOverex-pression pattern MicroRNA124a-mediated repression of c14orf24 was successfully monitored during neuronal differentiation Also, c14orf24 showed molecu-lar biological characteristics as follows: dominant expression in the cyto-plasm; a high level of expression in proliferating cells; and gradually decreased expression during neurogenesis Our noninvasive luciferease system was used for monitoring the functions of miRNAs, to provide imaging information on related neurogenesis and the miRNA-regulated molecular network in cellular metabolism and diseases
Abbreviations
AA, antibiotic ⁄ antimycotic solution; c14orf24, chromosome 14 ORF 24; CMV, cytomegalovirus; DAPI, 4¢,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2¢-deoxyuridine; Fluc, firefly luciferase; Gluc, Gaussia luciferase; LAMC1, laminin c1; LMNB1, lamin B1; MAP2, microtubule-associated protein 2; miRNA, microRNA; MSC, mesenchymal stem cell; Oct4, octamer4; PTBP1, polypyrimidine tract-binding protein 1; PTPN12, protein tyrosine phosphatase non-receptor type 12; qRT-PCR, quantitative RT-PCR; RA, retinoic acid; RBMS1, RNA-binding motif single-stranded interacting protein 1; ROI, region of interest; SD, standard deviation; USP48, ubiquitin-specific protease 48.
Trang 2induce either mRNA degradation or translational
inhi-bition [3–5] Recently, in animals, miRNAs have been
reported to destabilize the mRNA of their targets by
base paring with a continuous six or seven nucleotide
sequence in the 3¢-UTR of the target genes known as a
seed sequence, seed region, or seed match, in spite of
the partial base pairing between miRNAs and targets
[6]
The first reported miRNA, encoded by the
Caenor-habditis elegansgene lin-4, was found to be crucial for
the developmental timing and patterning of
postembry-onic stages [7] Since their identification, miRNAs have
been shown to play important roles in diverse
biolo-gical functions, such as cell differentiation, fat
meta-bolism, cell proliferation, and cell death [8–10] A
recent study found that many miRNAs, such as miR9,
miR9*, miR124a, miR134, miR23a, miR132, and
miR128, are expressed in neurons and regulate
neuro-nal development [11,12] Among them, miR124a is
present at undetectable or very low levels in neural
progenitors, but is expressed at a high level in
differen-tiating and mature neurons [13] A microarray study
of miR124a-treated HeLa cells (human carcinoma
cells) revealed 174 downregulated non-neuronal
tran-scripts [14] The endogenous targets directly bound
and repressed by miR124a include the genes encoding
small C-terminal domain phosphatase 1 [15],
poly-pyrimidine tract-binding protein 1 (PTBP1) (PTB⁄
hnRNP 1) [16], laminin c1, and integrin b1 [17]
Moreover, neurite outgrowth was promoted by
over-expression of miR124a during neuronal differentiation
[18]
It is a great challenge to study the expression and
function of endogenous miRNAs without killing the
animals The current methods, including northern
blot analysis and RT-PCR, used to investigate the
molecular regulation of endogenous miRNAs are
time-consuming, labor-intensive, nonrepeatable, and
not clinically relevant Recently, there have been
significant advances in optical imaging techniques
using multimodal reporter systems; this technology
has been used for noninvasive repeated quantitative
imaging of tumor and stem cells in living animals
[19–22] Previous articles from our laboratory have
reported the expression of miRNA and its target
genes in vitro and in vivo, using these luciferase
systems [21,23,24]
In our study, the function of miR124a in
neurogene-sis was analyzed using biomarker genes of stem cells
and neurons, and the expression level of miR124a
investigated by qRT-PCR during the neuronal
differ-entiation of P19 cells A bioinformatics analysis was
then performed to predict the targets of miR124a, and
showed, by RT-PCR, several genes that were directly regulated by miR124a One of these genes, chromosome
14 ORF 24 (c14orf24), which is of unknown function, was evaluated in our successfully developed luciferase reporter system, both in vitro and in vivo, to determine whether the 3¢-UTR of c14orf24 was directly regulated
by miR124a Also, for the first time, the biological functions of c14orf24 were investigated during cell proliferation
Results
MicroRNA124a is expressed at a high level during neurogenesis
MicroRNA124a is a small RNA composed of 22 nucleotides, and is well conserved from humans to aquatic species To determine and quantify the endoge-nous levels of miR124a during neuronal differentiation
of P19 cells, we performed quantitative RT-PCR (qRT-PCR) (Fig 1) cDNA was synthesized from small RNA of neuronal differentiated P19 cells, at 0,
1, 2, 3, 4, 5 and 6 days after retinoic acid (RA) treat-ment A pair of specific primers for miR124a was used, and the quantities of miR124a for each differentiation day were normalized with U6 small RNA The expres-sion of miR124a gradually increased during neuronal differentiation, and had increased more than three-fold
by the fifth day after RA treatment
4
3
2
1
0 Before 1 day 2 days 3 days
Day after RA treatment
4 days 5 days 6 days
Fig 1 The expression of miR124a during neuronal differentiation
of P19 cells (A) Quantitative RT-PCR analysis of the expression of mature miR124a Endogenous mature miR124a levels were increased during neuronal differentiation Data were normalized to U6 snRNA (DDC T = DC T-before – DC T-day , DC T-before = C T-miRNA⁄ before
– C T-U6RNA ⁄ before , DC T-day = C T-miRNA ⁄ day – C T-U6RNA ⁄ day ) Data are expressed as means ± SD in triplicate samples.
Trang 3Preneuronal characteristics of P19 cells were
induced by overexpression of miR124a
To investigate the role of miR124a during
neurogene-sis, overexpression of miR124a was examined in P19
cells We transfected exogenously derived miR124a, at
a concentration of 5 nm, into P19 cells, which are
believed not to be induced into neuronal differentiation
in the absence of RA The programming process of neuronal differentiation was induced by overexpression
of miR124a (Fig 2A) We followed the gradual acqui-sition of neuronal traits over time after transfection with exogenous miR124a Interestingly, 2 days after transfection, none of the P19 cells treated with miR124a showed neuronal morphology, whereas RA-treated P19 cells had the neuronal phenotype
Oct4 NeuroD MAP2
Before
Before
1 day 2 days 3 days 4 days Before 1 day 2 days 3 days 4 days 1 day 2 days 3 days 4 days Day after transfection with
miR124a
Day after anti-miR124a and RA treatment Day after RA treatment
β-actin
Oct4
NeuroD
MAP2
Day after transfection with miR124a
Day after transfection with miR124a
Day after RA treatment
Day after RA treatment
A
C
B
Fig 2 MicroRNA124a-induced preneurogenesis in P19 cells (A) Neuronal differentiation analysis in miR124a-transfected P19 cells Upper panel: P19 cells were changed to preneurons by overexpression of miR124a Lower panel: neuronal differentiation induced by RA treatment,
as a control (B) RT-PCR analysis of P19 cells transfected with miR124a and subjected to RA treatment Oct4, stem cell marker; NeuroD, preneuronal marker; MAP2, neuronal marker b-Actin was used as a control (C) Laser scanning confocal microscopy of immunofluorescence staining using Oct4, NeuroD and MAP2 for P19 cells transfected with miR124a, treated with RA, and treated with both anti-miR124a and
RA for 4 days Red fluorescence represents the cytoplasmic expression of Oct4 (top panel), NeuroD (middle panel), and MAP2 (bottom panel), and the blue fluorescence represents DAPI, which stained the nucleus.
Trang 4However, thereafter, miR124a-treated P19 cells
exhib-ited a marked change in cell morphology: there was a
gradual expansion of dendrites from the cells, even
though the rate of dendrite development from
miR124a-treated P19 cells was slower than in the
positive controls By RT-PCR analysis, it was shown
that, in undifferentiated P19 cells, expression of the
stem cell marker octamer 4 (Oct4) was upregulated, but
the differentiation markers NeuroD and
microtubule-associated protein 2 (MAP2) were not detected, as
previously reported [15] (Fig 2B) When P19 cells was
treated only with miR124a, the level of Oct4 transcript
was gradually decreased until 4 days, whereas RA
treatment of P19 cells resulted in the disappearance of
Oct4 expression 2 days after the treatment Both
miR124a-treated and RA-treated P19 cells showed a
significant increase in the expression of the preneuronal
marker NeuroD; however, this was less than in P19
cells with RA treatment Interestingly, the neuron
mar-ker MAP2 was present at high levels only in
RA-trea-ted P19 cells, and was not present at high levels in P19
cells treated with exogenous miR124a Additionally, to
inhibit the function of miR124a, we treated P19 cells
with both RA and miR124a antagomir (synthetic
oligonucleotides that fully complement the miR124a)
Oct4 was continuously expressed until 4 days after
anti-miR124a and RA treatment, but gradually
decreased in level, whereas MAP2 was undetectable
Moreover, NeuroD was detected only 3 days after
treatment with RA and miR124a antagomir These
results showed that miR124a antagomir retarded
RA-induced neuronal differentiation of P19 cells by
blocking miR124a function
To investigate how the protein levels of these
mark-ers were affected by miR124a or RA in P19 cells,
immunofluorescence staining was performed with each
antibody (Fig 2C) The confocal microscope image
showed that the fluorescent signals obtained with Oct4
was found in the cytoplasm of undifferentiated P19
cells, gradually decreased with the treatment with
miR124a, and was undetectable after RA treatment,
owing to the neuronal differentiation of P19 cells
Conversely, the cytoplasmic fluorescent activity of P19
cells with NeuroD gradually increased with treatment
with miR124a or RA by 4 days, with stronger signals
being seen in RA-treated P19 cells, whereas no signal
was found before the treatments The result of
immu-nofluorescence staining using MAP2 showed a gradual
increase of cytoplasmic fluorescence in RA-treated P19
cells for 4 days, but no significant signal either before
or after the miR124a treatment in P19 cells This
sug-gests that the sole function of miR124a could be to
trigger the initial neurogenesis program and that it is
not additionally involved in fully differentiating P19 cells into mature neurons
MicroRNA124a repressed multiple target genes during the neuronal differentiation of P19 cells
To find the genes that are directly regulated by miR124a during miR124a-directed neurogenesis and that contain miR124a seed sequences, microarray data from miR124a-treated HeLa cells [14] and bioinfor-matics data from miR124a-predicted targets from the PicTar database (http://pictar.mdc-berlin.de), an algo-rithm for the identification of miRNA targets using 3¢-UTR alignments, were compared Comparison of
174 microarray-analyzed genes that are significantly regulated by miR124a in HeLa cells and the PicTar database-predicted 787 genes showed 35 genes with overlapping coding sequences that might be directly regulated by miR124a (Fig 3A, Table S1)
For further analysis of miR124a-targeted gene expres-sion by RT-PCR, we randomly selected eight candidates after a review of the literature and determination of the neuronal correlation These included the following: c14orf24, laminin gamma 1 (LAMC1), PTBP1, RNA-binding motif single-stranded interacting protein 1 (RBMS1), hypothetical protein MGC5508 (transmem-brane protein 109), lamin B1 (LMNB1), protein tyrosine phosphatase non-receptor type 12(PTPN12), and ubiqu-itin-specific protease 48 (USP48) Unlike the gradual increase of miR124a expression during the neuronal dif-ferentiation of P19 cells treated with RA, the endogenous gene expression of five of the candidates was gradually decreased over the period of neuronal differentiation (Fig 3B) Unfortunately, three of the eight targets, transmembrane protein 109, Usp48, and RBMS1, could hardly be distinguished, owing to weak expression or technical problems with amplification of their amplicons (data not shown) To determine the direct correlation between the five candidates and miR124a, overexpres-sion analysis of exogenous miR124a was conducted in P19 cells in the absence of RA treatment We could more clearly predict that mRNA transcript levels of the c14orf24, LAMC1, PTBP1, PTPN12 and LMNB1 genes were significantly and directly repressed by miR124a, indicating that mRNA of the five target genes was destabilized by miR124a (Fig 3C)
C14orf24 was directly downregulated by miR124a Most of the miRNA target genes predicted in the Pic-Tar database have one to tens of different miRNA seed sequences at the 3¢-UTR of each mRNA This means that multiple miRNAs can regulate a single
Trang 5target in a cell or a tissue at the same time
Fortu-nately, the PicTar database showed that the 3¢-UTR of
c14orf24 has only two predicted miRNA seed
sequences, miR124a and miR128, both of which are
known to be expressed during neurogenesis [12] The
miRNA-related functions of these are unknown; we
sought to determine whether c14orf24 is directly
regu-lated by miR124a or not About 100 bp of the 3¢-UTR
of c14orf24 near the miR124a seed sequence was
cloned into our established luciferase reporter gene
sys-tem, CMV⁄ Gluc ⁄ c14ofr24-3¢UTR, to monitor whether
it is directly downregulated by miR124a (Fig 4A,B)
CMV⁄ Gluc ⁄ c14orf24-3¢UTR was first transfected into
HeLa cells, where miR124a expression is undetectable
The cotransfection with various doses of exogenous
miR124a showed a significant repression of Gaussia
luciferase (Gluc) activity as compared with the control,
CMV⁄ Gluc ⁄ c14orf24-3¢UTRmt construct, which
con-tained a completely mutated miR124 seed sequence of
c14orf24(Fig 4C)
To quantify the in vitro luciferase activity,
represent-ing the miR124a-directed endogenous expression level
of the c14orf24 gene during neurogenesis, CMV⁄
Gluc⁄ c14ofr24-3¢UTR was transfected into RA-treated
P19 cells The Gluc activity of CMV⁄ Gluc ⁄
c14ofr24-3¢UTR slightly decreased to the third day of neuronal
differentiation of P19 cells (Fig 4D) The significant
decrease in Gluc activity from CMV⁄ Gluc ⁄
c14ofr24-3¢UTR was detected 4 days after the neuronal
differen-tiation of P19 cells, as compared with CMV⁄ Gluc ⁄
c14orf24-3¢UTRmt
The in vivo imaging of 2.5· 106 implanted P19 cells
bearing CMV⁄ Gluc ⁄ c14ofr24-3¢UTR or CMV ⁄ Gluc ⁄
c14orf24-3¢UTRmt was monitored for 2 days of
neuro-nal differentiation, and then aneuro-nalyzed by a region of interest (ROI) analysis As compared with the control from the left thigh without RA treatment, the Gluc expression of CMV⁄ Gluc ⁄ c14ofr24-3¢UTR from the RA-treated P19 cells was significantly decreased, and almost disappeared by the second day of neuronal dif-ferentiation, due to the increased expression of miR124a (Fig 4E) Similar to what was found in the
in vitro luciferase assay of CMV⁄ Gluc ⁄ c14orf24-3¢UT-Rmt, the Gluc activity was slightly, but not signifi-cantly, decreased (Fig 4F) The fold ratio from the ROI analysis of CMV⁄ Gluc ⁄ c14ofr24-3¢UTR showed more dramatically decreased expression of c14orf24 in the RA-treated P19 cells than in the undifferentiated P19 cells (Fig 4G)
The c14orf24 gene was expressed in the cytoplasm as a cellular component and functioned biologically in the positive regulation of cell proliferation
Unfortunately, any potentially valuable biological functions of c14orf24 have not yet been studied, and even its antibody was not available Therefore, identifi-cation of the functions of c14orf24 is a considerable challenge To predict how the protein encoded by the c14orf24 mRNA regulated by miR124a functions in the intact cells, we introduced the coding sequence and the full 3¢-UTR containing miR124a seed sequences of c14orf24 into an expression vector, pcDNA3.1⁄ His vector (designated as Xp-c14orf24), that could express c14orf24 at abnormally high levels and represent the miR124a-mediated repression that takes place in cells
in the presence of miR124a First, to investigate the
752 genes
a
139 genes
C14orf24 LAMC1 PTBP1
PTPN12
Before 1 day 2 days 3 days 4 days 5 days 6 days
Untreated +miR124a
Day after RA treatment β-actin
LMNB1
C14orf24 LAMC1 PTBP1
PTPN12
β-actin
LMNB1
35 genes
Fig 3 The predicted target genes of miR124a in P19 cells (A) Prediction of miR124a target genes using bioinformatics By comparison with
787 genes obtained from the PicTar database (a) and 174 genes significantly downregulated in miR124a-treated HeLa cells (b), 35 genes with overlapping coding sequences (c) were discovered (B) RT-PCR analysis showing the expression of five predicted target genes The lev-els of c14orf24, LAMC1, PTBP1, PTPN12 and LMNB1 were repressed by increasing amounts of miR124a during the neuronal differentiation
of P19 cells (C) The target genes downregulated by overexpression of miR124a The five predicted genes (c14orf24, LAMC1, PTBP1, PTPN12, and LMNB1) were downregulated by miR124a transfection into P19 cells.
Trang 6A B
E
F
G
c14orf24 mRNA
3 ′UTR
Human Mouse
ORF
2360
* 1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
b
a
b
a
0 h 0.5 1.0
2.0
1.5
0
Time
2 days
10 8
4 6
2 0
400 300 200 100
8
4 6
2
10 n M Concentration of miR124a
*
1.2 1.0 0.8 0.6 0.4 0.2 0
20 n M
Before 1 day 2 days 3 days
Day after RA treatment
4 days 5 days
Seed sequence
ATTGAGC
CMV/Gluc/c14orf24-3 ′ UTR
CMV/Gluc/c14orf24-3 ′ UTRmt
3 ′ UTR
3 ′ UTRmt
Gluc CMV
Gluc CMV
Mutation of seed sequence
Fig 4 C14orf24 regulation by miR124a targeting (A) The genomic locus of the c14orf24 3¢-UTR containing the seed sequence that needs to
be recognized by miR124a The mutation of the seed sequence was designed to interrupt the binding of miR124a into the 3¢-UTR (B) Sche-matic diagram of CMV ⁄ Gluc ⁄ c14orf24-3¢UTR constructs The Gluc activity of CMV ⁄ Gluc ⁄ c14orf24-3¢UTR was downregulated when miR124a bound to the c14orf24 3¢-UTR However, the Gluc activity of the CMV ⁄ Gluc ⁄ c14orf24-3¢UTR mutant was not regulated by miR124a, because miR124a did not bind to the c14orf24 3¢-UTR mutant (C) Luciferase analysis to confirm whether the c14orf24 3¢-UTR was bound by exogenous miR124a in HeLa cells The Gluc activity of CMV ⁄ Gluc ⁄ c14orf24-3¢UTR (black bar) was significantly decreased as the concentration of miR124a increased CMV ⁄ Gluc ⁄ c14orf24-3¢UTRmt (gray bar) was used as a negative control Luciferase activity was normalized to the CMV ⁄ Gluc vector Data are expressed as means ± SD in triplicate samples *P < 0.05; **P < 0.005 (D) Luciferase analysis showing that endogenous miR124a binds to the c14orf24 3¢-UTR in P19 cells after RA treatment The Gluc activity of CMV ⁄ Gluc ⁄ c14orf24-3¢UTR (black bar) was decreased during neuronal differentiation The Gluc activity of CMV ⁄ Gluc ⁄ c14orf24-3¢UTRmt (gray bar), as a negative control, was not regulated Data are expressed as means ± SD in triplicate samples *P < 0.05 (E) Bioluminescence image showing that c14orf24 is the target gene of miR124a P19 cells (2.5 · 10 6 ) transfected with CMV ⁄ Gluc ⁄ c14orf24-3¢UTR were subcutaneously grafted onto the left side (a) and right side (b) of the nude mice On the right side, neuronal differentiation was induced by RA treatment Bioluminescence of grafted P19 cells on the right side was decreased in comparison with those on the left side (three mice) (F) Bioluminescence image showing that CMV ⁄ Gluc ⁄ c14orf24-3¢UTRmt, as a negative control, was not regulated by RT-induced neuronal differentiation We used the same method as that used to obtain the previous biolu-minescence image (three mice) (G) ROI analysis of the biolubiolu-minescence image The ratio differentiated ⁄ undifferentiated ratio reduced as time passed However, in the case of injected P19 cells transfected with CMV ⁄ Gluc ⁄ c14orf24-3¢UTR, the ratio remained unchanged.
Trang 7localization of expression of c14orf24 protein,
Xp-c14orf24 was transfected into P19 cells, and double
analysis was performed with the Anti-Xpress antibody
and 4¢,6-diamidino-2-phenylindole (DAPI), which
stains the nucleus As shown in Fig 5A, c14orf24
pro-tein was well expressed in the undifferentiated P19 cells
for 4 days, and showed even localization in the
cyto-plasm However, it was observed that the expression of
c14orf24 protein in the cytoplasm was almost
com-pletely lost 4 days after treatment with either RA or
miR124a of P19 cells These results implied that the
expression of c14orf24 protein is dominantly localized
in the cytoplasm and repressed directly by miR124a
during neuronal differentiation
Next, we investigated how the overexpression of
c14orf24 affects the proliferation and neuronal
differ-entiation of P19 cells Following Xp-c14orf24
transfec-tion, P19 cells displayed more rapidly achieved and
higher cell densities than the cells grown under normal
control conditions (Fig 5B) Additionally, the cellular
morphology demonstrated that treatment with both
RA and Xp-c14orf24 of P19 cells simultaneously
repressed the morphological changes of neuronal
dif-ferentiation, leading to less retraction of cytoplasm
towards the nucleus and a less spherical appearance of
cell bodies, whereas after a single treatment with RA
of P19 cells, the cells increasingly showed the neuronal
traits of a pyramidal and perikaryal appearance To
determine the effect of c14orf24 on P19 cells at the
molecular level, RT-PCR analysis was conducted with
total RNA from P19 cells 4 days after no treatment,
treatment with either RA or Xp-c14orf24, or both
treatments Transfection of Xp-c14orf24 into P19 cells
led to overexpression c14orf24 transcript (Fig 5C)
Similar to the results shown in Fig 4B, RA treatment
of P19 cells resulted in neuronal characteristics of P19
cells, which showed increased gene expression of the
neuron markers MAP2 and NeuroD, and a significant
decrease in that of the stem cell marker Oct4
However, additional treatment of RA-treated P19 cells with Xp-c14orf24 inhibited neuronal differentiation, resulting in lower levels of MAP2 and NeuroD tran-script, and higher levels of Oct4 trantran-script, than in P19 cells treated only with RA
To further verify the effect of c14orf24 on cell prolif-eration and neuronal differentiation of P19 cells, flow cytometry assay was performed with the mitotic marker, 5-ethynyl-2¢-deoxyuridine (EdU) EdU is incor-porated into replicating DNA similarly to 5-bromo-2¢-deoxyuridine (BrdU), and its terminal alkyne group reacts with fluorescent azide [25] The incorporation of the nucleoside analog EdU into cellular DNA of P19 cells was quantified, and demonstrated that the number
of EdU-labeled P19 cells with the overexpression of c14orf24revealed about 2.5-times higher than that with-out treatment (Fig 5D) The number of EdU-labeled P19 cells treated with both Xp-c14orf24 and RA showed 1.7-times higher than that of only RA-treated P19 cells These results indicated a higher rate of DNA synthesis in P19 cells with c14orf24 overexpression
To extend our understanding of the involvement of c14orf24in cellular proliferation and neuronal differen-tiation, the expression of the c14orf24 gene was investi-gated in various cells, including normal, cancer and neuronal precursor cell lines RT-PCR was conducted using total RNA from HT-ori3 cells (normal thyroid cells), L132 cells (lung normal cells), C6 cells (glioma cells), CT-26 cells (colon carcinoma cells), mesenchy-mal stem cells (MSCs), and G2 cells (neural stem cells) In normal cells, HT-ori3 cells, and L132 cells, the c14orf24 gene was expressed at a relatively low level, whereas a higher amount of the c14orf24 ampli-con was expressed in highly proliferating cells, C6 cells, and CT-26 cells (Fig 5E) Also, the c14orf24 gene was highly expressed in both MSCs and G2 Interestingly, when G2 cells were induced to undergo neuronal dif-ferentiation by the deletion of doxycycline from the growth medium, the transcript level of the c14orf24
Fig 5 The biological and cellular functions of the c14orf24 gene in cells (A) Immunocytostaining analysis of c14orf24 by laser scanning con-focal microscopy (red, c14orf24; blue, DAPI) The pcDNA3.1 ⁄ His_c14orf24 containing Xpress epitope was transfected into P19 cells and treated with RA, miR124a or nothing for 4 days The fluorescence image detected by Xpress antibody showed that c14orf24 proteins were predominantly present in the cytoplasm of P19 cells before the treatments, and that they disappeared during the neuronal differentiation of P19 cells induced by RA or after miR124a treatment Magnification: upper panel, · 400; lower panel, · 1000 (B) Cellular morphology charac-teristics of P19 cells affected by the c14orf24 gene Cell morphology was acquired 4 days after no treatment and treatment with either or both of RA or pcDNA3.1 ⁄ His_c14orf24 of P19 cells (C) RT-PCR analysis of P19 cells transfected with the c14orf24 gene Total RNA was extracted from P19 cells 4 days after no treatment and and treatment with either or both of RA or pcDNA3.1 ⁄ His_c14orf24 of P19 cells RT-PCR was conducted for MAP2, NeuroD, Oct4 and c14orf24 using a pair of primers listed in Tables S2 and S4 b-Actin was used as a con-trol (D) EdU-incorporated flow cytometry analysis of P19 cells affected by the c14orf24 gene EdU-labeled cells were measured 4 days after
no treatment and after treatment with either or both of RA or pcDNA3.1 ⁄ His_c14orf24 of P19 cells The y-axis indicates the percentage of EdU-labeled P19 cells Data are expressed as means ± SD in triplicate samples *P < 0.05; **P < 0.005 (E) RT-PCR analysis of c14orf24 expression in various cell lines: HT-ori3 cells (normal thyroid cell line), L132 cells (lung normal cell line), C6 cells (glioma cell line), CT-26 cells (colon carcinoma cell line), MSCs, and G2 cells (neural stem cell line) b-Actin was used as a control.
Trang 8gene was dramatically decreased, and the transcript
had almost disappeared on the fourth day of neuronal
differentiation This result showed that the c14orf24
gene must be positive regulator of cellular proliferation
and be downregulated during neurogenesis
Discussion
Hundreds of well-conserved miRNAs have been
reported to be evolutionarily well conserved over
spe-cies and related to cellular metabolism and various
diseases, including cancer, cardiovascular disease, and neurological disease [26] In particular, neuronal-specific miRNAs such as miR124a, miR9, miR128, miR131, miR178 and miR125b have been directly and indirectly shown to have relatively high expression levels during brain development, and are expected to regulate the various genes related to neuronal differen-tiation
Complicated intracellular and extracellular communi-cation could possibly be involved in the differentiation
of stem cells into mature neurons of the eukaryotic
sys-A
B
E
x1000 x1000
x1000 x1000
x1000 x1000
2 days
RA
c14orf24
RA c14orf24
RA
c14orf24
c14orf24
HT-ori3 L132 C6 CT26 MSC G2-un G2-4d G2-6d
+ + +
+ –
– –
–
MAP2 NeuroD
Oct4
β-actin
c14orf24
β-actin
+ +
+
– –
–
70 60 50 40 30 20 10 0
Day after transfection with miR124a
Trang 9tem In general, a single miRNA is believed to directly
target hundreds of genes, which may indirectly regulate
thousands of coding genes [14] Overexpression of miR1,
which is specifically expressed in cardiac and skeletal
muscle, induced the differentiation of C2C12 myoblasts
into myotubes [27] Transfection of exogenously derived
miR124a into P19 cells in the absence of RA resulted in
guidance of the neuronal program, partly explaining
why miR124a is specifically neuronally expressed
Unlike miR1, the solo miR124a could not sufficiently
induce P19 cells to fully differentiate into neurons, but
could induce only the preneuronal characteristics It is
possible that other, more complex, mechanisms are
required to complete the neuronal differentiation
pro-gram Surprisingly, some experiments in our laboratory
showed that transfection of single, dual or multiple
neu-ronal-specific miRNAs into P19 cells induced a more
neuronally differentiated status than single transfection
with miR124a, and that a cancer-related miRNA
delayed the neuronal differentiation of RA-treated P19
cells (data not shown) These results mean that miRNAs
play an important role in maintaining the stem cells
and inducing neuronal differentiation
As miRNAs have been shown to be correlated with
various diseases, many efforts in both experimental
and in silico studies have been focused on their gene
targets, in order to develop our understanding from
the simple concept of miRNA expression to the more
complicated regulatory interactions between miRNA
and target genes, which can decide cellular fate or
dis-ease progression [28,29] RT-PCR of genes selected by
the analysis from the comparison of microarray data
of miR124a-treated HeLa cells with miR124a-predicted
targets through the PicTar database showed that the
c14orf24, LAMC1, LMNB1, PTBP1 and PTPN12
genes had gradually decreased endogenous gene
expression during the neuronal differentiation of P19
cells when endogenous levels of miR124a were
gradu-ally increased Additiongradu-ally, excessive amounts of
exogenous miR124a in P19 cells resulted in
signifi-cantly direct regulation of these candidates PTPB1,
which is expressed at a high level in non-neuronal cells,
binds to pyrimidine-rich sequences in pre-mRNA and
inhibits the splicing of nearby neuron-specific
alterna-tive exons, but is known to be repressed in the nervous
system by miR124a, to allow the inclusion of
neuron-specific exons in mature mRNA [16] LAMC1, which
is one of the components of a heterodimeric molecule,
laminin-1, comprising laminin a1, laminin b1, and
lam-inin c1 subunits, has also been reported to be an
endogenous target of miR124a [17]
Our experiment using the established luciferase
sys-tem containing the putative 6 bp seed sequence matched
between the 5¢-end of miR124a and the 3¢-UTR of the c14orf24 gene showed a strong evidence of miR124a-mediated repression of c14orf24 during neurogenesis The coding region of c14orf24 (NM_173607), the func-tion of which is not known, possesses 213 amino acids and has a molecular mass of 23 kDa Our findings from PCR and in vitro⁄ in vivo luciferase assays show that c14orf24is expressed before and after neuronal differen-tiation of P19 cells, but its expression is significantly decreased on the day when the endogenous level of miR124a is at its peak Also, the strong suppression of c14orf24 transcript was caused by the exogenous miR124a, through direct binding of miR124a to the miR124a seed sequence of c14orf24 Unfortunately, the
in vivoGluc expression of transiently transfected lucifer-ase systems was almost gone after 3 days of neuronal differentiation For long-term noninvasive imaging, sta-ble cell lines or viral vectors such as lentiviral or adeno-viral vector will be helpful to image the dynamic changes of miR124a-regulated neurogenesis
Additional biological and cellular studies of the c14orf24 gene by immunocytostaining, flow cytometry and RT-PCR analysis showed that c14orf24 was domi-nantly expressed in the cytoplasm and highly expressed
in proliferating cells, but was repressed during neuro-nal differentiation In this study, for the first time, we showed that c14orf24 might function in maintaining cell proliferation, at least in P19 cells, and be involved
in the initial program of neurogenesis via the negative interaction with miR124a
Our noninvasive luciferase imaging systems for moni-toring the repression of the novel targets of miR124a will
be a useful tool for the study of the molecular regulation
of miRNAs related to cellular proliferation, differentia-tion, apoptosis, and various diseases In particular, our ongoing development of a dual luciferase reporter gene system to simultaneously monitor ectopically expressed miRNAs and their targets will provide bidirectional information for cellular therapy and disease diagnosis
Experimental procedures
Quantitative RT-PCR of miR124a With the mirVana miRNA isolation kit (Ambion, Austin,
TX, USA), small RNA was isolated during the neuronal differentiation of P19 cells qRT-PCR was performed using the mirVana qRT-PCR primer Set (Ambion) and mirVana qRT-PCR miRNA detection kits (Ambion), according to the manufacturer’s instructions The PCRs were performed
in triplicate using iCycer (Bio-Rad, Hercules, CA, USA) with SYBR Premix Ex Taq (2·) (Takara, Shiga, Japan) as follows: 95C for 3 min; and 40 cycles of 95 C for 15 s
Trang 10and 60C for 30 s) To normalize the experimental samples
for RNA content, the U6 snRNA primer set (Ambion) was
used as a control
Culture of HeLa cells and P19 cells
HeLa cells (cervical carcinoma cell line) were cultured
routinely in RPMI (Jeil Biotechservices Inc., Daegu, Korea)
containing 10% fetal bovine serum (Cellgro, Herndon, VA,
USA) and 1% antibiotics⁄ antimycotic solution (AA)
(Cell-gro) at 37C P19 cells were obtained from the ATCC In
brief, undifferentiated P19 cells were grown at 37C in
a-MEM (Gibco, Grand Island, NY, USA) supplemented
with 2.5% fetal bovine serum (Cellgro), 7.5% bovine calf
serum (Gibco) and 1% AA (Cellgro) in a 5% CO2humidified
chamber For induction of neuronal differentiation using the
monoculture differentiation method [21], P19 cells were
pla-ted on gelatin-coapla-ted culture plates at a density of
5· 103cellsÆcm)2in growth medium prior to growth factor
removal After 24 h, P19 cells were cultured under serum-free
conditions in DMEM⁄ 12 (1 : 1) medium (Gibco)
supple-mented with 1% insulin–transferrin–selenium (Gibco) and
1% antibiotics, and treated with 5· 10)7m all-trans-RA
(Sigma, St Louis, MO, USA) After 2 days, the RA was
removed from the medium, and the cells were cultured
further under serum-free conditions
RT-PCR
Using Trisol reagent (Invitrogen, Grand Island, NY, USA),
total RNA was isolated from P19 cells as during the day after
RA treatment Reverse transcription, for synthesis of the
first-strand cDNA, was carried out using random-hexamer
and SuperScript II reverse transcriptase (Invitrogen),
accord-ing to the manufacturer’s instructions, and this cDNA was
then used as a template for PCR amplification: 94C for
5 min, with 30 amplification cycles [94C for 30 s each Tm
for 30 s (Tables S2 and S4), 72C for 30 s], and 72 C for
4 min The sequences of the primers used for PCR
amplifica-tion are listed in Table S2 and Table S4
Development of vectors monitoring the
miR124a-directed repression of c14orf24
We constructed a Gluc reporter vector bearing a
cytomega-lovirus (CMV) promoter, and 99 nucleotides from the
3¢-UTR of the c14orf24 gene, containing an miR124a seed
sequence, identified through the PicTar database, were used
for imaging of miR124a-regulated repression They were
constructed by incubation with a pair of primers, sense and
antisense primers of the c14orf24 3¢-UTR, in annealing
buf-fer (· 1 TE bufbuf-fer + 50mm NaCl) for 10 min at 60 C,
cloned into CMV⁄ Gluc at the site between XhoI and XbaI,
and cloned into the CMV⁄ Gluc vector labeled
CMV⁄ Gluc ⁄ c14orf24-3¢UTR (Table S3) As a negative control, CMV⁄ Gluc ⁄ c14orf24-3¢UTRmt was constructed by complete mutation of the miR124a seed sequences in the 3¢-UTR of c14orf24, and annealing the oligonucleotides and sense and antisense primers of c14orf24-3¢UTRmt (Fig 4A,B)
Transfection and luciferase assay Transient transfection of various vectors of interest into undifferentiated⁄ differentiated P19 cells was performed by using 0.6 lg of DNA, 3 lL of Plus reagent (Invitrogen), and 1.5 lL of Lipofectamine (Invitrogen) per well After
3 h, the transfection medium was replaced with undifferen-tiated medium (a-MEM; 2.5% fetal bovine serum, 7.5% bovine calf serum, and 1% AA) from undifferentiated P19 cells and differentiated media (DMEM⁄ 12; 1% insulin– transferrin–selenium and 1% AA) from RA-treated P19 cells Gluc expression was analyzed 2 days after transfec-tion All transfections were carried out in triplicate The cells were washed with NaCl⁄ Piand lysed with 200 lL per well of passive lysis buffer (Promega, Madison, WI, USA) Next, 100 lL of cell lysate from each well was used to mea-sure luciferase activity with the Gaussia luciferase assay kit (Targetingsystems, San Diego, CA, USA), according to the manufacturer’s instructions, and measured on a Wallac1420 VICTOR3V (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) The data are presented as means ± standard deviation (SD) calculated from triplicate wells
Grafting of the cells with reporter gene constructs and in vivo visualization of miR124a and c14orf24 in nude mice All experimental animals were housed under specific patho-gen-free conditions and handled in accordance with the guidelines issued by the Institutional Animal Care and Use Committee of Seoul National University Hospital We performed transient transfection of P19 cells with CMV⁄ Gluc ⁄ c14orf24-3¢UTR and CMV⁄ Gluc ⁄ c14orf24-3¢UTRmt After 48 h of transfection, the cells were counted and resuspended in 100 lL of NaCl⁄ Pi(2.5· 106
cells per
100 lL of NaCl⁄ Pi) These cells were implanted subcutane-ously into male Balb⁄ c nude mice (6 weeks old, weighing 25–27 g) The cells containing CMV⁄ Gluc ⁄ c14orf24-3¢UTR
or CMV⁄ Gluc ⁄ c14orf24-3¢UTRmt were implanted in both thighs; the left thigh, without the RA treatment, was used
as a control, and the right thigh was treated with RA coin-cidently with cell injection Three mice in each group were subsequently anesthetized with 2.5% isofluorane, and trans-ferred into the chamber of an IVIS 100 imager (Xenogen, Alameda, CA, USA) To acquire images of Gluc, the mice were directly injected with 5 lg of coelenterazine The Gluc