MiRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma

The Journal of clinical investigation (Impact Factor: 13.22). 12/2012; 123(1). DOI: 10.1172/JCI63861
Source: PubMed
ABSTRACT
MicroRNAs (miRNAs) and methionine adenosyltransferase 1A (MAT1A) are dysregulated in hepatocellular carcinoma (HCC), and reduced MAT1A expression correlates with worse HCC prognosis. Expression of miR-664, miR-485-3p, and miR-495, potential regulatory miRNAs of MAT1A, is increased in HCC. Knockdown of these miRNAs individually in Hep3B and HepG2 cells induced MAT1A expression, reduced growth, and increased apoptosis, while combined knockdown exerted additional effects on all parameters. Subcutaneous and intraparenchymal injection of Hep3B cells stably overexpressing each of this trio of miRNAs promoted tumorigenesis and metastasis in mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Blocking MAT1A induction significantly reduced the antitumorigenic effect of miR-495 siRNA, whereas maintaining MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of these miRNAs increased total and nuclear level of MAT1A protein, global CpG methylation, lin-28 homolog B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. The opposite occurred with forced expression of these miRNAs. In conclusion, upregulation of miR-664, miR-485-3p, and miR-495 contributes to lower MAT1A expression in HCC, and enhanced tumorigenesis may provide potential targets for HCC therapy.

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Available from: Shelly C Lu, Nov 16, 2015
Research article
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 285
MicroRNAs regulate methionine
adenosyltransferase 1A expression
in hepatocellular carcinoma
Heping Yang,
1
Michele E. Cho,
2
Tony W.H. Li,
1
Hui Peng,
1
Kwang Suk Ko,
1,3
Jose M. Mato,
4
and Shelly C. Lu
1
1
Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases,
Southern California Research Center for Alcoholic and Pancreatic Diseases and Cirrhosis, Keck School of Medicine of University of Southern California,
Los Angeles, California, USA.
2
Department of Pediatric Gastroenterology and Nutrition, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3
Department of Nutritional Science and Food Management, College of Health Science, Ewha Womans University, Seoul, Republic of Korea.
4
CIC bioGUNE, Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (Ciberrehd), Technology,
Park of Bizkaia, Derio, Bizkaia, Spain.
MicroRNAs (miRNAs) and methionine adenosyltransferase 1A (MAT1A) are dysregulated in hepatocellular
carcinoma (HCC), and reduced MAT1A expression correlates with worse HCC prognosis. Expression of miR-
664, miR-485-3p, and miR-495, potential regulatory miRNAs of MAT1A, is increased in HCC. Knockdown
of these miRNAs individually in Hep3B and HepG2 cells induced MAT1A expression, reduced growth, and
increased apoptosis, while combined knockdown exerted additional effects on all parameters. Subcuta-
neous and intraparenchymal injection of Hep3B cells stably overexpressing each of this trio of miRNAs
promoted tumorigenesis and metastasis in mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and
miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Block-
ing MAT1A induction significantly reduced the antitumorigenic effect of miR-495 siRNA, whereas maintain-
ing MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of
these miRNAs increased total and nuclear level of MAT1A protein, global CpG methylation, lin-28 homolog
B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. The opposite
occurred with forced expression of these miRNAs. In conclusion, upregulation of miR-664, miR-485-3p,
and miR-495 contributes to lower MAT1A expression in HCC, and enhanced tumorigenesis may provide
potential targets for HCC therapy.
Introduction
Methionine adenosyltransferase (MAT) is an essential enzyme
that is responsible for the biosynthesis of S-adenosylmethionine
(SAMe), the principal biological methyl donor in all mammalian
cells (1). Mammals express 2 different genes, MAT1A and MAT2A,
that encode for 2 homologous MAT catalytic subunits, α1 and
α2, respectively (2). MAT1A is expressed mostly in adult liver and
serves as a marker for normal differentiated liver, whereas MAT2A is
expressed in all extrahepatic tissues and is induced during rapid liver
growth and liver dedifferentiation (1). In liver, the α1 subunit forms
dimer (MATIII) and tetramer (MATI) MAT isoenzymes (2). MAT1A
expression is reduced in about 60% of patients with cirrhosis due to
various etiologies (3) and is often silenced in human hepatocellular
carcinoma (HCC) (3, 4). In addition to reduced MAT1A expression,
the activity of the MATI/III isoenzymes is often reduced in patients
with chronic liver disease, resulting in chronic depletion of hepatic
SAMe level (5). The Mat1a-KO mouse model has illustrated the
many consequences of chronic hepatic SAMe depletion, the most
important of which is spontaneous development of HCC (6, 7).
The importance of hepatic SAMe depletion in HCC development
is supported by several rodent models of HCC using hepatocarcino-
gens in which hepatic SAMe depletion develops and HCC was pre-
vented by SAMe administration (8–10). The dominant mechanism
of SAMe chemopreventive effect was thought to be from preventing
hypomethylation of the promoter region of several protooncogenes,
as SAMe’s chemopreventive effect was blocked by 5-azacytidine (9,
10). However, in an orthotopic liver cancer model, SAMe adminis-
tration was also able to inhibit HCC establishment but was ineffec-
tive in blocking growth of already existing HCC (11). Part of this was
because of a compensatory response of the liver to induce methyl-
transferases that removed excess SAMe to prevent its accumulation
(11). A better strategy to maintain increased SAMe level in liver can-
cer cells is to induce the expression of MAT1A. Consistent with this,
liver cancer cells with forced expression of MAT1A doubled-cellular
SAMe levels grew slower in vitro and in vivo and exhibited reduced
angiogenesis, ERK, and AKT activation and increased apoptosis in
vivo (12). This proof of concept prompted the current work to inves-
tigate whether MAT1A expression is regulated by microRNAs (miR-
NAs) that are dysregulated in HCC, as miRNAs are potentially much
better therapeutic targets. In the course of our investigation, we
uncovered 3 miRNAs whose expression and function in HCC have
not been reported to be upregulated in HCC and contribute to the
downregulation of MAT1A, tumorigenicity, invasion, and metastasis.
Results
Expression of miRNAs in HCC and the effect of their knockdown on
MAT1A expression in HepG2 and Hep3B hepatoma cell lines. To
determine whether miRNAs can potentially regulate MAT1A
expression, we used an in silico approach to search for miR-
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2013;123(1):285–298. doi:10.1172/JCI63861.
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286 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
NAs that can bind to the 3 UTR of MAT1A. Using 3 differ-
ent miRNA prediction target databases (TargetScan, mirDB,
miRSVR), many miRNAs were identified (Supplemental Table
1; supplemental material available online with this article;
doi:10.1172/JCI63861DS1). We focused on those with high
scores (miRNA-664 [miR-664], miR-485-3p, miR-495, miR-
588, miR-766) whose expression has not been reported in HCC.
Figure 1A shows that miR-664, miR-485-3p, and miR-495 are
induced in HCC, while miR-588 and miR-766 are downregulat-
ed. Since the dominant mechanism of miRNA regulation is that
of downregulation of mRNA/protein levels (13), we focused
only on the 3 that are upregulated. To see whether these miR-
NAs regulate MAT1A expression and to determine the level of
regulation, HepG2 and Hep3B cells were treated with siRNA
targeting these miRNAs. The efficiency of knockdown is shown
in Figure 1B, which is comparable with 75% to 80% reduction
after 24 hours. Figure 1, C and D, shows that knockdown of
miR-664, miR-485-3p, and miR-495 individually raised MAT1A
mRNA and protein levels comparably and combined knock-
down exerted nearly an additive effect. This demonstrates that
these miRNAs negatively regulate MAT1A expression and the
effect is exerted at the mRNA level.
MAT1A 3 UTR contains functional binding sites for miR-664, miR-485-
3p, and miR-495. Figure 2A shows the MAT1A 3 UTR sequence con-
taining the putative binding sites for miR-495 (2 sites), miR-664,
and miR-485-3p. To determine functionality, each site was mutat-
ed alone or in combination and the effect of inserting the WT or
mutated MAT1A 3 UTR on reporter activity was measured in both
Hep3B and HepG2 cells after transient transfection. Figure 2, B and
C, shows that WT MAT1A 3 UTR reduced reporter activity by more
than 50% and mutation of each miRNA binding site led to slight
recovery, with incremental recovery as additional sites were mutat-
ed. When miR-664, miR-485-3p, and miR-495 were all mutated, the
inhibitory effect of the MAT1A 3 UTR was completely lost.
Figure 1
miR-664, miR-485-3p, and miR-495 are induced in HCC and negatively regulate MAT1A expression in liver cancer cell lines. (A) Northern blot
analysis showing expression of select miRNAs in HCC compared with adjacent nontumorous (NT) tissue. (B) Northern blot analysis conrming
siRNA knockdown efficiency of miR-664, miR-485-3p, and miR-495 in HepG2 and Hep3B cells as compared with scramble siRNA (SC) control.
(C and D) Northern (top) and Western (bottom) blot analyses showing the effect of siRNA knockdown of miR-664, miR-485-3p and miR-495,
alone or in combination, on MAT1A expression in HepG2 (C) and Hep3B cells (D). Numbers below the blots represent densitometric values
expressed as percentage of respective controls. Representative blots are shown for C and D from 3 experiments, *P < 0.01 vs. SC;
P < 0.05 vs.
SC, and triple knockdown;
P < 0.001 vs. SC and single knockdown.
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The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 287
Effect of siRNA against miR-664, miR-485-3p, and miR-495 on apopto-
sis and cell proliferation. We previously reported that forced MAT1A
expression in liver cancer cells reduced growth while inducing
apoptosis (12). To see if inducing MAT1A expression by target-
ing miRNAs also has the same effect, we measured apoptosis
and growth. Figure 3A shows that siRNA treatments (single,
double, and triple knockdown) in Hep3B cells induced apopto-
sis only after 72 hours of transfection and that, similar to the
effect on MAT1A expression, the effect on apoptosis was additive
with combined knockdown. To avoid toxicity causing nonspe-
cific effects, effect on growth was measured only after 24 hours
of siRNA treatment. Figure 3B shows that single knockdown
of these miRNAs reduced growth by about 20% and combined
knockdown of all 3 reduced growth by about 50%. Similar find-
ings on apoptosis and growth were observed in HepG2 cells (data
not shown). To determine the contribution of MAT1A induc-
tion on the growth inhibitory effect, Hep3B cells were stably
transfected with siRNAs targeting each miRNA. Control Hep3B
cells were stably transfected with scramble siRNA. These cells
were then transiently transfected with siRNA against MAT1A
or scramble siRNA for 24 hours. Figure 3C shows that Hep3B
cells with stable knockdown of each miRNA exhibited reduced
cell proliferation, but this effect was significantly blunted when
MAT1A was also knocked down (Figure 3C).
Forced lenti–miR-664, lenti–miR-485, and lenti–miR-495 expression
promotes and lenti-siRNAs against these miRNAs inhibit tumorigenesis
in xenograft. To determine whether miR-664, miR-485-3p, and
miR-495 are involved in liver tumorigenesis, stable Hep3B cell
lines expressing forced lenti-miRNAs or lenti-siRNAs against
these miRNAs were established. Supplemental Figure 1 shows
that forced expression of these miRNAs by 3-fold reduced
MATα1 levels by 50%, while knockdown of these miRNAs by 80%
Figure 2
MAT1A 3 UTR-driven reporter activity
and the effect of mutating miRNA bind-
ing sites. (A) Diagram of MAT1A 3 UTR
fragment containing the putative bind-
ing sites for miR-664, miR-485-3p, and
miR-495. Mutations created for each
miRNA site are denoted in bold ital-
ics. Transient transfection assays were
performed using a luciferase reporter
system with WT and mutated MAT1A
3 UTR constructs as described in
Methods in (B) HepG2 and (C) Hep3B
cells. *P < 0.05 vs. control;
P < 0.05
vs. MAT1A 3 UTR;
P < 0.05 vs. triple
miRNA siRNA knockdown. n = 3 experi-
ments, done in triplicate.
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288 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
doubled MATα1 levels. Injection of cells expressing high miRNA
levels subcutaneously into the flanks of nude mice resulted in
more rapid growth of tumors (Figure 4, A and C), whereas tumor
growth was significantly inhibited in cells expressing siRNAs
against these miRNAs (Figure 4, B and C). Manipulating the
expression of miR-495 resulted in the most dramatic effect. At
8 weeks, tumors from mice injected with forced lenti–miR-664,
lenti–miR-485-3p, and lenti–miR-495 showed tumor volumes
that were 160%, 170%, and 360% higher as compared with empty
vector (EV), respectively (Figure 4A). Tumors from mice injected
with cells expressing stable knockdown of miR-664, miR-485-
3p, and miR-495 showed tumor volumes that were 50%, 52%,
and 79% lower as compared with scramble control, respectively
(Figure 4B). Immunohistochemistry staining for proliferating
cell nuclear antigen (PCNA) in these tumors showed increased
staining in tumors with forced miRNA expression and reduced
staining in those with siRNA against these miRNAs (Figure 4C).
Stability of the MAT1A expression in tumors is demonstrated
using immunohistochemistry, and low MATα1 staining corre-
lated with higher PCNA staining and more rapid growth, where-
as high MATα1 staining correlated with lower PCNA staining
and slower growth (Figure 4C).
Forced lenti–miR-664, lenti–miR-485, and lenti–miR-495 expression
promotes and lenti-siRNAs against these miRNAs inhibit tumor invasion
and metastasis in an orthotopic liver cancer model. In order to examine
whether these miRNAs play a role in invasion and metastasis, we
switched to an orthotopic liver cancer model where cancer cells are
directly injected into the left hepatic lobe of nude mice. Figure 5A
shows that at the end of day 45, Hep3B cells expressing forced lenti-
miRNAs developed larger hepatic tumors as in the xenograft model,
higher PCNA staining, lower MATα1 staining, and importantly,
exhibited metastasis to the lung in at least 50% of the mice. Hep3B
cells stably expressing siRNA against these miRNAs had smaller
tumors, lower PCNA staining, and higher MATα1 staining. Since
scramble siRNA control cells did not metastasize to the lung at this
time point, these results indicate that forced expression of these
miRNAs increased invasion and metastatic potential of Hep3B cells.
Effect of reducing the expression of miR-495, miR-485-3p, and miR-
664 in an invasive liver cancer cell line in vivo. Since Hep3B cells did
not metastasize to the lung, we switched to HepG2 cells, which
can invade adjacent structures and metastasize to the lung in the
same orthotopic liver cancer model (Figure 5B). Mice were treated
with siRNA targeting miR-495, miR-485-3p, miR-664, or scramble
control for up to 8 weeks. At week 8, there was a significant (35%
Figure 3
Increased cellular apoptosis and decreased cell growth
in Hep3B cells by miRNA knockdown requires MAT1A
induction. (A) Apoptosis rates were determined by
Hoechst staining at 24, 48, and 72 hours after tran-
sient miRNA knockdown in Hep3B cells. *P < 0.01,
**P < 0.05 vs. SC;
P < 0.05 vs. single or triple siRNA
knockdown. n = 3 experiments, each with 12 determi-
nations. (B) BrdU incorporation assay was measured
at 24 hours after transient miRNA knockdown in Hep3B
cells. *P < 0.05, **P < 0.01 vs. SC;
P < 0.05 vs. single
or triple siRNA knockdown. n = 3 experiments, each
with 8 determinations. (C) To determine the role of
MAT1A induction on growth, Hep3B cells stably trans-
fected with miR-664, miR-485-3p, and miR-495 siRNA
or scramble siRNA (stable SC) were transiently trans-
fected with MAT1A siRNA or SC, and BrdU incorpo-
ration and MAT1A protein levels were measured 24
hours later. *P < 0.05, **P < 0.01 vs. SC;
P < 0.05 vs.
SC+MAT1Asi and each respective miRNA siRNA+SC.
n = 3 experiments, each with 8 determinations for BrdU.
Numbers below the Western blot represent mean den-
sitometric values expressed as percentage of control.
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The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 289
to 62%) reduction in tumor volume (Figure 5B) and reduced inci-
dence of metastases to the lung, abdominal wall, pancreas, and dia-
phragm in mice treated with siRNAs (Figure 5B and Supplemental
Table 2). Reducing miR-495 expression had the most significant
inhibitory impact on tumor growth, invasion, and metastasis.
Role of MAT1A in HCC invasion and antitumorigenic effect of miR-
495 siRNA. To examine the role of MAT1A directly in tumori-
genesis and treatment efficacy of the most potent miRNA
siRNA, miR-495 siRNA, mice were treated with siRNA against
MAT1A, miR-495, and scramble control alone or together using
the same invasive HepG2 cells orthotopic liver cancer model.
Figure 6 shows that reducing MAT1A expression with MAT1A
siRNA more than doubled the tumor volume after 8 weeks and
significantly blunted the treatment efficacy of miR-495 siRNA
on tumor growth. MAT1A expression also had a direct effect on
invasion and metastasis, as MAT1A siRNA treatment increased
Figure 4
Effect of stably transfected miR-664, miR-485, and miR-495 and their siRNAs on tumor growth in a xenograft mouse model. Nude mice were
injected with Hep3B cells subcutaneously containing either (A) stably transfected miR-664, miR-485, and miR-495 (*P < 0.05, **P < 0.01 vs. EV;
P < 0.05 vs. miR-664 or miR-485; n = 8) or (B) stably transfected siRNAs against miR-664, miR-485-3p, and miR-495 (*P < 0.05, **P < 0.0001
vs. SC;
P < 0.05 vs. miR-664-si or miR-485-3psi; n = 8), and tumor volumes were measured over time. (C) Representative pictures of subcutane-
ous tumors at 8 weeks following injection of cells containing stably transfected miRNAs (left) and siRNAs (right) (top row), immunohistochemistry
stained for PCNA (middle row) and MAT1A protein expression (bottom row) Original magnication, ×200. Numbers below PCNA staining repre-
sent percentage of positively stained cells. *P < 0.01 vs. miR-485, miR-664 and EV;
P < 0.05 vs. EV;
P < 0.05 vs. miR-485-3psi, miR-664si and
SC;
§
P < 0.05 vs. SC.
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290 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
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The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 291
invasion to adjacent organs and distant metastasis and blunted
the therapeutic efficacy of miR-495 siRNA on the same param-
eters (Figure 6 and Supplemental Table 3).
MAT1A is the key mediator of miR-664, miR-485, and miR-495 on
tumorigenesis and metastasis. To further evaluate the role of MAT1A
in the tumorigenic effect of miR-664, miR-485, and miR-495,
we examined the effect of overexpressing these 3 miRNAs (in 1
vector) in the presence or absence of a MAT1A expression vec-
tor that does not have the 3 UTR so that these miRNAs cannot
lower MAT1A expression from this vector. Altering the expression
of all 3 miRNAs by combining them or their siRNAs into 1 vec-
tor raised or lowered MAT1A expression, respectively, more than
altering the expression of single miRNAs (Figure 7A). Absence of
the 3 UTR did not alter MAT1A expression from that of the vec-
tor that included it (Figure 7B). In vitro, overexpression of these
miRNAs increased, while overexpression of MAT1A (with or with-
out 3 UTR) decreased cell growth. However, when combined,
overexpression of MAT1A without the 3 UTR was able to reduce
the growth-inductive effect of the miRNAs, while overexpression
of MAT1A with 3 UTR was not (Figure 7C). This translated to
in vivo tumorigenesis so that overexpression of miRNAs greatly
increased tumor size, invasion, and metastasis, while overexpres-
sion of either MAT1A or siRNAs targeting all 3 miRNAs inhibited
tumor growth by 40% and 58%, respectively. Most importantly,
overexpressing the 3 miRNAs failed to increase growth and metas-
tasis in cases in which MAT1A that could not be inhibited by the
3 miRNAs was also overexpressed (Figure 7D). MAT1A protein
levels in these tumors confirmed that the miRNAs were not able
to inhibit MAT1A expression, as the construct did not contain the
MAT1A 3 UTR (Figure 7D).
Molecular mechanism of MAT1A-dependent effect on tumorigenesis,
invasion, and metastasis. Increasing MAT1A expression is expected
to increase SAMe levels (14), which may alter DNA methylation.
Consistent with this, tumors derived from Hep3B cells express-
ing lower miRNAs (hence higher MAT1A) have higher global
DNA methylation and vice versa (Figure 8A). Increased MAT1A
expression resulted in higher nuclear levels of H3K27me3 and
SAMe (Figure 8B). Although many genes are deregulated in HCC,
LIN28B/let-7 (where LIN28B indicates lin-28 homolog B [Cae-
norhabditis elegans]) axis is of particular interest because LIN28B
is overexpressed in HCC and it can repress let-7, a well-studied
tumor suppressor often repressed in cancers including HCC (15).
There are multiple CCGG sites in the promoter region of LIN28B
(Figure 8C). Southern blotting following digestion of DNA with
HpaII or MspI shows that tumors expressing high levels of miR-
664, miR-485, or miR-495 have a hypomethylated LIN28B pro-
moter region as compared with EV (much stronger band at 1300,
weaker or absent band at 4000), while those expressing reduced
levels of these miRNAs have a hypermethylated LIN28B promoter
region as compared with scramble control (absent band at 1300)
(Figure 8C). This correlated with LIN28B expression, so that hypo-
methylation of LIN28B (with forcing miRNA and lower MAT1A)
resulted in higher LIN28B expression, while hypermethylation of
LIN28B (with miRNA knockdown and higher MAT1A) resulted in
lower LIN28B expression (Figure 8D). LIN28B can repress let-7a
(15) and, consistent with this, reducing LIN28B expression result-
ed in higher let-7a expression and vice versa (Figure 8D). Most
interestingly, increasing MAT1A expression by reducing miR-495,
miR-485-3p, or miR-664 increased nuclear content of MAT1A pro-
tein (MATα1) dramatically (Figure 8E).
Discussion
HCC ranks as the fifth most common cancer and the third most
frequent cause of cancer death worldwide (16). Most patients
(70% to 85%) with HCC are diagnosed with advanced disease, and
the overall 5-year survival rate is less than 12% (17). Even in those
HCC patients that undergo surgical resection, the recurrence rate
is about 50% at 3 years (18). Sorafenib, a multikinase inhibitor,
represents a major breakthrough in treatment of advanced HCC,
which was shown to increase the median overall survival from 7.9
to 10.7 months in a randomized, placebo-controlled phase III trial
(SHARP) (19). However, sorafenib did not delay time to symptom-
atic progression, and its cost can be prohibitive. Thus, the search
continues for better treatment strategies against HCC.
MAT1A is predominantly expressed in normal liver, and it is
often silenced in human HCC (3, 4, 20, 21). In both animal mod-
els and human HCC, decreased MAT1A expression correlates
with more aggressive disease and worse prognosis (21). Consis-
tent with an important role of MAT1A in HCC pathogenesis,
mice deficient in Mat1a develop spontaneous HCC (7). Liver can-
cer cells forced to express MAT1A grew slower in vitro and when
tested in xenograft model in vivo (3, 12). In animal models of
HCC, increasing MAT1A expression achieved stable higher tumor
SAMe levels than exogenous SAMe treatment, since chronic
administration of SAMe led to an increase in the expression of
methyltransferases that prevented SAMe accumulation (11, 12).
Thus, developing strategies to increase MAT1A expression in
HCC may be effective in slowing HCC growth.
Both transcriptional and posttranscriptional mechanisms
have been reported to help explain reduced MAT1A expression
in HCC. Promoter hypermethylation correlated with reduced
MAT1A expression in cirrhotic patients and HCC (3, 21). In addi-
tion, methylation of the coding region near the translational
start site can also inhibit MAT1A transcription (22). Recently we
Figure 5
Effect of varying miR-664, miR-485-3p, and miR-495 expression on
tumorigenesis, invasion, and metastasis in an orthotopic liver can-
cer model. (A) Hep3B cells stably transfected with lenti–miR-664,
miR-485, and miR-495/EV or lenti-siRNA against these miRNAs or
SC were injected into the left hepatic lobe, and mice were sacriced
after 45 days. The top row shows H&E staining of liver tumors, and
tumor volume at the site of injection are shown below for each con-
dition (*P < 0.05 vs. miR-485, miR-664 and EV;
P < 0.05 vs. EV;
P < 0.05 vs. miR-485-3psi, miR-664si, and SC;
§
P < 0.05 vs. SC).
Second row shows H&E staining of lung tissue and incidence of lung
metastasis. Arrows point to lung metastasis. Third and fourth rows show
immunohistochemistry for PCNA and MAT1A protein. Numbers below
PCNA represent percentage of positive cells, *P < 0.01 vs. miR-485,
miR-664 and EV;
P < 0.05 vs. EV;
P < 0.05 vs. miR-485-3psi, miR-
664si and SC;
§
P < 0.05 vs. SC. Original magnication, ×100 (rst row);
×200 (second through fourth rows). (B) HepG2 cells capable of inva-
sion and metastasis were injected into the left hepatic lobe as above,
and lentiviral vectors containing miRNA siRNA or SC were injected into
the spleen at the time of HepG2 cell injection. Two weeks later, lentiviral
siRNA was injected into the tail vein, and this was repeated every 2
weeks until sacrice at 8 weeks. H&E staining showing the effect of
miR-495, miR-485-3p, and miR-664 siRNAs on tumor invasion and
metastasis. Arrows point to tumor at the site of injection, and tumor vol-
umes and invasion incidences are shown below each image. *P < 0.01
vs. scrambled siRNA (SC);
P < 0.05 vs. miR-485-3psi and miR-664si.
Original magnication, ×100.
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292 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
showed that the MAT1A 3 UTR binds to the AU-rich RNA bind-
ing factor 1 (AUF1), which is one of the hnRNP proteins known
to destabilize target mRNAs (23). HCC specimens express higher
AUF1 protein levels, and knockdown of AUF1 increased MAT1A
mRNA level (23). However, these mechanisms are difficult to tar-
get, since they can affect numerous other genes. This prompted
us to consider the possibility that miRNAs might also regulate
MAT1A expression.
miRNAs are small noncoding RNAs that regulate gene expres-
sion by targeting the 3 UTR of mRNAs, leading to reduced protein
translation and/or increased mRNA degradation in most cases
(13). Dysregulation of miRNA expression plays an important role
in the pathogenesis of HCC, and miRNA signatures may serve
as biomarkers for HCC classification and prognostic risk strati-
fication as well as therapy (24, 25). We used the miRWalk web-
site (http://www.ma.uni-heidelberg.de/apps/zmf/mirwalk/index.
html) to simultaneously search 8 different established miRNA
prediction programs to see which miRNAs have the most positive
prediction matches. Using the most popular search algorithms —
miRSVR (uses miRANDA), TargetScan and mirDB — we generated
a list of miRNAs that gave the best scores for MAT1A. Interestingly,
while some of these are known to have altered expression in HCC,
the expression of most of these miRNAs in HCC is unknown. Of
the 5 best-matched miRNAs whose function in HCC is unknown,
3 are induced in 4 sets of HCC samples as compared with adjacent
nontumorous liver, and we focused on these for the current work,
since the majority of miRNAs downregulate expression of their
targets. Indeed, knockdown of miR-664, miR-485-3p, or miR-495
in both HepG2 and Hep3B cells raised MAT1A mRNA and protein
levels comparably (Figure 1), which is consistent with the notion
that the mechanism of these miRNAs on MAT1A is to increase its
mRNA degradation. Reporter assay confirmed the presence of
functional miRNA-binding sites in the MAT1A 3 UTR (Figure 2).
Each miRNA exerted a significant influence on growth and apop-
tosis in vitro and tumor growth in vivo; however, although the
miRNAs all have comparable effects on MAT1A expression, miR-
495 exerted the most pronounced effect on tumor growth, inva-
sion, and metastasis. This suggests miR-495 has other important
targets besides MAT1A. Nevertheless, knocking down miR-664 or
miR-485-3p reduced tumor growth by over 50% as well as invasion
and metastasis, supporting an important role for MAT1A. Consis-
tent with this, lowering MAT1A expression increased the tumori-
genicity, invasion, and metastatic potential of HCC and blunted
the therapeutic efficacy of miR-495 siRNA on these parameters.
Figure 6
Role of MAT1A in tumorigenesis and therapeutic effect of miR-495 siRNA. HepG2 cells were injected into the left hepatic lobe of male BALB/c
nude mice and lentiviral vectors containing MAT1A siRNA (MAT1Asi), miR-495 siRNA (miR-495si), and scramble siRNA (SC), alone or together,
were injected into the spleen at the time of HepG2 cell injection (n = 8 per group). Control group received only HepG2 cell injection. Two weeks
later, lentiviral siRNAs were injected into the tail vein, and this was repeated every 2 weeks until sacrice at 8 weeks. First row: arrows point
to tumors at the site of injection, and tumor volumes are shown below. *P < 0.005 vs. SC+SC;
P < 0.005 vs. MAT1Asi+SC;
P < 0.05 vs. miR-
495si+MAT1Asi. Second and third rows show metastasis to lung and pancreas (indicated by arrows) in the various treatment groups, with the
incidence shown below. Original magnication, ×200.
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The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 293
Furthermore, forced expression of MAT1A that cannot be inhib-
ited by these 3 miRNAs completely eliminated the inductive effect
of these miRNAs on growth and metastasis. Taken together, these
results indicate that MAT1A is the key target for these miRNAs in
exerting their influence on HCC tumorigenesis.
miR-485-3p has been shown to mediate topoisomerase IIα
downregulation in part via altered regulation of the transcription
factor nuclear factor YB and to have a role in drug responsiveness
in CEM and CEM/VM-1-5 cells (human leukemic lymphoblastic
cells) and in Rh30 and Rh30/v1 cells (human rhabdomyosarcoma
cells) (26). miR-495 has been shown to be upregulated in breast
cancer stem cells, where its overexpression promoted tumorigenic
activity by downregulating E-cadherin and REDD1 (regulated
in development and DNA damage responses 1), an inhibitor of
mTOR signaling (27). miR-495 is also thought to have a role in
liver and pancreas development (28). Increase of miR-664 expres-
sion was found in atrial fibrillation patients with rheumatic heart
disease (29). However, as of today, the expression and function of
these miRNAs in HCC has not been reported. Recently, Kotur-
bash et al. demonstrated that miR-29b might play a role in down-
regulating Mat1a in the preneoplastic liver tissue of rats treated
with the hepatocarcinogen 2-acetylaminofluorene (30). However,
miR-29a-c was found to be downregulated in HCC, and it has
been shown to promote apoptosis by lowering the expression of
Bcl-2 and Mcl-1 (31). Human MAT1A has not been shown to be
regulated by miRNA. Our study clearly demonstrates an impor-
tant role for miR-495, miR-485-3p, and miR-664 in regulating
MAT1A expression and that also shows that downregulating their
expression inhibited tumor growth, invasion, and metastasis of
HCC. The next question is, how does increasing MAT1A expression
impact on tumorigenesis, invasion and metastasis?
Since MAT is responsible for SAMe synthesis and MATI/III are
the products of the MAT1A gene (1), increasing MAT1A expres-
sion would increase steady state SAMe levels (3). Changes in DNA
methylation, particularly hypermethylation of tumor suppressors,
play a critical role in cancer pathogenesis, including HCC (32).
This prompted us to examine whether MAT1A expression in liver
cancer cells influences DNA methylation. Indeed, when MAT1A
expression was increased by knocking down miR-664, miR-485-3p,
or miR-495, nuclear SAMe levels and global DNA methylation
increased, and the opposite occurred when MAT1A expression
was reduced by forcing the expression of these miRNAs. Of the
many genes deregulated in HCC, we focused on LIN28B because
of a recent report that demonstrated LIN28B (not LIN28) is
overexpressed in HCC and promotes transformation and inva-
sion in HCC in part via repression of let-7 (15). The let-7 family of
miRNAs regulates factors that control cell-fate decisions, includ-
ing oncogenes and cell-cycle factors (33–35). LIN28B (and LIN28)
exerts a reciprocal regulation with let-7 (33). let-7 suppresses the
expression of LIN28 through let-7–binding sites in the LIN28 3
UTR, while LIN28/LIN28B suppress the production of mature
let-7s at multiple levels as well as enhancing let-7 degradation via
3 terminal uridylation of let-7 precursors (33). This opposing
expression pattern of Lin28 and let-7 can be found throughout
development and in oncogenesis and has been compared with a
yin-yang balancing act by Ji and Wang (33). This can be illustrated
by activation of LIN28 by c-Myc and NF-κB, leading to let-7 repres-
sion and cell transformation (33). The LIN28B promoter region
has multiple CpG sites, including 3 CpG islands (as determined
by CpG island searcher: cpgislands.usc.edu), where Viswanathan
et al. correlated the loss of DNA methylation of a downstream
CpG island with that of its expression found in HepG2 and K562
erythromyeloblastoid leukemia cells (36). Interestingly, we found
that forced miRNAs reduced MAT1A expression and LIN28B pro-
moter methylation and increased LIN28B expression. This cor-
related with a fall in let-7a expression. Increasing MAT1A expres-
sion by knocking down miR-664, miR-485-3p, and miR-495 led
to LIN28B promoter hypermethylation, reduced LIN28B expres-
sion, and increased let-7a expression (Figure 8D). Thus, enhanc-
ing MAT1A expression shifted the balance of LIN28B/let-7 toward
let-7 and inhibited tumor growth, invasion, and metastasis.
Recently Reytor et al. reported finding MATI/III in the nuclei
(37). The authors speculated that presence of nuclear MAT might
provide a continuous source of nuclear SAMe, since SAMe is
charged and whether or not it can traverse the nuclear membrane
is in debate. In support of this, nuclear accumulation of the active
MAT1A protein correlated with higher levels of histone H3K27
trimethylation, an epigenetic modification associated with gene
repression and DNA methylation (37). Our findings are consistent
with this report. Interestingly, we found that knocking down miR-
664, miR-485-3p, and miR-495 increased total cellular MAT1A pro-
tein level, particularly the nuclear fraction (Figure 8E). This may
explain the dramatic effect that MAT1A expression has on DNA
methylation.
In summary, we have identified 3 miRNAs that are increased
in HCC that can negatively regulate MAT1A expression at the
mRNA level. Reducing the expression of these miRNAs raised
MAT1A expression, which we believe is a novel strategy to shift
the LIN28B/let-7 balance toward let-7. We suspect this increase in
MAT1A expression also has an impact on the expression of many
other genes involved in tumorigenesis. This will be the subject of a
future investigation. Our results also help to explain why decreased
MAT1A expression in HCC is a poor prognostic indicator (21). Few
therapeutic options currently exist to treat HCC. These miRNAs
are potential therapeutic targets and offer substantial promise in
expanding treatment options for patients with HCC.
Methods
Materials and reagents. α-
32
P-dCTP and γ-
32
P ATP (3,000 Ci/mmol) were pur-
chased from PerkinElmer. Antibodies used for either Western blot and/
or immunohistochemistry to PCNA, LIN28B, and β-actin were purchased
from Cell Signaling Technology. MATα1 antibody was purchased from
Novus Biologicals, whereas H3K27me3 and GFP antibody were purchased
from Abcam. Lipofectamine 2000 and RNAimax were purchased from
Invitrogen, whereas the MethylFlash Methylated DNA quantification kit
was purchased from Epigentek. Lenti–miR-664, lenti–miR-485 (lenti–miR-
485-3p is not available), lenti–miR-495, lenti–miR-664 siRNA, lenti–miR-
485-3p siRNA, lenti–miR-495 siRNA, and a lentiviral purification kit
were purchased from SBI System Biosciences. Lenti-MAT1A siRNA was
purchased from Applied Biological Material Inc. siRNA to hsa–miR-664
(AGGCTGGGGATAATTGAAT), hsa–miR485-3p (AGAGGAGAGCCGTG-
TATGAC), and hsa–miR-495 (5-AGAAGTGCACCATGTTTGTT-3) were
purchased from EXIQON. pMir-Target vector for MAT1A 3 UTR clone
was purchased from OriGene Technologies. GFP expression after injection
of lenti-miRNAs or lenti-siRNAs was visualized on paraffin sections by
immunohistochemistry with mouse anti-GFP antibody (1:200; Clontech,
BD Biosciences) using the ABC method (Vector Laboratories). All other
reagents were of analytical grade and obtained from commercial sources.
Source of normal and cancerous liver tissue. Normal and cancerous liver tis-
sues were obtained as described (38).
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294 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
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The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 295
containing the 2 CMV promoters miR-485-3p and miR-495 was sub-
cloned into the miR-664 containing pGreenPuro EcoRI site. Each shRNA
and its CMV promoter was confirmed by sequencing.
MAT1A expression vector cloning was described previously (12). MAT1A
3 UTR fragment was subcloned into pCDH-CMV-MCS-EF1-copGFP vec-
tor (SBI System Biosciences) EcoRI site to create MAT1A without 3 UTR.
Lenti-reporter-luciferase vector containing only the MAT1A 3 UTR (ABM)
was excised and subcloned into the BamHI site of pCDH-CMV-MCS-EF1-
copGFP vector to generate MAT1A with 3 UTR. Stable Hep3B cell lines
expressing these vectors were established as described above.
DNA constructs and dual luciferase assay. A partial forward and reverse MAT1A
3 UTR fragment from +1822 bp to +1961 bp and mutant fragments con-
taining Sgf1 and MluI linkers were synthesized. Two base pair mutants were
performed for miR-664 (AATGAATAAATAAT, where italics represent the
targeted nucleotides within the miRNA sequence for mutation), miR-485-3p
(TGTATGATGTTGGA), and miR-495 (TTTGTTTTATTT) putative tar-
get site(s) in MAT1A 3 UTR (Figure 2A). The annealed fragments and pMir-
Target vector were digested with Sgf1 and MluI. WT and mutant MAT1A 3
UTR were cloned into the pMirTarget vector containing a luciferase reporter
(OriGene Technologies). Hep3B cells were placed in 24-well plates the day
before transfection. The WT, mutated 3 UTR of MAT1A pMirTarget vector,
or pMirTarget EV (200 ng) and a control Renilla luciferase expression vector
(2.5 ng) were cotransfected into Hep3B cells with Superfect (QIAGEN) fol-
lowing the manufacturer’s instructions. Luciferase assays were performed 24
hours later using the Dual Luciferase Reporter Assay System (Promega) as
directed by the manufacturer’s suggested protocol. Firefly luciferase activity
was normalized to Renilla luciferase activity.
Xenograft model. Sixty-four 4-week-old male BALB/c nude mice from Jack-
son ImmunoResearch Laboratories Inc. were divided equally into 8 groups
(n = 8 per group) and given the following Hep3B stable cell line injection:
group 1, lenti-EV; group 2, lenti–miR-664; group 3, lenti–miR-485; group
4, lenti–miR-495; group 5, lenti-miRNA scramble siRNA; group 6, lenti–
miR-664 siRNA; group 7, lenti–miR-485-3p siRNA; group 8, lenti–miR-495
siRNA. Hep3B cells (1 × 10
7
) in 100 μl PBS were injected subcutaneously
into the right flank of each nude mouse. From week 3 on, xenograft tumor
size was measured by calipers. The tumor volume was calculated according
to the formula: π/6 (length × width
2
) (39). Animals were sacrificed at week
8. Parts of the tumor tissues were used for RNA and protein analysis; the
rest were fixed in 4% formalin for histology and immunohistochemistry.
Orthotopic liver cancer model using Hep3B cells expressing varying levels of
miRNAs and MAT1A. Hep3B cells stably transfected with lentiviral vectors
expressing miRNAs or siRNAs against the miRNAs (1.5 × 10
6
cells/50 μl)
were slowly injected into the left hepatic lobe of 4-week-old male BALB/c
nude mice (n = 8 per group). Animal groups and number were the same as
described for the xenograft model. The tumor size in liver tissues was mea-
sured as above at day 45 (pilot experiment showed 50% mice died at day 56
in the miR-495 group, but all mice in different groups survived at day 45)
and the tumor volume was calculated. Lung, liver, and adjacent tissues were
harvested for DNA, RNA, and protein assays as well as standard pathologi-
cal studies as described for the xenograft model.
In separate experiments, 4-week-old male BALB/c nude mice (n = 8 per
group) were injected in the left hepatic lobe as above with Hep3B cells
(1.5 × 10
6
cells/50 μl) and stably transfected with MAT1A expression vector
that does not have the 3 UTR (MAT1A-no 3 UTR) or EV. Concurrently, mice
were also injected into the spleen with lentiviral vector that expresses either
all 3 miRNAs (miRNAs), siRNAs against all 3 miRNAs (miRNAsi), or EV.
The packaging was done using the Trans Lentiviral pGIPz Packaging system
(TLP4614; Open Biosystems). Viral harvesting was done as described in the
Open Biosystems protocol. A total of 1 × 10
5
Hep3B cells were infected at
a multiplicity of 20 PFU/cell for 24 hours. 2 × 10
9
transducing units (final
Cell lines and stable transfection of lenti-miRNAs and lenti-siRNAs. 293T,
Hep3B, and HepG2 cell lines were obtained from Cell Culture Core of the
USC Research Center for Liver Diseases and cultured in DMEM supple-
mented with 10% fetal bovine serum.
To establish stable expression of miRNA or its siRNA, 10
5
Hep3B cells
were seeded in a 24-well plate 1 day prior to infection. To generate cells
stably expressing miRNAs or siRNAs, Hep3B cells were transfected with
lenti–miR-664, lenti–miR-485, lenti–miR-495, lenti-EV, lenti–miR-664
siRNA, lenti–miR-485-3p siRNA, lenti–miR-495 siRNA, and lenti-scramble
siRNA vector for 3 hours by using Lipofectamine 2000 (Invitrogen). Fol-
lowing selection with puromycin (Invitrogen), stable clonal cell lines were
established and examined for the expression of miRNA or siRNA and GFP
expression by Northern analysis.
In separate experiments, stable cell lines expressing siRNA against miR-
495, miR-485-3p, or miR-664 were transiently transfected with scramble
siRNA or siRNA against MAT1A (SI00009387; QIAGEN) in Hep3B for 24
hours using RNAimax (Invitrogen). MAT1A expression, apoptosis, and
BrdU incorporation were measured as described below.
Construction of vectors and stable cell lines expressing multiple miRNAs or their
siRNAs and MAT1A with or without its 3 UTR. Single miRNA expression vec-
tors for premiR-485 (PMIRH485PA-1), premiR-495 (PMIRH495PA-1), and
preMIR-664 (PMIRH664PA-1) were obtained from SBI System Biosciences.
The multiple miRNA expression vector was constructed by sequentially
cloning the premiR-485 and premiR-664 insert into the premiR-495 lenti-
viral expression vector BamHI and EcoRI sites, respectively.
Oligonucleotides used to construct the lenti-vector containing siRNAs
targeting miR-664, miR-485-3p, and miR-495 are shown in Supplemental
Table 4. miR-664 siRNA oligonucleotide was inserted into the BamHI site
of pGreenPuro (SBI System Biosciences). CMV promoter was amplified
by PCR using the primers 5-TAGTTATTAATAGTAATCAATTACGGG-3
(forward primer) and 5-GATCTGACGGTTCACTAAACCAG-3 (reverse
primer) from pGreenPuro and cloned into pCR 2.1 vector by TA clon-
ing (Invitrogen). miR-485-3p siRNA oligonucleotide was inserted at the
EcoRV, followed by miR-495 inserted at the SacI and SpeI sites. A second
CMV promoter was cloned into the HindIII and KpnI sites. The fragment
Figure 7
MAT1A is the key mediator of miR-664, miR-485, and miR-495 on
tumor growth and metastasis. (A) MAT1A Northern and Western
blots of Hep3B cells stably transfected with lenti–miR-664, lenti–
miR-485, and lenti–miR-495/EV singly or all 3 miRNAs together
(miRs), lenti-siRNA against miRNAs (alone or together, miRsi), or
SC. *P < 0.05 vs. respective controls;
P < 0.05 vs. individual miRNA
or miRNAsi. (B) MAT1A Western blots of Hep3B cells stably trans-
fected with lenti-MAT1A with or without 3 UTR. *P < 0.05 vs. EV.
(C) BrDU measurement in Hep3B cells stably expressing MAT1A
with or without 3 UTR, then transiently transfected with lentiviral
vector containing all 3 miRNAs, or EV for 24 hours and expressed
as percentage of control (EV+EV). Results are mean ± SEM from 3
experiments done in triplicate. *P < 0.01 vs. EV+EV;
P < 0.05 vs.
EV+MAT1A no 3 UTR; **P < 0.05 vs. EV+miRNAs and MAT1A with
3 UTR+miRNAs;
#
P < 0.05 vs. EV+MAT1A with or without 3 UTR.
(D). Hep3B cells stably transfected with MAT1A without 3 UTR or
EV were injected into the left hepatic lobe and treated with lentiviral
vector expressing all miRNAs, siRNA against all 3 miRNAs (miR-
NAsi), or EV. Tumor volumes at the site of injection 45 days later are
shown below for each condition, *P < 0.05 vs. EV+EV;
#
P < 0.05 vs.
EV+miRNAs. MAT1A protein levels and incidence of lung metastasis
are shown below. *P < 0.05 vs. EV+EV. Original magnication, ×200.
Numbers below all blots refer to densitometric values expressed as
percentage of respective controls.
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296 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
Page 12
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The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013 297
lular RNA was extracted by using TRIzol reagent (Invitrogen) according to
the manufacturer’s instructions. Equal amounts of total RNA (15 μg) were
denatured, fractionated by electrophoresis on a 15% polyacrylamide–8 M
urea gel, electroblotted, and cross-linked onto a nylon membrane. North-
ern blot analysis was performed as described using Ultrahyb-Oligo (Ambi-
on). As a control for normalization of RNA expression levels, blots were
hybridized with an oligonucleotide probe complementary to the U6 RNA
(5-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3).
Xenograft and liver tissues isolated from the different treatment groups
were subjected to Western blot analysis. Fifteen micrograms of total pro-
tein extract were resolved on 12.5% SDS-polyacrylamide gels. Membranes
were probed with antibodies to LIN28B, H3k27me3, and MATα1. To
ensure equal loading, membranes were stripped and reprobed with anti–
β-actin antibodies. Semi-quantitative analysis was performed for both
Northern and Western blots using Quantity One (Bio-Rad).
Histology and immunohistochemistry. Sections from xenograft, liver, lung,
and pancreas were fixed with formalin for 4 hours, embedded in paraffin,
sectioned, and stained with H&E, as previously described (12). Staining
and counting of PCNA were performed according to the manufacturer’s
suggested protocol (Invitrogen), whereas MATα1 antibody was diluted to
1:200. Immunohistochemical staining of MATα1 was performed with the
Vector ABC Kit according to the manufacturer’s method. For quantifying
immunohistochemical staining, a total of 5 fields at ×100 magnification
were randomly selected (minimum of 1000 cells total), and positive nuclei
or cells were counted and expressed as a percentage of the total using Meta-
Morph imaging software. Control with no antibody showed no staining.
Apoptosis and BrdU incorporation. Apoptosis was measured as described
(12). BrdU incorporation was measured with BrdU Detection Kit accord-
ing to the manufacturer’s protocol (BD Biosciences — Pharmingen).
Nuclear SAMe levels. SAMe levels were measured as described (11) in puri-
fied nuclear fractions from tumors expressing varying levels of miRNAs
or their siRNAs.
Statistics. Data are given as mean ± SEM. Statistical analysis was per-
formed using ANOVA followed by Fisher’s test for multiple comparisons.
Significance was defined as P < 0.05.
Study approval. The study protocol conformed to the ethical guidelines
of the 1975 Declaration of Helsinki as reflected in a priori approval by
Keck School of Medicine University of University of Southern California
Health Science Institutional Review Board (Los Angeles, California, USA).
All procedure protocols, use, and the care of the animals were reviewed and
approved by the Institutional Animal Care and Use Committee at UCLA.
Acknowledgments
This work was supported by NIH grants R01DK51719 (to S.C.
Lu and J.M. Mato), R01AT01576 (to S.C. Lu and J.M. Mato), and
Plan Nacional of I+D SAF 2008-04800, and HEPADIP-EULSHM-
CT-205 (to J.M. Mato). 293T, HepG2, and Hep3B cells were pro-
vided by the Cell Culture Core of the USC Research Center for
Liver Diseases (P30DK48522). Pathological sections and staining
were done by the Imaging Core of the USC Research Center for
Liver Diseases (P30DK48522).
Received for publication June 6, 2012, and accepted in revised
form October 18, 2012.
Address correspondence to: Shelly C. Lu, Division of Gastrointes-
tinal and Liver Diseases; HMR Bldg., 415, Department of Medi-
cine, Keck School of Medicine, USC, 2011 Zonal Ave., Los Angeles,
Calfornia 90033, USA. Phone: 323.442.2441; Fax: 323.442.3234;
E-mail: shellylu@usc.edu.
volume 0.1 ml) were injected into the tail veins of mice. In order to maintain
a high level of miRNA knockdown, repeated tail-vein injections were done at
week s2 and 4. Tumor volumes in the liver and presence or absence of lung
metastasis were documented at day 45 as above.
Orthotopic liver cancer model using HepG2 cells and treatment with siRNAs
against miR-495, miR-485-3p, miR-664, or MAT1A. HepG2 cells have the ability
to invade and metastasize (40). To test the effect of knocking down miR-
NAs, HepG2 cells (1.5 × 10
6
cells/50 μl) were injected into the left hepatic
lobe of 4-week-old male BALB/c nude mice following spleen injection of
miRNA siRNA. Then tail-vein injection was done every 2 weeks. Animal
groups (n = 8 per group) were as follows: group 1, lenti-scramble siRNA;
group 2, lenti–miR-495 siRNA; group 3, lenti-485 siRNA; and group 4,
lenti-664 siRNA. Mice were sacrificed at day 56. Lentiviral packaging and
harvesting were as described above except HeG2 cells were used and tail
vein injection was repeated at week 6. Immunohistochemistry and Western
blot were done to assess transduction efficiency using GFP.
To examine the role of MAT1A expression on tumorigenicity and the
therapeutic effect of miR-495 siRNA, in separate experiments, mice were
injected with HepG2 cells directly into the left lobe as above and treated with
lenti-MAT1A siRNA and lenti–miR-495 siRNA alone or in combination as
described above. Animal groups (n = 8 per group) were as follows: group 1,
lenti-scramble vector and lenti-scramble siRNA; group 2, HepG2 injection;
group 3, lenti-MAT1A siRNA+lenti-scramble siRNA; group 4, lenti–miR-
495 siRNA+lenti-MAT1A siRNA; and group 5, lenti–miR-495 siRNA+lenti-
scramble siRNA. Mice were sacrificed at day 56; liver tumor volume at the
site of original injection was measured and tissues (lung, liver, pancreas) were
harvested for pathological exam as described above.
Global DNA and LIN28B promoter methylation assay. Levels of 5-methylcyto-
sine (5-mC) in hepatic tumors derived from Hep3B cells stably expressing
miR-664, miR-485, and miR-495 or their siRNAs were measured by the
MethylFlash Methylated DNA Quantification Kit.
DNA samples of tumor tissues from mice injected with Hep3B cells
expressing forced miR-664, miR-485, and miR-495 and their respective
siRNAs were extracted and digested by MspI, HpaII, PvuII, and NdeI. South-
ern blot was done as described previously (41).
Northern and Western blot analysis. Northern blotting probes for miR-664,
miR485-3p, miR-495, and let-7a were purchased from EXIQON. Total cel-
Figure 8
Possible mechanism of miR-664, miR-485-3p, miR-495, and MAT1A
involvement in tumorigenesis, invasion and metastasis. (A) 5-mC levels
in tumors derived from Hep3B cells stably expressing lower (with siRNA
or si) or higher levels of miRNAs. *P < 0.05; **P < 0.01; ***P < 0.0001 vs.
SC;
P < 0.05 vs. EV. n = 8 per condition. (B) Effect of varying miRNA
expression on nuclear H3K27me3 levels in tumors (Western blots) and
SAMe levels. Results are mean ± SEM from 8 per condition. *P < 0.05;
**P < 0.01 vs. respective controls. (C) Diagram shows HpaII and MspI
sites between PvuII and NdeI in human LIN28B promoter. Black
squares, CCGG sites; TSS, transcriptional start site. Numbers are rela-
tive to TSS. Southern blot analysis of LIN28B promoter region between
–1576 and +2432 (right). DNA samples from liver tumor derived from
stably transfected Hep3B containing miR-664, miR-485, and miR-495
and their siRNAs were digested as indicated. MspI digestion results in a
band size of 1359 bp as control for HpaII digestion. (D) Effect of overex-
pressing miR-664, miR-485, and miR-495 and their siRNAs on MAT1A
and LIN28B protein expression (top) and let-7a mRNA expression (bot-
tom). Numbers below the blots are densitometric values expressed as
percentage of respective controls. *P < 0.01 vs. EV;
P < 0.05 vs. miR-
495;
P < 0.01 vs. SC;
§
P < 0.05 vs. miR-495si. (E) Increased nuclear
localization of MAT1A protein is seen after knockdown of miR-664, miR-
485-3p, and miR-495. Original magnication, ×630 (oil immersion).
Page 13
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298 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
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  • Source
    • "Hsa-miR485-3p was found to be upregulated in Beijing/W infected macrophages. Hsa-miR-485-3p has been shown to be involved in cell survival [24] and knockdown of this miRNA in hepatic cells increased apoptosis [25]. Previous report indicated that miR-485-3p post-transcriptionally targeted NF-YB [24], a direct transcriptional repressor of Top2α gene and of MDR1 and CCNB2 genes [26] in regulation of the cell cycle, Our results suggest that high miR-485-3p possibly facilitates survival of the Beijing/W strains in macrophages and evades apoptosis or alters macrophage lysis and subsequent downstream immune response toward clearance of MTB. "
    [Show abstract] [Hide abstract] ABSTRACT: The role of microRNAs in association with Mycobacterium tuberculosis (MTB) infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI), and from healthy controls. The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (P<0.05). A unique signature of 11 microRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-β signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems. We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.
    Full-text · Article · Jun 2015 · PLoS ONE
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    • "The results showed that miR-664 was up-regulated in the 43 T-ALL children samples and its predicted target PLP2 was down-regulated in the 49 children with T-ALL. Increased expression of miR-664 has been found in hepatocellular carcinoma [17]. The relationship between miR-664 and T-ALL has never been reported before. "
    [Show abstract] [Hide abstract] ABSTRACT: MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. However, the role of microRNAs in leukaemia, particularly T-cell acute lymphoblastic leukaemia(T-ALL), has remained elusive. Here, we identified miR-664 and its predicted target gene PLP2 were differentially expressed in T-ALL using bioinformatics methods. In T-ALL cell lines, CCK-8 proliferation assay indicated that the cell proliferation was promoted by miR-664, while miR-664 inhibitor could significantly inhibited the proliferation. Moreover, migration and invasion assay showed that overexpression of miR-664 could significantly promoted the migration and invasion of T-ALL cells, whereas miR-664 inhibitor could reduce cell migration and invasion. luciferase assays confirmed that miR-664 directly bound to the 3'untranslated region of PLP2, and western blotting showed that miR-664 suppressed the expression of PLP2 at the protein levels. This study indicated that miR-664 negatively regulates PLP2 and promotes proliferation and invasion of T-ALL cell lines. Thus, miR-664 may represent a potential therapeutic target for T-ALL intervention. Copyright © 2015. Published by Elsevier Inc.
    Preview · Article · Feb 2015 · Biochemical and Biophysical Research Communications
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    • "We did not notice any significant cancer biology related publications for miR-561. miR-664 has a potential tumor suppressive activity in hepatocellular carcinoma and has been documented to downregulate methionine adenosyltransferase 1A (MAT1A) [29]. miR-548 is implied in regulating pancreatic cancer progression and downregulation of low-density lipoprotein receptor-related protein (LRP1B), in thyroid cancer [30,31]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis. Method Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses. Results Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β. Conclusion Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.
    Full-text · Article · Aug 2014 · Molecular Cancer
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