MiR-93 enhances angiogenesis and metastasis by targeting LATS2.
ABSTRACT Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17-92 cluster, was significantly upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-93's effects and found that the large tumor suppressor, homology 2 (LATS2) is a target of miR-93. Higher levels of LATS2 were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.
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ABSTRACT: In our previous study, miRNA-183, a miRNA in the miR-96-182-183 cluster, was significantly over-expressed in esophageal squamous cell carcinoma (ESCC). In the present study, we explored the oncogenic roles of miR-183 in ESCC by gain and loss of function analysis in an esophageal cancer cell line (EC9706). Genome-wide mRNA microarray was applied to determine the genes that were regulated directly or indirectly by miR-183. 3'UTR luciferase reporter assay, RT-PCR, and Western blot were conducted to verify the target gene of miR-183. Cell culture results showed that miR-183 inhibited apoptosis (p < 0.05), enhanced cell proliferation (p < 0.05), and accelerated G1/S transition (p < 0.05). Moreover, the inhibitory effect of miR-183 on apoptosis was rescued when miR-183 was suppressed via miR-183 inhibitor (p < 0.05). Western blot analysis showed that the expression of programmed cell death 4 (PDCD4), which was predicted as the target gene of miR-183 by microarray profiling and bioinformatics predictions, decreased when miR-183 was over-expressed. The 3'UTR luciferase reporter assay confirmed that miR-183 directly regulated PDCD4 by binding to sequences in the 3'UTR of PDCD4. Pearson correlation analysis further confirmed the significant negative correlation between miR-183 and PDCD4 in both cell lines and in ESCC patients. Our data suggest that miR-183 might play an oncogenic role in ESCC by regulating PDCD4 expression.Molecules and Cells 12/2014; · 2.24 Impact Factor
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ABSTRACT: MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.Oncotarget 11/2014; · 6.63 Impact Factor
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ABSTRACT: Melanoma is one of the fastest-rising types of cancer in North American. Accumulating evidence suggests that anti-tumor immune tolerance plays a critical role in tumor development. B16 melanoma cells were injected into wild type and miR-17 overexpressing transgenic mice. Tumor growth was monitored and tumor bearing mice were sacrificed by the end of the forth week. Peripheral blood and spleen cells were subject to flow cytometry analysis and tumor samples were subject to immunohistochemistry staining. Meanwhile, Jurkat cells transfected with mock-control or miR-17 overexpressing plasmid were co-cultured with B16 cells. The influence of miR-17 on cell cycle, proliferation and survival was evaluated. The melanoma tumors formed in mice overexpressing miR-17 were less than that in wild type mice. In addition, the miR-17 tumors were less invasive and less angiogenic. The percentage of CD8+ T cells was suppressed in miR-17 transgenic mice before melanoma cell injection. Its level was significantly increased upon tumor grafting. More tumor infiltrating CD8+ cytotoxic T lymphocyte could be found in transgenic mice with tumor formation. Luciferase assay and protein analysis indicated that STAT3 was the target of miR-17. Decreased levels of STAT3 were associated with miR-17 over-expression. Down-regulation of STAT3 in Jurkat cells promoted cell proliferation and mitosis. MiR-17 inhibits melanoma growth by stimulating CD8+ T cells mediated host immune response, which is due to its regulation of STAT3.Oncoscience. 01/2014; 1(7):531-9.
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Cell Cycle 11:23, 1–14; December 1, 2012; © 2012 Landes Bioscience
MiR-93 enhances angiogenesis and metastasis
by targeting LATS2
*Correspondence to: Burton B. Yang; Email: firstname.lastname@example.org
Submitted: 08/17/12; Revised: 10/24/12; Accepted: 10/25/12
MicroRNAs (miRNA) are single-stranded, non-coding RNAs,
18 to 25 nucleotides in length. They are transcribed from
genomic DNA to make long primary transcripts, which are
modified by RNase III-type enzymes Drosha and Dicer to pro-
duce precursor miRNAs and then mature miRNAs.1 More than
1,000 miRNAs have been detected in human cells. Mature
miRNAs can bind to the complementary sequences in the
3'-untranslated regions (3'UTR) of target mRNAs,2 resulting in
post-transcriptional repression. On the other hand, the 3'UTR
has been shown to regulate miRNA functions.3-5 As a new class
of regulatory molecules, miRNAs have diverse functions in
regulating cell activities associated with cell proliferation,6-8 dif-
ferentiation,9 invasion,10 tissue morphogenesis and growth,11,12
tumor formation,13-15 angiogenesis16-19 and metastasis.20-22 The
largest functional group of miRNAs are the ones involved in
cancer development, and among these, some have been reported
to function as oncogenic miRNAs or tumor suppressors, while
others exert diverse functions.23-25 A primary transcript usually
consists of a miRNA cluster that gives rise to multiple precursors
Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17–92 cluster, was significantly
upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell
line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control
cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells
and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a
mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell
culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-
93’s effects and found that the large tumor suppressor, homology 2 (LATS2) was a target of miR-93. Higher levels of LATS2
were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation
and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that
miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the
inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.
Ling Fang,1,5 William W. Du,1,5 Weining Yang,2 Zina Jeyapalan Rutnam,1,5 Chun Peng,2 Haoran Li,1,5 Yunxia Q. O’Malley,3
Ryan W. Askeland,3 Sonia Sugg,3 Mingyao Liu,4 Tanvi Mehta,1,5 Zhaoqun Deng1,5 and Burton B. Yang1,5,*
1Sunnybrook Research Institute; Sunnybrook Health Sciences Centre; Toronto, ON Canada; 2Department of Biology; York University; Toronto, ON Canada;
3Division of Surgical Oncology and endocrine Surgery; University of Iowa Carver College of Medicine; Iowa City, IA USA; 4University Health Network;
University of Toronto; Toronto, ON Canada; 5Department of Laboratory Medicine and Pathobiology; University of Toronto; Toronto, ON Canada
Keywords: microRNA, siRNA, KPM, angiogenesis, tumorigenesis
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; GFP, green fluorescent protein; siRNA, small interfering RNA; LATS2 (or KPM), the large
tumor suppressor homology 2
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
and mature miRNA.26 These miRNAs can form polycistronic
clusters or exist individually.
One of the most intensively studied clusters is miR-17~92,
which has paralogs, miR-106A~363 and miR-106B~25, that
play important roles in cancer development through the repres-
sion of many tumor-associated genes.27-30 The overexpression
of miR-17~92 enhances cell proliferation and reduces apoptosis
by regulating cell cycle progression.31-33 The oncogenic func-
tions of mir-17~92, mir-106A~363 and mir-106B~25 have been
extensively reported.31,34-36 Most recently, mir-106B~25 cluster
was reported to exert oncogenic effects in hepatocellular car-
cinoma.37 However, the precise functions of each miRNA in
the mir-106B~25 cluster are not clear. This cluster of miRNAs
contains three pre-miRNAs: mir-106B, mir-93 and mir-25.
Interestingly, miR-106B and miR-93 share identical seed regions,
suggesting that these two miRNAs may exert the prevailing
functions in this cluster. Previous studies indicated that miR-93
can repress the tumor suppressor TP53INP1 in human T-cell
leukemia virus 1-transformed human T-cells38 and FUS-1 in
human lung cancer cell lines.39 We have also found that expres-
sion of miR-93 promoted tumor growth and angiogenesis in a
©2012 Landes Bioscience. Do not distribute.
2 Cell Cycle Volume 11 Issue 23
we studied the roles of miR-93 in breast cancer development.
RNAs were isolated from paraffin-embedded tumor tissues and
benign tissues of patients with breast carcinoma. Analysis of
miR-93 with real-time PCR indicated that there was a signifi-
cant upregulation of miR-93 levels in the tumor tissues compared
with benign tissues (Fig. 1A, p = 0.0279). We also isolated RNAs
from the tumors and adjacent benign tissues of 20 patients that
were lymph node-positive, an indication of metastasis. Analysis
of miR-93 in these patients showed significant higher levels of
miR-93 in the tumor tissues than the benign tissues (Fig. 1B, p
To study how miR-93 might affect breast cancer develop-
ment, we stably transfected breast carcinoma cell line MT-1 with
human brain tumor cell model.16 To study the role of miR-93 in
other types of cancers, we analyzed levels of miR-93 in human
breast carcinoma specimens and found significant upregula-
tion of miR-93 in the tumor tissues. This study was designed to
explore the function of miR-93 in breast cancer angiogenesis and
MiR-93 affects the interaction of tumor and endothelial cells
and angiogenesis. We have previously reported that the human
brain tumor cell line, U87, transfected with miR-93 can grow
faster and form more blood vessels in nude mice.16 In this study,
Figure 1. expression of miR-93 in human breast carcinoma specimens. (A) RNAs were isolated from paraffin blocks of human breast carcinoma speci-
mens and the benign breast tissues, followed by real-time PCR analysis of miR-93 levels. The breast carcinoma tissues expressed significantly higher
levels of miR-93 than the benign tissues. (B) RNAs were isolated from paraffin blocks of human breast carcinoma specimens with lymph-positive
(metastasis) and the benign breast tissues followed by analysis of miR-93 levels. The tumor tissues expressed significantly higher levels of miR-93.
©2012 Landes Bioscience. Do not distribute.
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injections, indicating the number of mice injected and the num-
ber of mice developing lung tumors. Lung sections were obtained
and subjected to H&E staining. Nodules were detected, indicat-
ing metastasis of the tumor cells to the lungs (Fig. 3B). Lung
sections were also analyzed for Ki67 reactivity, a marker of pro-
liferation. It was found that the miR-93 tumor displayed greater
number of Ki67-positive cells than the control (Fig. 3C). The
sections were also probed for E2F4, a tumor suppressor. Since
the antibody used was specific for human E2F4, we could only
detect positive cells in the tumor sections but not in the mouse
lung sections. Interestingly, the E2F4-positive cells were either
unhealthy or were localized in the areas showing extensive cell
death (Fig. 3D). DNA was isolated from lung tissues and sub-
jected to PCR to amplify the CMV promoter, which was used
to drive the expression of miR-93 in the construct, to indicate
metastasis of the cancer cells. Expression of miR-93 significantly
increased CMV levels (Fig. 3E). The level of CMV signal in each
mouse was also provided (Fig. S2C).
MiR-93 promotes tumor cell survival and invasion. The in
vivo data suggest that miR-93 may enhance tumor cell survival
and invasion. We therefore performed in vitro studies to test this
possibility. MT-1 cells transfected with miR-93 or mock were
seeded in tissue culture dishes or Petri dishes. The cells were
maintained in serum-free conditions. Transfection with miR-93
enhanced cell survival compared with the controls both in tis-
sue culture dishes and Petri dishes (Fig. 4A; Fig. S3A). We then
conducted cell invasion assays. Mock- and miR-93- transfected
MT-1 cells were loaded into the insert with serum-free medium
and then incubated at 37°C for 24 h. The invasive cells were
stained and counted in six randomly selected fields under a light
microscope. We found that cells transfected with miR-93 dis-
played higher levels of invasion than the mock-transfected cells
(Fig. 4B; Fig. S3B).
To confirm the function of miR-93, we transfected MT-1 cells
with a construct expressing an antisense sequence against miR-93
(anti-miR-93) followed by culturing for 5 d. Cell survival assays
showed a significant reduction in survival rate in the miR-93-ex-
pressing cells transfected with anti-miR-93 as compared with the
control (Fig. 4C; Fig. S3C). We also performed invasion assays,
and found that expression of anti-miR-93 significantly decreased
the activity of invasion (Fig. 4D; Fig. S3D).
MiR-93 represses LATS2 expression. We investigated the
target of miR-93 in mediating the observed effects focusing
on tumor suppressors. The large tumor suppressor homology 2
(LATS2 or KPM) was identified as a potential target of miR-93.
The 3'UTR of LATS2 harbored two typical target sequences for
miR-93 at nucleotides 3955–3977 and nucleotides 4058–4078
(Fig. 5A). Cell lysates prepared from the miR-93- and mock-
transfected cells were analyzed on protein gel blot probed with
anti-LATS2 antibody. We found that expression of LATS2 was
repressed in the miR-93 transfected cells (Fig. 5B).
To obtain direct evidence that the 3'UTR of LATS2 was a tar-
get of miR-93, we generated two luciferase expression constructs
harboring fragments of the LATS2 3'UTR containing the miR-
93 target sites, which produced the constructs Luc-Lats-3955 and
Luc-Lats-4058 (Fig. 5C, upper; Fig. S4A). Two mutant constructs
a miR-93 expression construct or a control vector that harbored
a random sequence. The cells were injected into CD-1 nude
mice. Tumors excised from the mice were cut into sections. To
evaluate angiogenesis or the blood vessel density, the tumor sec-
tions were immunohistochemically stained for CD34 expression,
which served as a marker for the blood vessel endothelium. It was
noted that tumors formed by the miR-93-expressing cells showed
a greater number of blood vessels than the mock tumors (Fig. 2A,
upper). The blood vessels were counted in four randomly selected
fields for each cell type and a significant difference was obtained
(Fig. 2A, lower).
To understand how expression of miR-93 affected angiogen-
esis, we tested the effect of miR-93 on endothelial cell activities.
The miR-93- or mock-transfected cells were mixed and co-cul-
tured with rat endothelial YPEN cells. Two to three days after cell
inoculation, the cultures were fixed and stained for examination
of cell morphology. It was found that the mock-transfected MT-1
cells did not interact with YPEN cells: each group of cells tended
to group together. On the other hand, the miR-93 MT-1 cells
could mix and interact well with the YPENcells (Fig. 2B). The
YPEN cells linked together or squeezed into stretches when co-
cultured with the mock MT-1 cells, while the cells were squeezed
into small island-like nodules when co-cultured with the miR-93
MT-1 cells (Fig. S1A). When the mock and miR-93 MT-1 cells
were co-cultured with mouse endothelial cells EOMA, similar
results were obtained (Fig. 2B; Fig. S1B). We also co-cultured
the mock and miR-93 MT-1 cells with human bronchial epithe-
lial cells BEAS-2B, and found that the miR-93 MT-1 cells could
mix well with the BEAS-2B cells (Fig. 2B; Fig. S1C), suggesting
stronger cell-cell interactions.
MT-1 cells stably transfected with miR-93 or mock vectors
were seeded on tissue culture plates at different cell densities over-
night followed by inoculation of endothelial cells YPEN on top of
the existing cultures. The mixed cultures were examined under
a light and fluorescent microscope. We observed that at lower
cell densities of MT-1 cells, endothelial cells tended to attach to
empty areas in the mock culture plates. However, endothelial
cells could adhere well to the miR-93-transfected MT-1 cells. At
higher MT-1 cell densities, endothelial cells attached on top of
the MT-1 cell cultures. After an additional overnight culture, the
endothelial cells were able to spread on the miR-93 cultures, but
not on the mock-transfected cells (Fig. 2C; Fig. S2A)
The miR-93- or mock-transfected cells were also mixed with
the YPEN cells and cultured in Matrigel to examine tube forma-
tion. In the presence of the miR-93-transfected cells, YPEN cells
formed larger complexes and longer, tube-like structures com-
pared with the mock-transfected cells (Fig. 2D; Fig. S2B). These
results indicated that miR-93-expressing MT-1 cells supported
endothelial cell activities.
MiR-93 enhances breast cancer metastasis. We then tested
the role of miR-93 in a different mouse model by injecting the
cells into NOD-SCID mice via the tail vein. Seven weeks after
the injection, the mice were sacrificed and examined. It was
noted that five mice in the miR-93 group developed cachectic
lung tumors. Typical metastatic lesions in the lungs are shown
(Fig. 3A). The numbers in the figure were combined from two
©2012 Landes Bioscience. Do not distribute.
4 Cell Cycle Volume 11 Issue 23
Figure 2. For figure legend, see page 5.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Cell Cycle 5
from MT-1 cells transfected with siRNA-2439 or a control oligo
were analyzed on protein gel blot to confirm the silencing effect
of the siRNA targeting LATS2 (Fig. S6C). These results sug-
gested that the LATS2-mediated pathway was essential for miR-
93-enhanced cell survival and invasion.
To confirm that miR-93 promoted cell survival and inva-
sion by targeting LATS2, rescue experiments were performed.
MT-1 cells stably transfected with miR-93 were transfected with
LATS2 expression construct or a control vector. Confirmation of
the expression of LATS2 was assayed on protein gel blot probed
with anti-LATS2 antibody (Fig. S6D). Survival assay showed
that reintroduction of LATS2 into the miR-93-expressing cells
reversed the effect of miR-93 on cell survival (Fig. 7C; Fig. S6E).
In cell invasion assays, decreased invasion was found in cells
transfected with LATS2 (Fig. 7D; Fig. S6F). In tube formation
assays, we found that ectopic expression of LATS2 decreased tube
formation (Fig. 7E), suggesting its role in reducing angiogenesis.
Thus, re-expression of LATS2 was sufficient to cause cell death,
and decrease both cell invasion and tube formation. This sug-
gested that the effects of miR-93 on enhanced angiogenesis and
metastasis were at least partly taking place through repression of
MiR-93 is expressed in the miR-106B~25 cluster, along with other
miRNAs such as miR-106B and miR-25. Recent studies have
demonstrated that the miR-106B~25 cluster is involved in onco-
genic activities. Consistent with other groups, we also reported
that the expression of miR-93 promotes tumor growth.16,39 The
seed region of miR-93 (nucleotides 2–8 of the miRNA) is identi-
cal to miR-106B in the same cluster, and to miR-17, miR-20a,
miR-20b, and miR-106A in two paralogs (miR-17~92 and miR-
106A~363 clusters) of the miR-106B-25 cluster. Since the seed
regions are the critical sites for gene targeting, it suggests that
these six miRNAs could target the same mRNAs and play simi-
lar roles. Indeed, these three paralogs have been reported to play
a critical role in cancer development.16,31,40
In this study, we utilized two model systems to study the
roles of miR-93 in breast cancer development: a local tumor for-
mation assay to examine angiogenesis and a metastasis assay to
analyze tumor invasion and metastasis to the lungs. In the local
tumor formation assay, although expression of miR-93 did not
enhance tumor growth, it promoted angiogenesis significantly.
Luc-Lats-3955-mut and Luc-Ltas-4058-mut were also generated
(Fig. 5C, upper; Fig. S4B). Expression of Luc-Lats-3955 and
Luc-Lats-4058 showed that luciferase activities were significantly
repressed when the constructs were co-transfected with miR-93
(Fig. 5C, lower). Mutations of the miR-93 target sites abolished
the effects of miR-93. Examination of the target sequences indi-
cated that the miR-93 target sites at nucleotides 3955–3977 (Fig.
5D) and nucleotides 4058–4078 (Fig. 5E) were highly conserved
across different species. In all species obtained, the seed regions
that were critical for miR-93 targeting were 100% homologous.
Sections from the miR-93 and mock tumors were probed with
anti-LATS2 antibody. In the miR-93 tumor sections, LATS2 lev-
els were much lower than the mock tumor sections (Fig. 6A).
Interestingly, LATS2 expression was co-localized with cell death
in both mock and miR-93 tumor sections. The downregulation
of LATS2 expression was also seen in the miR-93-metastatic lung
sections compared with the stromal tissues (Fig. 6B). We also
examined LATS2 levels in human breast carcinoma specimens.
Tumor sections from a number of randomly picked patients
were immunostained with anti-LATS2 antibody. We could only
detect LATS2 expression in the benign breast tissues, but not in
the tumor mass (Fig. 6C; Fig. S5A).
To test whether or not LATS2 expression was correlated with
miR-93 levels, a number of human breast cancer cell lines were
analyzed for LATS2 expression by protein gel blot analysis, and
miR-93 levels were analyzed by real-time PCR (Fig. S6). We
found that only the benign breast cell line MCF-7 expressed
detectable LATS2 (Fig. 6D). Consistent with this, the MCF-7
cells expressed the lowest level of miR-93. Similar results were
obtained by using mouse breast cancer cell lines (Fig. S5B).
Confirmation of the targeting effects by silencing and res-
cue experiments. To confirm that LATS2 played an important
role in mediating miR-93’s effects in regulating cell survival and
invasion, we introduced siRNA to suppress expression of LATS2.
MT-1 cells were transfected with four siRNAs targeting LATS2
and maintained in serum-free medium. Cells transfected with
the siRNAs displayed increased survival rates as compared with
cells transfected with the control oligo (Fig. 7A; Fig. S6A).
Since all siRNAs appeared to have functioned efficiently,
one of them, the siRNA-2439, was used for further analysis.
MT-1 cells transfected with siRNA-2439 were subjected to inva-
sion assays. It was found that the siRNA-2439-transfected cells
showed a higher ability in invading the Matrigel than the control
oligo-transfected cells (Fig. 7B; Fig. S6B). Cell lysates prepared
Figure 2 (See opposite page). expression of miR-93 enhanced breast cancer angiogenesis. (A) Top: sections of tumor from mice injected with miR-
93- or mock-MT-1 cells were probed with CD34 primary rat antibody in 10% goat serum with TBS, followed by probing with anti-rat IgG (positive).
The negative or control tumor sections were probed with anti-rat polyclonal IgG antibody only. There were increased levels of tumor-associated
vascularization (as shown with arrows), where the miR-93 expression in MT-1 cells was enhanced. Scale bars, 50 μm. Bottom: the average count of
the number of the blood vessels found in the tumor sections at four randomly selected fields. error bars, SD (n = 4), **p < 0.0001. (B) Mock- and miR-
93-transfected cells were co-cultured with endothelial cells YPeN or eOMA or lung cells BeAS-2B at the ratio of 2:1. After 2 d of culture, the co-cultured
cells were photographed. The miR-93 cells displayed higher capacities in expansion than the vector-transfected cells. As a result, the endothelial cells
were squeezed into small islands by the miR-93 cells. The miR-93 cells could mix well with the lung cells compared with the mock control. (C) MT-1 cells
stably transfected with miR-93 or mock were seeded on tissue culture plates at a cell density of 1.5 x 105 cells/well on 6-well plates. After overnight
culture, endothelial cells YPeN were inoculated on top of the existing cultures (6 x 104 cells/well). After an additional overnight culture, the endothe-
lial cells were able to spread over the miR-93 cultures, but could not spread over the mock cultures. (D) The miR-93- and mock-transfected cells were
mixed with YPeN cells and inoculated in Matrigel, followed by examination of tube formation. The YPeN cells formed larger complexes and longer
tubes when mixed with the miR-93 expressing cells compared with the mock-transfected cells.
©2012 Landes Bioscience. Do not distribute.
6 Cell Cycle Volume 11 Issue 23
miR-93 tumor cells promoted angiogenesis, similar to the results
we reported previously.16
The function of miR-93 in the promotion of angiogenesis was
corroborated with in vitro experiments. We found that miR-93
The miR-93 tumor sections showed much more blood vessel
formation than the mock tumor sections. This could be clearly
seen in the sections with H&E staining and the slides stained for
CD34, a marker of blood vessels. These results suggested that
Figure 3. MiR-93 enhanced breast cancer metastasis to the lung. (A) Mock- or miR-93-transfected MT-1 cells (2 × 105) were injected into the tail vein of
CD-1 nude mice (n = 20). Six weeks after the injection, six mice in the miR-93 group developed visible tumors in the lungs, but only one in the control
group. Typical metastatic lesions in the lungs are shown (arrows). (B) H&e staining of lungs from mock and miR-93 mice showed metastasis lesions
in the miR-93 lungs (arrows). (C) The sections were immunohistochemically stained with antibody against Ki67. The miR-93 tumor sections showed
higher levels of Ki67 staining than the control group. (D) The sections were also probed with antibody against e2F4. The miR-93 tumor sections
showed e2F4 staining, which was not detected in the control group. (E) DNA was isolated from lung tissues and subjected to PCR to amplify the CMV
promoter to indicate metastasis of the tissues. expression of miR-93 promoted metastasis.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Cell Cycle 7
and more complex tubes in Matrigel by YPEN cells co-cultured
with the miR-93-transfected MT-1 cells was an indication of
enhanced angiogenesis. When the cell number was low, no exten-
sive tubes were formed. Larger complexes were seen in the pres-
ence of miR-93-expressing cells. This result further confirmed
that the miR-93-transfected cells were able to interact well with
endothelial cells facilitating blood vessel formation.
In co-culture experiments, the miR-93- and mock-transfected
MT-1 cells were mixed with YPEN or EOMA endothelial cells.
In both cases, the mock MT-1 cells did not interact well with
either of the endothelial cells. However, the miR-93 MT-1 cells
could adhere well with both types of endothelial cells. This indi-
cated that the miR-93 MT-1 cells allowed extension of the endo-
thelial cells into the tumor mass. In other words, the miR-93
MT-1 cells could facilitate angiogenesis of the endothelial cells.
enhanced endothelial cell activities, including cell spreading and
tube formation. In the cell-cell interaction experiments, YPEN
endothelial cells were able to spread over the miR-93-transfected
cells better than on the mock-transfected cells. These results sug-
gested that the surface of the miR-93-transfected cells were dif-
ferent from that of the mock-transfected cells. MiR-93 may have
repressed cell surface proteins. We have previously demonstrated
that miR-93 could repress integrin β8,16 allowing the spreading
of endothelial cells and close contact of endothelial cells to miR-
93-transfected cells. These would favor the process of blood ves-
sel extension or angiogenesis.
To understand how expression of miR-93 affected angiogen-
esis, we performed tube-formation and co-culture experiments,
and found that miR-93-transfected cells displayed greater activ-
ity in forming tube-like structures than the mock-transfected
cells when co-cultured with YPEN cells. The formation of longer
Figure 4. MiR-93 promotes cell survival and invasion. (A) The MT-1 cells transfected with miR-93 or mock (2.5 x 105 cells per 1 mL) were seeded onto
Petri dishes and tissue culture dishes and were maintained in a serum-free medium at 37°C and 5% CO2 for 7 d. The cells were counted at the end of
seventh day. The miR-93 transfected cells were seen to survive better. error bars indicated standard deviation (SD) with n = 5. (B) The miR-93 and mock
cells (1 x 105) suspended in 100 μl serum-free medium were loaded onto the insert and incubated at 37°C for 48 h. The invasive cells were stained blue
and were counted in six fields of views/membrane using a light microscope. error bars indicate SD (n = 6). (C) MT-1 cells were transiently transfected
with anti-miR-93 oligos or control oligos with random sequence. The cultures were maintained in tissue culture dishes in serum-free DMeM for 5 d, fol-
lowed by microscopic examination and photographed. The number of cells was counted for statistical analysis. error bars indicate SD (n = 6). (D) MT-1
cells were transiently transfected with anti-miR-93 oligos or control oligos with random sequence or with anti-miR-93 plasmid or control vector. The
cells (1 x 105) were subjected to invasion assays at 37°C for 64 h. Transfection with anti-miR-93 inhibited cell invasion.
©2012 Landes Bioscience. Do not distribute.
8 Cell Cycle Volume 11 Issue 23
allowed identification of the metastatic mass compared with the
surrounding stroma. In addition, the stromal cells displayed a
limited capacity in cell proliferation, as shown by weak Ki67 stain-
ing. On the contrary, the metastatic MT-1 cells displayed strong
Ki67 immunoreactivity, an indication of extensive cell prolifera-
tion. Interestingly, we detected significant levels of Ki67-positive
cells with small nuclei, mixing with the tumor cells that had large
nuclei. This may have been the consequence of inflammatory cell
These results may partly explain why the miR-93 tumors con-
tained much more vasculature than the mock tumors.
In tumor metastasis experiments, we found that the miR-
93-transfected cells had a greater activity in metastasis to the
lung than the mock-transfected cells. As the nuclei of the MT-1
breast cancer cells appeared much larger than the lung cells,
the cancer cells could be readily distinguished from the stromal
cells. Morphologically, the large complex of the tumor mass also
Figure 5. Targeting analysis of LATS2 by miR-93. (A) Computational analysis indicated that miR-93 potentially targeted LATS2 located at nucleotides
3955–3977 and nucleotides 4058–4078. (B) Cell lysate prepared from miR-93- or mock-transfected MT-1 cells was analyzed on protein gel blot for
LATS2 expression. LATS2 expression was repressed by miR-93 transfection. Staining for β-actin from the same membrane confirmed equal loading.
(C) Top: two luciferase constructs were generated, each containing a fragment harboring the target site of miR-93, producing Luc-Lats-3955 and
Luc-Lats-4057. Mutations were also generated on the potential target sequence (red color), resulting in two mutant constructs Luc-Lats-3955-mut
and Luc-Lats-4057-mut. Bottom: MT-1 cells were co-transfected with miR-93 and a luciferase reporter construct. The luciferase reporter vector was
used as a control. Asterisks indicate significant differences. error bars, SD (n = 3). (D) Top: alignment of the miR-93 target sites on LATS2 located at
nucleotides 3955–3977 across Homo sapiens (NM_014572), Pan troglodytes (XM_509566), Bos taurus (XM_584953), Mus musculus (NM_015771), Gallus
gallus (XM_417143), Xenopus (Silurana) tropicalis (NM_001102704), Rattus norvegicus (NM_001107267) and Taeniopygia guttata (XM_002191071). The
seed regions for miR-93-LATS2 interactions are boxed. Bottom: conservation of the sequences is shown across all species. (E) Top: alignment of the
miR-93 targeting LATS2 located at nucleotides 4058–4078 across the same species. The seed regions for miR-93-LATS2 interactions are boxed. Bottom:
conservation of the sequences is shown across all species.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Cell Cycle 9
a series of experiments. Silencing LATS2 expression by siRNA
indicated that LATS2 played important roles in cell survival and
invasion. In rescue experiments, we demonstrated that ectopic
expression of LATS2 decreased cell survival, cell invasion and
tumor formation, all of which were associated with angiogen-
esis and metastasis. Nevertheless, further studies are needed to
dissect the signal pathways underlying the effects of LATS2 on
angiogenesis and metastasis.
In summary, we have demonstrated that miR-93 functions as
an oncogene by enhancing tumor cell survival, blood vessel for-
mation and tumor metastasis by targeting LATS2. MiR-93 can
potentially target a great number of genes, some acting directly
on tumorigenesis and angiogenesis. Others may only indirectly
affect tumorigenesis and angiogenesis. Thus, we cannot exclude
the possibility that other miR-93 targeting proteins may also
play a role in the system we studied. Future investigation would
involve elucidating the network by which miR-93 functions and
understanding its contribution to cancer development.
Materials and Methods
Materials and cell culture. The monoclonal antibodies against
LATS2 were purchased from Santa Cruz Biotechnology. The
monoclonal antibody against β-actin used was obtained from
Sigma. Horseradish peroxidase-conjugated goat anti-mouse IgG
and horseradish peroxidase-conjugated goat anti-rabbit IgG were
obtained from Bio-Rad. Immunoblotting was performed using
the ECL protein gel blot detection kit. High Pure PCR Template
Preparation Kits were obtained from Roche Applied Science. The
breast cancer MT-1 cell line was used in this experiment. The
cells were cultured in RPMI-1640 supplemented with 10% fetal-
bovine serum, FBS (Invitrogen). These cells were maintained in
an incubator at 37°C under an atmosphere of 5% CO2. The cul-
ture medium was changed twice a week. The LATS2 expression
construct is a kind gift from Dr. Nicholas Dyson in Massachusetts
General Hospital Cancer Center, Harvard Medical School.47
Construct generation. A cDNA sequence containing two
human precursor miR-93 units was inserted into a mammalian
expression vector pEGFP-N1 in the restriction enzyme sites BglII
and HindIII. The sequence of the precursor miR-93 in the con-
struct is the same as the endogenous sequence.
We used a luciferase reporter vector (pMir-Report, Ambion)
to generate luciferase reporter constructs. There are two poten-
tial binding sites for miR-93 in the LATS2 3'UTR. These two
sites are close to each other, which made the analysis of each site
individually difficult. It is essential to develop a strategy to distin-
guish the effect of each site separately. The LATS2 fragment was
cloned with two primers HuLats2-R93-SacI (5' gg gagctc tag atg
ggg gcc agg cac ccc cac) and HuLats2-R93-MluI (5'mL acgcgt
cct aaa tgtg aat aagt gct atgg). The PCR product was digested
with SacI and MluI, followed by insertion into a SacI- and MluI-
opened pMir-Report vector. To generate a mutant containing
a mutation in the second site of the 3'UTR (leaving the other
site active), the primer HuLats2-R93-SacI was combined with
a primer HuLATS2-R93-MluI-mut2 (5'mL acgcgt cct aaa tgtg
aat ttca gct) in a PCR, followed by cloning of the fragment in
infiltration of the tumors. We also observed that some areas in
the tumor mass had no Ki67-positive cells. This may have been
a consequence of cell death, since necrosis typically accompanies
tumor growth, due to lack of nutrition. Corroboration of these
results were obtained by immunohistochemical analysis of E2F4,
a tumor cell death-associated molecule. We detected extensive
staining of E2F4 in the metastatic tumor mass.
To understand how expression of miR-93 promoted tumor
metastasis, we performed tumor cell survival and invasion assays.
By cell survival assays, we found that the miR-93-transfected
MT-1 cells were able to survive significantly longer compared
with the mock-transfected MT-1 cells when maintained in
serum-free conditions. Since the miR-93-expressing cells did not
grow significantly faster than the controls, the promotion of cell
survival did not appear as a consequence of tumor cell growth.
To allow metastasis to occur, single cancer cells or a limited num-
ber of cancer cells must be able to survive while invading to a
new location. The ability of miR-93 cells to survive may play an
important role in the metastasis of cancer cells.
MiR-93 appeared to play roles in enhancing invasion of MT-1
cells. The miR-93-MT-1 cells were able to penetrate through
the Matrigel as compared with the control cells. The invasive-
ness of transformed cells represents an essential step in tumor
progression. The transition from tumor growth to metastatic
disease is defined by the ability of tumor cells from the primary
site to invade local tissues and cross tissue barriers. To initiate
the metastatic process, cancer cells must first penetrate the epi-
thelial basement membrane, They invade the interstitial stroma
by active proteolysis of the dense matrix containing collagens,
glycoproteins and proteoglycans, among others. Degradation of
these matrix molecules allows cancer cells to break through the
established adhesion barriers of the local tissues. It appeared that
the miR-93 MT-1 cells were able to digest the matrix molecules,
which facilitated cell invasion and led to metastasis.
In order to understand how miR-93 functioned, it was essen-
tial to identify the target of miR-93 in our experimental system.
Since expression of miR-93 promoted angiogenesis and metasta-
sis, the target(s) were thought to be a tumor suppressor or may
have functioned as negative regulators of angiogenesis and metas-
tasis. Computational analysis showed that many potential targets
of miR-93, were associated with tumor growth, including the
large tumor suppressor homolog 2 (LATS2/KPM).
LATS2 is a member of the LATS tumor suppressor family. It
plays a central role in the Hippo pathway in the inhibition of cell
growth and in tumor suppression.41,42 Other proteins in the Hippo
pathway can trigger LATS2 activation, resulting in the inhibition
of cell growth.43,44 LATS2 can regulate mitotic progression and
p53 activity, leading to suppression of tumor growth.45,46 In addi-
tion, LATS2 plays roles in regulating Retinoblastoma protein
(pRB) activity associated with senescence, cell cycle arrest and
inhibition of tumor growth.47 Our experiments demonstrated
that expression of miR-93 repressed LATS2 levels, leading to the
promotion of angiogenesis and metastasis. Since the effects of
LATS2 on tumor angiogenesis and metastasis are not known,
we confirmed that LATS2 played important roles in mediating
miR-93 functions associated with angiogenesis and metastasis by
©2012 Landes Bioscience. Do not distribute.
10 Cell Cycle Volume 11 Issue 23
Figure 6. Repression of LATS2 expression in the miR-93 tumors. (A) Sections of tumor from mice injected subcutaneously with miR-93- or mock-MT-1
cells were probed with LATS2 primary goat antibody in 10% goat serum, followed by probing with anti-goat IgG. There were higher levels of LATS2
staining (arrows) in the mock tumor section compared with the miR-93 tumor sections. (B) To examine the metastatic tumors, sections from the lungs
were probed with anti-LATS2 antibody. The levels of LATS2 were lower in the miR-93 group than in the control group. (C) Sections from human breast
carcinoma and normal tissues (N) were subjected to immunohistochemistry for LATS2 expression. LATS2 was detected in the duct structure (open ar-
rows) but not in the tumor mass (closed arrows). (D) Top: Cell lysates prepared from different human breast cancer cell lines were subjected to protein
gel blot analysis probed with anti-LATS2 and anti-actin antibodies. LATS2 was detected in the benign breast cell line MCF-7. Right: the levels of miR-93
were analyzed by real-time PCR. The MCF-7 cells expressed significantly lower levels of miR-93 than the other breast cancer cell lines.
the reporter vector. To generate a mutant containing mutations
in both sites, the primer HuLats2-R93-SacI was combined with
the primer HuLATS2-R93-MluI-mut (5'mL acgcgt cct aaa tgtg
aat ttca gct atgg ataaaatacaaa tgtagaaaataacagcagca tgattt gtcaaa
gtt aat ccc tat aattta gtaa gaaaaaa tgg atat aaac aaaa t ttca gctc)
in a PCR. To generate a mutant containing a mutation in the
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Cell Cycle 11
Kit (Ambion) or from freshly frozen tissues. Quantification of
miRNAs was performed with real-time PCR.
Cell invasion assay. Cell invasion assay was performed with
the modified chemotactic Boyden chamber assays as described.48
Briefly, cells were loaded into 8-μm cell culture inserts and
placed in 24-well cell culture plates. The upper chamber of the
Polyethylene Terephthalate (PET) membrane was coated with
100 μl diluted Matrigel (1 mg/ml). The lower chamber was filled
with 600 μl 10% FBS/DMEM medium. Cell suspension (100
μL containing 3 × 105 cells) was transferred to the upper cham-
ber. The transwell was incubated at 37°C for 4 h. Cells (1 × 105)
in 100 μl serum-free DMEM medium were gently loaded onto
each filter insert (upper chamber) and then incubated at 37°C
for 24–72 h. The filter inserts were removed from the chambers,
fixed with methanol for 5 min and stained with Harris’ hemo-
toxylin for 20 min. Samples were subsequently washed, dried and
first site, the primers HuLats2-R93-SacI and HuLats2-R93-MluI
were used in a PCR with the construct containing both mutation
sites as a template. Thus, all inserts had exactly the same size for
comparison in luciferase activity assays.
Clinical specimens. Breast carcinoma specimens were col-
lected at the time of mastectomy from previously untreated
patients. Primary tumors and surrounding normal breast tis-
sues were freshly excised and fixed in 10% formalin overnight,
immersed in 70% ethanol, embedded in paraffin and sectioned.
The sections were subjected to immunohistochemistry probed
with antibodies against LATS2. The work was conducted fol-
lowing a protocol approved by the Clinical Research Ethics
Committee at the University of Iowa Carver College of Medicine.
The paraffin-fixed specimens were also used to isolated miRNAs.
In brief, small RNAs were isolated from formalin-fixed, paraffin-
embedded tissue blocks, using the Total Nucleic Acid Isolation
Figure 7. LATS2 mediates the effects of miR-93. (A) The MT-1 cells (1 × 105 cells/ml/well) were transfected with siRNA oligos targeting LATS2 or a
control oligo. The cells were seeded onto a 12-well plate and incubated in serum-free medium at 37°C for 7 days. The survived cells were counted.
Transfection with siRNAs increased cell survival. Data are mean ± SD (n = 4). (B) The MT-1 cells transfected with siRNA oligos targeting LATS2 or a con-
trol oligo were subjected to invasion assays at 37°C for 48 h. Transfection with anti-miR-93 inhibited cell invasion. Transfection with siRNAs increased
invasion. error bars, SD (n = 4). (C) ectopic expression of LATS2 in the miR-93 cells reversed miR-93 effect on cell survival. (D) The miR-93-expressing
cells were transfected with LATS2 and a control vector. The cells were suspended in 100 ml serum-free medium and loaded in the transwell insert
containing Matrigel, followed by incubation at 37°C for 60 h for invasion assays. Transfection with LATS2 partially reversed the effect of miR-93 on cell
invasion. (E) ectopic expression of LATS2 in the miR-93 cells reversed miR-93 effect on tube formation.
©2012 Landes Bioscience. Do not distribute.
12 Cell Cycle Volume 11 Issue 23
purchased from Qiagen. The primers used as real-time PCR con-
trols were human-U6RNAf and human-U6RNAr as described.12
Protein gel blot analysis. To prepare cell lysates, the miR93-
or mock-transfected MT-1 cells were seeded onto five different
35-mm cultures dish at a density of 1.5 × 105 cells/plate. When
the culture reached sub-confluence, the cells in each dish were
lysed with 100 μL of lysis buffer containing protease inhibitors
(150 mM NaCl, 25 mM TRIS-HCl, pH 8.0, 0.5 M EDTA, 20%
Triton X-100, 8 M urea, and 1x protease inhibitor cocktail).
Protein concentration was measured by Bio-Rad Protein Assay
Kit (#5000–0006). The lysates were subjected to SDS-PAGE
(sodium dodecyl sulfate PAGE). After being separated, the pro-
teins were transferred onto a nitrocellulose membrane followed
by immunostaining with anti-LATS2 primary antibody at the
dilution of 1:400 at 4°C overnight. The following day, this mem-
brane was washed in TBST (Tris-Buffered saline and Tween 20)
and then incubated with HRP-conjugated goat-anti-mouse sec-
ondary antibody for 2 h at room temperature. The protein bands
were detected by enhanced chemiluminescence (ECL) detection.
The blot was re-probed with anti-β-actin mouse primary anti-
body and goat-anti-mouse secondary antibody to confirm equal
loading of samples.
Luciferase activity assay. Luciferase activity assay was per-
formed by using a dual-luciferase reporter system developed by
Promega (E1960). The cells were cultured on 12-well tissue cul-
ture plates at a density of 2 × 105 cells per well in DMEM con-
taining 10% FBS. The cultures were maintained at 37°C for 24
h, until sub-confluence. The cultures were co-transfected with
the luciferase reporter constructs, corresponding miRNA mimics
and Renilla luciferase construct (as an internal control) mixed
with Lipofectamine 2000. The cells were then collected and
lysed with 150 μl of passive lysis buffer per well from Luciferase
Assay Kit (Promega) on a shaker for 20 min. The cell lysates was
centrifuged at 3,000 rpm for 5 min. The supernatants were trans-
ferred into a black 96-well plate (3 × 10 μl) for luciferase activity
measurement and into a transparent 96-well plate (3 × 50 μl) for
Renilla activity analysis. For the luciferase activity measurement,
luciferase assay reagent (70 μl) was added to each well, and the
luciferase activities were detected by using microplate scintilla-
tion and luminescence counter (Packard, Perkin Elmer). For the
internal control of Renilla activities, 90 μl of assay reagent (4
mg/ml ONPG, 0.5 M MgSO4, β-mercaptoethanol and 0.4 M
sodium phosphate buffer) were added to each well. The plate was
then incubated at 37°C for 60 min. Renilla luciferase activity
was then measured in the same tube. The absorbance at 410 nm
was measured by using a microplate reader (Bio-Tek Instruments,
Inc.). Luciferase activities between different treatments were
compared after normalization with Renilla luciferase activities.
Tumorigenicity assays in nude mice. Tumor formation assay
was performed as previously described.51 In brief, 5-week-old
CD1 strain nude mice were injected with the miR-93- or mock-
transfected MT-1 cells at the cell number of 5 × 106 cells in 150
μl PBS per mouse. Tumor sizes were measured weekly thereafter.
When the sizes of the tumors were above the limit described by
the animal protocol approved by the Animal Care Committee
at Sunnybrook Research Institute, the mice were sacrificed and
mounted onto slides. The invasive cells were stained blue and
visualized under a microscope (Axiover Inverted Microscope,
Zeiss) then counted in six random fields for statistical analysis.
Cell survival assay. The method was described in detail previ-
ously.49 In brief, cells (1.5 × 105 cells per well or 2 × 105 cells per
well) were seeded on 35-mm Petri dishes or 12-well tissue cul-
ture dishes in DMEM containing 0–10% FBS and maintained
at 37°C for 12 h. After cell attachment, the medium was changed
to serum-free DMEM medium or 10% FBS/DMEM medium.
Cells were harvested daily, and cell number was determined by a
Co-culture experiments. MiR-93- or mock-transfected MT-1
cells were mixed with stromal cells, YPEN, BEAS-2B or EOMA
(1.5 × 105 cells/ml for each) and seeded on 3.5-cm culture dishes
in DMEM supplemented with 10% FBS (2 ml). The morphol-
ogy of two types of cells was examined under a light microscope
daily. The images were captured at different intervals.
In YPEN cell-spreading experiments, the miR-93- or mock-
transfected MT-1 cells were cultured at different cell densities
(0.5–1.5 × 105 cells/well) in tissue culture plates overnight. Next
day, YPEN cell were inoculated on top of the MT-1 cell cultures
(6 × 104 cells/well). YPEN cell spreading on top of the MT-1 cells
was examined under a light and fluorescent microscope.
To test the effect of miR-93 on tube formation, we mixed the
miR-93- or mock-transfected MT-1 cells with YPEN cells. The
mixture was cultured in Matrigel. The interaction of both types
of cells and the formation of tube-like structures were examined
under a light and fluorescent microscope.
Transfection of MT-1 cells with siRNAs. The siRNAs were
designed and synthesized by GenePharma. Four siRNAs that were
synthesized to target LATS2 expression were used in this experi-
ment: siRNA-1 (LATS2–2439) sense 5'-cua ugu uug uca aga uca
att; siRNA-2 (LATS2–4369) sense 5'-gca gau uuc uuc uau uau
att; siRNA-3 (LATS2–339) sense 5'-gaa agu aug uuu aca aca att;
siRNA-4 (LATS2–5268) sense 5'-gaa guu uau cag ugu uua att;
and control sense 5'-uuc ucc gaa cgc guc acg utt. siRNAs transfec-
tion was performed using Lipofectamine 2000 (Invitrogen). In
brief, 4 × 104 cells in 2 mL of RPMI-1640 (10%FBS) were plated
in each of five different 35-mm tissue culture dishes and were
incubated overnight at 37°C and 5% CO2 atmosphere. For each
dish, 10 μL siRNA was added into 150 μL of serum-free medium
and mixed with 3 μL of Lipofectamine. The mixture was added
to cells and incubated for 6 h before replacing the medium with
RPMI-1640 containing 10% FBS. Total protein assay was pre-
pared 72 h after transfection for protein gel blot analysis.
Real-time PCR. Total RNAs were extracted from cell cultures
with mirVana miRNA Isolation Kit according to the instructions
of the manufacturer (Ambion). Real-time PCR was performed
as previously described.50 For mature miRNA analysis, the total
RNAs were extracted from ~1 × 106 cells. The first strand cDNA
was synthesized using 1 μg RNA. Real-time PCR was performed
with QuantiMir-RT Kit using 1 μl cDNA as template. To per-
form these experiments, other kits were also needed, including
Qiagen, miScript Reverse Transcription Kit, cat#218060, miS-
cript Primer Assay, cat#218411, and miScriptSYBR GreenPCR
Kit, cat#218073. The primer specific for mature miR-93 was
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Cell Cycle 13
10% formalin. The fixed organs were subsequently immersed
in 70% ethanol, embedded in paraffin and sectioned. The sec-
tions were subjected to H&E staining and immunohistochemis-
try. Briefly, tissue sections were de-paraffinized with xylene and
ethanol and then boiled in a pressure cooker. After washing with
Tris-Buffered-Saline (TBS) containing 0.025% Triton X-100,
the sections were blocked with 10% goat serum for 1 h. The sec-
tions were then incubated with primary antibody in TBS con-
taining 10% goat serum albumin overnight. The sections were
washed and labeled with biotinylated secondary antibody, fol-
lowed by avidin conjugated horseradish peroxidase provided by
the Vectastain ABC kit (Vector, PK-4000). The slides were sub-
sequently stained with DAB followed by Mayer’s Hematoxylin
for counter staining and slide mounting.
Statistical analysis. The results (mean values ± SD) of all the
experiments were subjected to statistical analysis by t-test. The
level of significance was set at p < 0.05, p < 0.01 and p < 0.0001.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was supported by grants from Canadian Institutes of
Health Research (MOP-102635 and MOP-111171) to Burton
B. Yang who is the recipient of a Career Investigator Award (CI
7418) from the Heart and Stroke Foundation of Ontario. W.Y.
was a recipient of NSERC Undergraduate Summer Research
Supplemental material may be downloaded here:
the tumors were removed. Tumors were fixed in 10% buffered
formalin (Histochoice Tissue Fixative MB, Amresco), processed
and embedded in paraffin. Immunohistochemistry was per-
formed on 5-μm paraffin sections mounted on charged slides.
The sections were stained with H&E, and immunostained with
CD34 to detect blood vessels. Sections were also immunostained
for expression of LATS2.
Lung metastasis in nude mice. The mock- and miR-93-tans-
fected MT-1 cells were cultured in 10% FBS/RPMI 1640 media
at 37°C with 5% CO2 until sub-confluence. The cells were given
fresh 10% FBS/RPMI 1640 media 24 h before being harvested
for injection into the mice. Cell viability was determined, and
cells were suspended with greater than 95% viability without cell
clumping. Five-week-old CD-1 nude mice were injected via tail
vein using the above transfected cells (2 × 105 cells in 150 μl 10%
FBS/ RPMI 1640 medium). Mice were grouped at random, 10
mice in each group. All mice were sacrificed seven weeks after
injection. At necroscopy, lungs, liver, spines were dissected and
examined carefully. Half the organs were fixed in 10% formalin,
and the other half were frozen in liquid nitrogen for subsequent
To analyze metastasis, mouse lung tissues were homogenized,
and the genomic DNAs were isolated with High Pure PCR
Template Preparation Kit according to the instructions of the
manufacturer. Tumor burden for each individual tissue was mea-
sured using real-time PCR. Primers used were as follows: CMV
forward (5'-gtc atc gct att acc atg gtg atg cgg) and CMV reverse
(5'-agc tct gct tat ata gac ctc cca ccg) for genotyping; β-actin
forward (5'-ccg gca tgt gca aag ccg gct tcg) and β-actin reverse
(5'-ctc att gta gaa ggt gtg gtg cc) for loading control.
Tissue processing, H&E staining and immunohistochem-
istry. Primary tumors or lungs were freshly excised and fixed in
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