Journal of Cancer Therapy, 2012, 3, 379-387
http://dx.doi.org/10.4236/jct.2012.324050 Published Online September 2012 (http://www.SciRP.org/journal/jct)
379
Involvement of miR-214 and miR-375 in Malignant
Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
Megumi Ishimura1, Mika Sakurai-Yageta1, Tomoko Maruyama1, Tomoko Ando2,
Masashi Fukayama3, Akiteru Goto1,4, Yoshinori Murakami1*
1Division of Molecular Pathology, The University of Tokyo, Tokyo, Japan; 2Laboratory of Pathology, Institute of Medical Science,
The University of Tokyo, Tokyo, Japan; 3Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo,
Japan; 4Department of Cellular and Organ Pathology, Graduate School of Medicine, Akita University, Akita, Japan.
Received July 31st, 2012; revised August 30th, 2012; accepted September 13th, 2012
ABSTRACT
A tumor suppressor gene, CADM1, encoding an immunoglobulin superfamily cell adhesion molecule, is inactivated in
various cancers, including non-small-cell lung cancer (NSCLC). Although promoter methylation is one of the mecha-
nisms to suppress CADM1 expression, about half of tumors lacking CADM1 expression do not show methylation of the
gene promoter. We herein investigated the possible involvement of microRNA (miRNA) in the down-regulation of
CADM1. Using computational algorithms, miR-214 and miR-375 were identified as candidate miRNAs targeting
CADM1. A luciferase reporter assay demonstrated that miR-214 and miR-375 repressed the promoter activity through
3’-UTR of CADM1. Quantitative RT-PCR analysis demonstrated that miR-214 and miR-375 was highly expressed in
21 (62%) and 17 (50%) cases of 34 primary NSCLCs. Notably, increased expression of miR-214 was preferentially
observed in tumors with advanced pathological stages and in those lacking CADM1 expression but were not associated
with the promoter methylation, suggesting that miR-214-mediated silencing would be another mechanism to suppress
CADM1 expression. On the other hand, introduction of miR-214 or miR-375 into NSCLC cells decreased CADM1 pro-
tein expression. Furthermore, overexpression of miR-214 enhanced anchorage-independent growth of NSCLC cells,
A549, whereas transfection of miRNA inhibitors of miR-214 or miR-375 significantly suppressed the in vitro wound
healing activity of HCC827 cells. These findings suggest that overexpression of miR-214 and miR-375 could partici-
pate in the malignant features of NSCLC through down-regulating CADM1 and would provide a potential target for the
treatment of a subset of NSCLC.
Keywords: CADM1; miR-214; miR-375; Non-Small-Cell Lung Cancer
1. Introduction
Lung cancer is the leading cause of cancer death in the
world. More than 80% of lung cancer is categorized as
non-small-cell lung cancer (NSCLC). We previously
identified a tumor suppressor gene, CADM1/TSLC1, in
NSCLC by functional cloning [1]. CADM1 encodes an
immunoglobulin superfamily cell-cell adhesion molecule
and is expressed in various tissues, including the brain,
testis, lung, kidney, and breast [2]. CADM1 expression
was frequently lost or reduced in concordance with
tumor progression in NSCLC and various other cancers
[3]. Moreover, loss or reduction of CADM1 expression
indicated worse clinical prognosis of the cases in lung
adenocarcinoma, hepatocellular carcinoma, and esopha-
geal squamous cell carcinoma [4-7]. However, the me-
chanisms underlying the loss of CADM1 expression in
cancers are not fully clarified yet. So far, a major postu-
lated mechanism is promoter methylation of the CADM1
gene through bi-allelic methylation or mono-allelic me-
thylation associated with loss of the other allele. In fact,
44% of NSCLC as well as 30% - 60% of various other
cancers showed promoter methylation of the CADM1
gene [8]. However, considerable numbers of tumors lac-
king CADM1 expression do not show promoter methy-
lation of the CADM1, suggesting that additional un-
known mechanisms would be involved in the suppression
of CADM1 expression.
MicroRNA (miRNA) is a group of small non-coding
RNAs that suppress gene expression by interacting with
the 3’-UTR of target mRNAs for translational supp-
*Corresponding author.

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
380
ression and mRNA degradation [9]. Numbers of mi-
RNAs were identified to regulate cell proliferation, diffe-
rentiation, and apoptosis by targeting different gene sets,
thereby acting in an either oncogenic or tumor-supp-
ressor manner. Several oncogenic and tumor-suppressor
miRNAs have been implicated in the regulation of tumor
progression in NSCLC, such as miR-21 and let-7 [10-12].
Therefore, it is tempting to speculate that suppression of
CADM1 expression could be mediated by miRNAs in
NSCLC.
In the present study, we identified miRNAs, miR-214
and miR-375, to target 3’-UTR of CADM1 and examined
their functional roles in anchorage-independent cell
growth and tumor cell migration/invasion in NSCLC cell
lines. Furthermore, we analyzed the expression of miR-
214 and miR-375 in primary NSCLC. Enhancement of
the malignant phenotype of NSCLC by these miRNAs,
as well as high incidence of their overexpression in pri-
mary NSCLC, suggests that miR-214 and miR-375 act as
oncomiRs of NSCLC by suppressing CADM1 expres-
sion.
2. Materials and Methods
2.1. Cell Lines, Transfection and Clinical Samples
A549 and Caco-2 cells were obtained from RIKEN Cell
Bank (Ibaraki, Japan); HEK293 from the Health Science
Research Resources Bank (Osaka, Japan); and NCI-H441
and HCC827 from The American Type Culture Collec-
tion (Manassas, VA). Cells were cultured according to
the supplier’s recommendation. Control siRNA (ON-
TARGETplus siCONTROL Non-targeting pool, D-
001810-10), siRNA against CADM1 (siGENOME ON-
TARGETplus Human IGSF4, J-016565-05), microRNA
mimics (miRIDIAN Mimic Human hsa-miR-214, C-
300569-07; miRIDIAN Mimic Human hsa-miR-375, C-
300682-05) and microRNA inhibitors (miRIDIAN Hair-
pin Inhibitor Human hsa-miR-214, IH-300569-08; mi-
RIDIAN Hairpin Inhibitor Human hsa-miR-375, IH-
300682-07) were obtained from Thermo Fisher Scientific
(Waltham, MA). Cells were transfected with 50nM of
siRNA or miRNA using LipofectamineTM LTX reagent
(Invitrogen). We collected 34 cancer tissues and adjacent
non-cancerous lung tissues from NSCLC patients who
underwent surgical resection at the University of Tokyo
Hospital (Tokyo, Japan) after receiving approval from
the Institutional Ethics Review Committee and obtaining
informed consent from all patients.
2.2. Real-Time Quantitative PCR (qRT-PCR)
Oligonucleotide primer sequences for real-time PCR are
shown in Table 1. Total cellular RNA was extracted us-
ing an RNeasy Mini kit (QIAGEN Sciences, German-
town, MD) and first-strand cDNA was synthesized using
a Transcriptor first-strand cDNA synthesis kit (Roche
Diagnostics, Basel, Switzerland). Then, real-time PCR
was carried out using Light Cycler® 1.5 with light cycler
Taq Man master and universal probe of #21 for miRNAs
(Roche Diagnostics). Expression of miRNAs was nor-
malized to that of small nuclear RNA U6.
2.3. Immunoblot Analysis
Immunoblotting was carried out as described previously
[2] using a rabbit anti-CADM1 polyclonal antibody (pAb)
(C-18) [13] or a goat anti-GAPDH pAb (V-18, Santa
Cruz Biotechnology, Santa Cruz, CA) as a control. Quan-
tification of signal intensities was performed using Image
J Software Ver. 1.44.
2.4. Luciferase Assay
CADM1 3’-UTR of 2,848 bp was cloned by RT-PCR
Table 1. Primers for cloning of CADM1 3’-UTR and real-time PCR of miRNAs.
Target Primer sequence (5’ → 3’) PCR product size (bp)
F 5’-GCTAGCATCAGCCTTTTTGTTTCAATGAGG-3’
CADM1
3’-UTR R 5’-ACTAGTCACTTTGTAACATTAATTTTTTTTTATTAAG -3’ 2848
RT 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACTGCC-3’
miR-214
F 5’-CGGCGGACAGCAGGCACAGACA-3’ 62
RT 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCACGC-3’
miR-375 F 5’-CGGCGGTTTGTTCGTTCGGCTC-3’ 62
miR Universal R 5’-GTGCAGGGTCCGAGGT-3’
RT 5’-CGCTTCACGAATTTGCGTGTCAT-3’
F 5’-GTGCTCGCTTCGGCAGCACATATAC-3’ U6
R 5’-CCTTGCGCAGGGGCCATGCTAA-3’
72
RT: primer for reverse transcriptase reaction; F: forward primer for PCR; R: reverse primer for PCR.

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
381
from RNA of Caco-2 cells using primers described in
Table 1. The amplified fragment was inserted down-
stream of the Firefly luciferase gene of pGL3-Basic
(Promega, Madison, WI). Then, a CMV promoter cleaved
from pcDNA3.1/Hygro(+) (Invitrogen) was inserted up-
stream of the luciferase gene to obtain pCMV-GL3-
CADM1-3’-UTR. HEK293 cells were transfected with 25
nM of microRNA mimic, 0.25 μg of pCMV-GL3-
CADM1-3’-UTR, and 2.8 ng of pRL-TK (Promega). The
luciferase activities of Firefly and Renilla were measured
after 48 h with a dual luciferase reporter assay system
(Promega) using a Lumat LB9507 luminometer (Ber-
thold Technologies, Bad Wildbad, Germany).
2.5. Colony Formation Assay in Soft Agar
After 48 h of transfection, A549 cells were trypsinized
and plated at 1 × 104 in 0.36% soft agar on the top of a
base layer containing 0.5% agar and grown at 37˚C for
15 days. The numbers of colonies per dish with diame-
ters over 100 m formed in soft agar were counted under
the microscope and normalized to those in a liquid cul-
ture for 7 days.
2.6. Wound Healing Assay
HCC827 cells transfected with 50 nM of microRNA in-
hibitors or 1 μg of the CADM1 expression vector were
cultured to obtain 90% confluency. A wound was created
by scraping the cells using a P200 pipette tip and images
were captured immediately (0 h) and at 10 h and 20 h.
Migration of cells was assessed by measuring the aver-
age width of the wounds at three different points.
2.7. Immunohistochemical Analysis
Immunohistochemistry was performed using anti-CADM1
polyclonal antibody, CC2, as described previously [5].
2.8. Methylation-Specific PCR (MSP)
Methylation-specific PCR of the CADM1 promoter was
performed as described elsewhere [14]. The primers used
were: 5’-AGTGACGGAAATTTGTAACG-3’ and 5’-
AAAAACTCGAACTCCAAAAAACG-3’ for the me-
thylated DNA and 5’-AGTGATGGAAATTTGTAATG-
3’ and 5’-AAAAACTCAAACTCCAAAAAACA-3’ for
unmethylated DNA.
2.9. Statistical Analysis
Statistical differences were determined by the two-tailed
Student’s t-test with SigmaPlot software Ver. 11 (Systat
Software, San Jose, CA). Fisher’s exact tests were app-
lied to test significant associations between the expres-
sion state of miRNAs and the clinicopathological char-
acteristics of primary NSCLC. A p-value of <0.05 was
considered as statistically significant.
3. Results
3.1. Identification of miRNAs Targeting CADM1
A couple of computational algorithms TargetScan [15]
and PicTar [16], were used to identify a numbers of po-
tential miRNAs targeting CADM1. The search program
TargetScan identified miR-375 and miR-214 (Table 2)
as the strongest candidate miRNA targeting CADM1,
while another program, PicTar, identified miR-214 (Ta-
ble 3). Database analysis showed that three and two pre-
dicted binding sites of miR-214 and miR-375 were pre-
sent, respectively, in the 3’-UTR of the CADM1 (Figure
1(a)). The seed sequences (SS) of miR-214 and miR-375
were well conserved between mouse and human, except
for the third predicted seed sequences of miR-214-SS3
located about the 1200 bp from the starting site of the
CADM1 3’-UTR (Figure 1(b)).
Table 2. Prediction of miRNAs targeting CADM1 by Tar-
getScan Human 5.2.
miRNA Aggregate PCT
a
miR-375 0.50
miR-214/761 0.42
miR-124/506 0.37
miR-129/129-5p 0.33
miR-205 0.30
miR-148/152 0.28
miR-101 0.27
miR-138 0.26
miR-200bc/429 0.22
miR-208/208ab 0.19
aPCT, Preferentially conserved targeting.
Table 3. Prediction of miRNAs targeting CADM1 by Pic-
Tar.
miRNA PicTar Score
miR-214 10.25
miR-182* 8.57
miR-144 3.94
miR-199a* 3.94
miR-101 3.60
miR-190 2.47
miR-186 2.47
miR-10b 2.29
miR-10a 2.09
miR-195 1.87

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
382
(a)
(b)
(c)
Figure 1. Targeting CADM1 by miR-214 and miR-375. (a)
Predicted binding sites for the seed sequences of miR-214
and miR-375 in the 3’-UTR of CADM1; (b) Sequence align-
ments of miR-214 and miR-375 with 3’-UTR of CADM1 as
indicated in A. The species abbreviations are Hs: Homo
sapiens; Mm: Mus musculus; (c) Suppression of the luci-
ferase activity of luciferase-CADM1 3’-UTR reporter gene
by miRNA mimics of miR-214 and miR-375. Data are the
mean ± SEM of four independent experiments in duplicate.
*p < 0.01.
To determine whether miR-214 and miR-375 target
the 3’-UTR of the CADM1 mRNA, we performed a luci-
ferase assay using a reporter construct bearing the 3’-
UTR of the CADM1 at the downstream of the luciferase
gene. Co-transfection of the reporter plasmid with a mi-
RNA mimic, miR-214 or miR-375, into HEK293 cells
decreased the luciferase activity to 72% and 60% of that
from siControl-transfected cells, respectively. It indicates
that the 3’-UTR of the CADM1 is a target of miR-214
and miR-375 (Figure 1(c)).
3.2. Up-Regulation of miR-214 and miR-375 in
Human Primary NSCLC
Next, we examined the expression of miR-214 and miR-
375 by qRT-PCR in 34 pairs of cancerous and adjacent
non-cancerous lung tissues from primary NSCLC. Over-
expression of miR-214 and miR-375 was defined by
more than two fold expression in tumors compared with
the adjacent non-cancerous region. According to this
criteria, miR-214 and miR-375 were overexpressed in 21
(62%) and 17 cases (50%) of primary NSCLC, respect-
tively (Table 4). When expression of CADM1 was ana-
lyzed in the same series of samples by immunohisto-
chemistry, loss of CADM1 expression was observed in
20 of 34 (59%) primary NSCLC, as representative im-
ages were shown in Figures 2(a) and (b) [5]. Overex-
pression of miR-214 and miR-375 was observed in simi-
lar incidence (55% and 50%, respectively) in tumors
lacking CADM1 expression. However, when focused on
the pathological stages of the tumors lacking CADM1
expression, miR-214 was overexpressed in all 5 tumors
with pathological stages II and III but only in 6 of 14
(43%) tumors with pathological stage I (P < 0.05). These
findings suggest that overexpression of miR-214 is in-
volved in the suppression of CADM1 expression in the
(a) (b)
(c)
Figure 2. Expression and promoter methylation state of
CADM1 in primary NSCLC. (a) and (b) Representative
images of immunohistochemical analysis of CADM1. Mem-
branous staining of CADM1 in a well-differentiated lung
adenocarcinoma (a); Loss of CADM1 in a moderately dif-
ferentiated lung adenocarcinoma (b); Magnification ×40
(objective); (c) Representative results of methylation-speci-
fic PCR of the CADM1 promoter. U and M indicate un-
methylated and methylated DNA, respectively. Samples 187,
172, 19, and 258, derived from primary NSCLC. Bladder
cancer cell lines, KK47 with hypermethylation and RT112
without methylation of the CADM1 promoter, were used as
controls.

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
383
Table 4. Overexpression of miR-214 and miR-375 and clinicopathological characteristics of primary NSCLCs.
No. of tumors examined No. of tumors lacking CADM1 expression
Total Overexpression of
miR-214a
Overexpression of
miR-375a Total Overexpression of miR-214a
Overexpression of
miR-375a
34 21 (62) 17 (50) 20 11 (55) 10 (50)
Age
<65 16 11 (67) 7 (44) 8 6 (75) 3 (38)
≧65 18 10 (56) 10 (56) 12 5 (42) 7 (58)
Sex
Male 17 8 (47) 8 (47) 12 6 (50) 7 (58)
Female 17 13 (76) 9 (53) 8 5 (63) 3 (38)
Histology
Adenocarcinoma 25 16 (64) 13 (52) 12 7 (58) 6 (50)
Squmos cell carcinoma 9 5 (56) 4 (44) 8 4 (50) 4 (50)
Pathological stage b
I 24 13 (54) 12 (50) 15 6 (40) 6 (40)
II and III 10 8 (80) 5 (50) 5 5 (100) 4 (80)
*
P-value was calculated using Fisher’s exact test. *p < 0.05; aHigh expression was defined by >2-fold expression compared with the adjacent noncancerous
region; bAccording to the TNM pathological classification.
were used as recipient cells because they expressed sig-
nificant amounts of CADM1 protein and did not show
methylation of the gene promoter. Introduction of miR-
375 or miR-214 reduced CADM1 expression in both
NCI-H441 and A549 cells relative to that in cells trans-
fected with siControl (Figure 3).
late stage of tumor progression. On the other hand, any
other clinicopathological factors examined were not sig-
nificantly correlated with overexpression of miR-214 or
miR-375.
We next examined the status of promoter methylation
of CADM1, which is one of the major mechanisms to
suppress CADM1 expression. Methylation-specific PCR
demonstrated that, among 20 NSCLCs lacking CADM1
expression, 14 (70%) tumors showed promoter methyla-
tion of the CADM1 gene, supporting the previous find-
ings that promoter methylation is an important mecha-
nism to inactivate CADM1 in NSCLC (Figure 2(c))
[1,17]. However, 6 (30%) tumors lacking CADM1 ex-
pression did not show promoter methylation of the
CADM1. It should be noted that, among the 6 tumors,
overexpression of miR-214 and miR-375 was detected in
5 (83%) and 1 (17%) tumors, respectively (Table 5). In
these tumors, overexpression of miR-214 or miR-375
would be causally involved in the down-regulation of
CADM1.
Then, the biological functions of these miRNAs were
examined in vitro assays related to the tumor suppressive
activities of CADM 1. Since A549 is known to have a
moderate activity of colony formation in soft agar [18],
we examined the effect of these miRNAs on colony for-
mation in soft agar using A549 as recipient cells. As
shown in Figures 4(a) and (b), introduction of miR-214
significantly enhanced the colony formation of A549
cells. In addition, miR-375 slightly promoted colony for-
mation, although not in a statistically significant manner.
These results suggest that miRNA-mediated suppression
of CAD M1 expression enhances anchoring-independent
growth of NSCLC cells, although biological activities by
other target molecules of each miRNA could also modify
the degree of colony-forming ability in each cell.
Next, we examined the effect of the inhibitors of miR-
214 and miR-375 on CADM1 expression by introducing
these inhibiters into a NSCLC cell line, HCC827. As
shown in Figures 5(a) and (b), introduction of the in-
hibitor of miR-214 or miR-375 significantly enhanced
the protein expression of CADM1 . Since HCC827 cells
showed sheet-like morphology with significant migration
ability, we examined whether miRNA-mediated suppres-
sion of CADM1 could affect the collective cell migration
of HCC827 cells. For this purpose, we transfected these
3.3. Biological Functions of miR-214 and
miR-375 in Malignant Features of NSCLC
Cells
We then examined the effect of miR-214 and miR-375
on the expression of CADM1. For this purpose, each
mature miRNA-mimic of these miRNAs was transfected
into NSCLC cell lines, NCI-H441 and A549, and the
introduction of an excess amount of each miRNA was
confirmed by qRT-PCR (data not shown). These cells

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
384
Table 5. Overexpression of miR-214 and miR-375 and expression and promoter methylation state of CADM1 in primary
NSCLCs.
No. of tumors examined No. of tumors lacking CADM1 expression
Total Overexpression of
miR-214 a
Overexpression of
miR-375 a Total Overexpression of miR-214 a
Overexpression of
miR-375 a
Methylation state
Methylated 17 9 (53) 10 (59) 14 6 (43) 9 (64)
Unmethylated 17 12 (71) 7 (41) 6 5 (83) 1 (17)
a High expression was defined by >2-fold expression compared with the adjacent noncancerous region.
(a)
(b)
Figure 3. Effects of miRNA mimics and inhibitors of miR-
214 and miR-375 on CADM1 expression in NSCLC cells.
Immunoblotting (a) and its quantification (b) of NCI-H441
(left) and A549 cells (right) transfected with siControl, si-
CADM1, or miRNA mimics as indicated; (b) Intensities of
CADM1 were normalized to those of GAPDH in NCI-H441
(black) and A549 (white) cells. Relative intensities to siCon-
trol-transfected cells are shown. Data are the mean ± SD of
four independent experiments. **p < 0.05; *p < 0.01.
inhibitors of miRNAs as well as an expression vector of
CADM1 into HCC827 cells and assessed their wound-
healing activity. As shown in Figures 5(c) and (d), the
velocity of HCC827 cells transfected with inhibitors of
miR-214 and miR-375 significantly decreased to 64%
and 66% of that of siControl-transfected cells, respect-
tively, with values that are almost equivalent to those
seen in HCC827 transfected with CADM1 (50%). These
results suggest that CADM1 is one of target genes of
miR-214 and miR-375 for their enhancement in collec-
tive cell migration of an NSCLC cell, HCC827.
4. Discussion
In this study, using a reporter assay, suppression of
CADM1 expression by miR-214 was demonstrated for
(a)
(b)
Figure 4. Soft agar colony formation of A549 cells trans-
fected with miRNA mimics of miR-214 or miR-375. (a)
Representative images of A549 cells with miRNA mimics as
indicated. Bars, 200 m; (b) Numbers of relevant colonies
are shown relative to those of control cells. Data are the
mean ± SD of three independent experiments. **p < 0.05.
the first time. Moreover, miR-375-mediated CADM1
suppression is consistent with the previous report that
CADM1 is one of the 10 targets of miR-375 in pancre-
atic islet cells derived from miR-375-deficient mice [19].
It is noteworthy that CADM1 gives a pair of transcript
with the molecular size of 4.4 kb and 1.6 kb by Northern
blot analysis [1], but that the shorter transcript does not
contain the sequences in its 3’-UTR targeted by miR-214
or miR-375. In normal lung, as well as many other tis-
sues, the transcript of 4.4 kb is predominantly expressed
and could be physiologically regulated by these miRNAs.
On the other hand, the ratio of 4.4 kb-transcript to 1.6
kb-transcript was significantly decreased in several
NSCLC cells in comparison with normal lung tissues,
suggesting that CADM1 transcript of 4.4 kb is selectively
degraded by overexpressed miR-214 and miR-375 in
NSCLC cells.

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
385
(a) (b) (c)
(d)
Figure 5. Collective cell migration activity analyzed by wound-healing assay using HCC827 cells transfected with expression
vector of CADM1 or miRNA inhibitors of miR-214 or miR-375. Immunoblotting (a) and its quantification (b) of cells trans-
fected with siControl, siCADM1, or miRNA inhibitors as indicated; (b) Intensities of CADM1 were normalized to those of
GAPDH. Data are the mean ± SD of four independent experiments; (c) Representative image of HCC827 cells with miRNA
inhibitors indicated at 0 h and 10 h after scratching. The dashed lines indicated the edge of the gap. Bars, 100 μm; (d) Veloc-
ity of relevant cells is shown relative to that of control cells. Data are the mean ± SEM of two independent experiments in
triplicate. **p < 0.05; *p < 0.01.
Using in vitro assays, miR-214 consistently conferred a
malignant phenotype to NSCLC cells, including an an-
chorage-independent growth ability to A549 cells and a
collective cell migration activity to HCC827 cells. These
effects could be explained by the functions of CADM1 as
a tumor suppressor. However, these results also suggest a
partial implication of other targets by miR-214 in these
assays. In this connection, it is noteworthy that PTEN
and AP-2 are reported to be additional targets by miR-
214. In ovarian cancer, it was demonstrated that the in-
troduction of miR-214 targeted PTEN and activated Akt
pathways for the survival of cancer cells [20]. Another
study showed that Twist1, a key transcription factor in-
volved in epithelial-mesenchymal transition (EMT),
upregulated miR-214 expression, resulting in the proli-
feration and survival of ovarian cancer cells [21]. In me-
lanoma, miR-214 was shown to participate in tumor pro-
gression by targeting a transcription factor, AP-2, and
modulating various proteins implicated in cell invasion
and blood vessel extravasation [22]. These findings sug-
gest that miR-214 could act as an oncomiR of NSCLC
and that CADM1, as well as additional targets of miR-
214, would cooperatively regulate the malignant features
of NSCLC.
On the other hand, miR-375 confers the malignant
phenotype to NSCLC cells in rather restricted manner.
Although the miR-375 inhibitor significantly suppressed
the collecting cell migration activity of HCC827 cells,
the introduction of miR-375 only slightly enhanced the
colony formation activity of A549 cells in soft agar. Con-
sidering that CADM1 consistently suppresses the malig-
nant phenotype in these cells, the target genes of miR-
375 other than CADM1 appear to be deeply involved in
these features of NSCLC cells. In fact, the activities of
miR-375 are controversial in terms of tumor progression
or suppression. In the pancreas, gene-deficient mice of
miR-375 demonstrated that miR-375 was essential for
the growth of islet cells, where CADM1 was one of the
possible targets of this miRNA [19]. In breast cancer,
miR-375 was shown to be upregulated to promote cell
growth by targeting RASD1, an activator of G-protein
signaling [23]. In contrast, miR-375 targeted PDK1, and
14-3-3zeta and suppressed growth of gastric cancer cells
[24,25]. These findings suggest that the functions of
miR-375 in tumor progression or suppression are de-
pendent on the cell types and the sets of target genes and
that miR-375 has a restricted activity as an oncomiR in
NSCLC.

Involvement of miR-214 and miR-375 in Malignant Features of Non-Small-Cell Lung Cancer by
Down-Regulating CADM1
386
In addition to the cell biological analyses, we exam-
ined the expression of miR-214 and miR-375 in 34 pri-
mary NSCLCs in this study. As discussed above, it is
necessary to consider the patho-biological significance of
these miRNAs from two viewpoints: one, as an inde-
pendent oncomiR with multiple distinct target molecules,
and the other, as miRNA that targets a tumor suppressor,
CADM1. The high incidences of overexpression of miR-
214 (62%) and miR-375 (50%) in primary NSCLC
strongly suggest that both miR-214 and miR-375 act as
oncomiRs in human NSCLC. MiR-214 was upregulated
in primary ovarian cancer [20], whereas higher expres-
sion of miR-375 was associated with poor prognosis of
esophageal adenocarcinoma [26]. MiR-375 was also re-
ported as a candidate sputum biomarker of lung adeno-
carcinoma [27].
On the other hand, with regard to the ability of mi-
RNAs to target a tumor suppressor, CADM1, overex-
pression of these miRNAs could provide a novel mo-
lecular mechanism to suppress CADM1 expression in
NSCLC in addition to the methylation of the gene pro-
moter. In fact, among the tumors lacking CADM1 ex-
pression, miR-214 overexpression is observed in much
higher incidence in tumors without the CADM1 methyla-
tion in comparison with tumors with the CADM1 methy-
lation. Furthermore, the high incidence of overexpression
of miR-214 in advanced NSCLC with pathological stages
II and III (P < 0.05). It suggests that disruption of
miRNA expression could be a rather late event in multi-
stage tumorigenesis of NSCLC.
5. Conclusion
We demonstrate a novel mechanism to suppress the ex-
pression of a tumor suppressor CADM1 by miRNAs,
miR-214 and miR-375, in NSCLC. Overexpression of
miR-214 and miR-375 in more than half of primary
NSCLC, as well as the enhancement in cell motility and
anchorage-independent cell growth of NSCLC cells by
miR-214 or miR-375 through suppression of CADM1
expression, suggests that miR-214 and miR-375 would
provide potential targets for the treatment of a subset of
NSCLC.
6. Acknowledgements
The authors express their gratitude to Dr. Junichiro Inoue,
Division of Cellular and Molecular Biology, Department
of Cancer Biology, Institute of Medical Science, the
University of Tokyo, for his generous help with the Lu-
mat LB9507 luminometer. We thank members of Divi-
sion of Molecular Pathology for constructive criticism
and fruitful discussion. This work was supported in part
by a Grant-in-Aid for Young Scientists (B) [24790310
for M.SY.] and a Grant-in-Aid for Scientific Research (B)
[22300336 for Y.M.] from the Ministry of Education,
Culture, Sports, Science, and Technology, Japan; a
Grant-in-Aid for the Third Term Comprehensive Control
Research for Cancer from the Ministry of Health, Labor,
and Welfare, Japan [Y.M.]; and a Grant from the Smok-
ing Research Foundation, Japan [Y.M.].
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