miR-138-5p Promotes Proliferation of Human Melanoma Cells by Inhibiting hTERT Expression

39
0884-6812/19/4102-0039/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
A
RTICLES
An Official Periodical of The International Academy of Cytology and the Italian Group of Uropathology
AQCH
ANALYTICAL and
QUANTITATIVE
CYTOPATHOLOGY and
HISTOPATHOLOGY®
OBJECTIVE: Melanoma is one of the most aggressive
and fatal forms of skin cancer. MicroRNAs (miRNAs),
a series of small noncoding RNAs, function through
translation repression and/or mRNA degradation. In
the present study we aimed to explore the potential role
and underlying mechanism of miR-138 in the regula-
tion of melanoma development.
STUDY DESIGN: We suggested that miR-138 expres-
sion was strongly upregulated in melanoma tissues and
human melanoma cells by comparison with their corre-
sponding controls.
RESULTS: Specific overexpression of miR-138-5p pro-
moted Me45 melanoma cell proliferation. By contrast,
specific knockdown of miR-138 prevented its prolifera-
tion. Bioinformatics analysis indicated that miR-138-5p
potentially targets the 3’-untranslated region (3’-UTR)
of human telomerase reverse transcriptase (hTERT),
which was confirmed by luciferase activity assay. Fur-
ther, this study revealed that miR-138-5p regulated the
proliferation of human melanoma cells by directly reg-
ulating hTERT expression as shown by the result that
knockdown of hTERT reversed the promotive effects of
miR-138-5p on proliferation of human melanoma cells.
CONCLUSION: Taking all these results together, this
study demonstrated that miR-138-5p acts as a carcino-
genesis factor by targeting hTERT during human mel-
anoma development. (Anal Quant Cytopathol Hist-
pathol 2019;41:39–46)
Keywords: hTERT, malignant melanoma, melano-
ma, microRNA, miRNA, miR-138-5p, proliferation,
skin cancer, skin neoplasms.
Analytical and Quantitative Cytopathology and Histopathology®
miR-138-5p Promotes Proliferation of
Human Melanoma Cells by Inhibiting hTERT
Expression
Tingting Ye, M.S., Yexuzi Li, B.S., Junying Ye, B.S., and Chengzhong Zhang, B.S.
From the Departments of Dermatology and Venereology and of Intensive Care Medicine, the First People’s Hospital of Wenling, Wenling;
and the Department of Obstetrics and Gynecology, Wenling Women’s and Children’s Hospital, Wenling, Zhejiang Province, China.
Tingting Ye, M.S., and Yexuzi Li, B.S., are joint first authors.
Drs. T. Ye and Zhang are Dermatologists, Department of Dermatology and Venereology, the First People’s Hospital of Wenling.
Ms. Li is Nurse, Department of Intensive Care Medicine, the First People’s Hospital of Wenling.
Ms. J. Ye is Nurse, Department of Obstetrics and Gynecology, Wenling Women’s and Children’s Hospital.
Address correspondence to: Chengzhong Zhang, B.S., Department of Dermatology and Venereology, the First People’s Hospital of
Wenling, No. 333, Chuan’an Nan Street, Chengxi District, Wenling 317500, Taizhou, Zhejiang Province, China (zhangczphd@163.com).
Correspondence may also be addressed to Junying Ye, B.S., Department of Obstetrics and Gynecology, Wenling Women’s and Chil-
dren’s Hospital, No. 102, Xiabao Road, Chengdong Street, Wenling 317500, Taizhou, Zhejiang Province, China (msyejunying@sina.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

Malignant melanoma is one of the most aggres-
sive malignant tumors, accounting for about 4%
of human skin cancers and even causing 80% of
skin cancer–related deaths.1,2 It remains the most
aggressive type of skin cancer and one of the
most difficult cancers to treat.3 The prognosis of
metastatic melanoma patients is quite underde
veloped yet, which leads to its low 5-year sur-
vival rate of around 10–15%.4 Furthermore, the
poor prognosis of patients with advanced mela-
noma, such as stage IV melanoma, inevitably pro-
gresses to death.5 As of now, therapeutic strate-
gies for advanced melanoma are still limited.
miRNAs play an indispensable role in the
maintenance of normal intracellular homeostasis
by regulating gene expression.6 Strong evidence
has shown that short noncoding RNA can par-
ticipate in tumorigenesis and tumor progression
through a variety of mechanisms.7 The link be-
tween miRNAs and the development of meta-
static melanoma has been confirmed in numer-
ous studies, among which is that demonstrating
miRNAs mainly affect proliferation of melanoma
cells through various signaling pathways.8 For
example, miR-26b inhibited proliferation of mel-
anoma cells by suppressing the activation of
MAPK mediated by TRAF5.9 Xu et al pointed
out that miR-205 functioned as a tumor suppres-
sor in melanoma occurrence through regulating
CCL18, thus inhibiting melanoma cell prolifera-
tion and inducing cancer cell senescence.10 Zhang
et al proved that miR-767 could be a tumor sup-
pressor and the knockdown of miR-664 expres-
sion promoted melanoma proliferation by up-
regulating CYLD.11 In addition, Li et al recent-
ly found that miRNA-488-3p might enhance the
sensitivity of malignant melanoma to cisplatin
by targeting PRKDC. Downregulation of PDK4
mediated by miR-211 in melanoma cells inhib-
ited the invasion of amelanotic melanoma by
destabilizing HIF-1α.12 However, the role of miR-
138-5p in human melanoma has rarely been in-
vestigated.
In the present study we investigated the ex-
pression and functional role of miR-138-5p in mel-
anoma tissues and cell lines, which will provide a
novel therapeutic target for human melanoma.
Materials and Methods
Clinical Specimens
Melanoma tissues were obtained from 38 patients
histopathologically diagnosed at the First People’s
Hospital of Wenling. Ethical approval for this
study was given by the ethics committee of the
First People’s Hospital of Wenling. All samples
were collected and analyzed with prior written,
informed consent of the patients.
Cell Culture
Human melanoma cells lines, including SK-Mel-2,
UACC-239, B16-F10, A875, A375, M14, and
Me45, were obtained from the American Type
Culture Collection (ATCC, Manassas, Virginia,
USA), grown and maintained in DMEM medium
(Gibco/Thermo Fisher Scientific, Waltham, Mas-
sachusetts, USA) supplemented with 10% fetal
bovine serum (FBS) (Sigma, USA) and antibiotics
(penicillin-streptomycin, Invitrogen, Carlsbad,
California, USA). Human melanocytes (NGM)
were maintained with DMEM/F-12 medium sup-
plemented with 20% FBS and 1% HMGS (Life
Technologies/Thermo Fisher Scientific, Waltham,
Massachusetts, USA). All cells were cultured in a
humidified incubator at 37°C with 5% CO2.
qRT-PCR
According to the instructions, total RNA was ex-
tracted by Trizol reagent (Invitrogen). After con
version of mRNA into cDNA, qRT-PCR was then
performed by SYBR Mix (TaKaRa) and Bio-Rad
iCycler iQ Multicolor Real-time PCR Detection
System (iQTM5, Bio-Rad). The sequence of PCR
primers synthesized by GeneCopoeia was as fol-
lows: Cyclin D1: forward: 5’-ACACCATTCCTA-
AGCTTCACCCAGGAC-3’ and reverse: 5’-TAG-
GTCAGATCCGTTACATGCGGGCATC-3’; Myc:
forward: 5’-TTTCGCCTGTTGGCTCCC-3’ and re-
verse: 5’-TGGTTGGTCTGTAATGGGCG-3’; and
GAPDH: forward: 5’-TTCGGCACTCGCGCACA-3’
and reverse: 5’-AACGCTGCGTTTCACGAATT-3’.
GAPDH was used as a reference gene, and the
target genes were normalized and calculated by
2-ΔΔCT.
Western Blotting
Total protein extracts were boiled with buffer
containing 0.125 M Tris/HCl, pH 6.8, and 2.5%
sodium dodecyl sulphate (SDS). 20 µg proteins
were loaded onto 10% SDS-PAGE gel and elec
trophoresed followed by transferring to PVDF
membranes (Millipore, Billerica, Massachusetts,
USA). The membrane was incubated overnight
with rabbit polyclonal anti-hTERT (1:1000, Abcam,
Cambridge, Massachusetts, USA), anti-Cyclin D1
40 Analytical and Quantitative Cytopathology and Histopathology®
Ye et al

(1:1000, Cell Signaling Technology, Beverly, Mas-
sachusetts, USA), anti-c-Myc (1:1000; Cell Signal-
ing Technology) antibodies, and mouse monoclo-
nal anti-Tubulin (1:2000, CWBIO, Peking, China)
antibody. After washing 3 times in TBS-T, the
membranes were incubated with HRP-conjugated
secondary antibody (Santa Cruz Biotechnology,
Santa Cruz, California, USA). Image J software was
further used for the densitometry analysis of each
band acquired from signal detection.
Luciferase Assays
hTERT-3’UTR wild type (wt) was cloned into
pGL3 luciferase reporter gene vector (Promega,
USA). Cells were co-transfected with miR-138-
5p, miR-138-5p inhibitor, or miR-138-5p-mutant,
respectively, using Lipofectamine 2000 reagent (In-
vitrogen). The lysate was collected 48 hours after
transfection. According to the manufacturer’s in
formation, Dual-Luciferase Reporter Gene Assay
kit (Promega, Madison, Wisconsin, USA) was used
for detection.
MTT and Colony Formation Assay
MTT (Sigma-Aldrich) was used to detect the ac
tivity of Me45 cells. For each transfection group
including miR-138-5p, miR-138-5p inhibitor, and
corresponding control mimics, 5000 Me45 cells
were grown onto the 96-well plate. Cells were
incubated in 37°C and 5% CO2 incubator. After 1,
2, 3, 4, and 5 days, 20 mL 5 mg/mL MTT was
added to each well for 4 hours. Methylamine was
dissolved in 150 mL dimethyl sulfoxide (DMSO)
for 10 minutes, and the optical density (OD) was
measured at 495 nm. For colony formation assay,
Me45 cells transfected with miR-138-5p, Mi-138-
5p inhibitor, or control (500 cells per well) were
grown onto the 96-well plate and incubated for
14 days and then fixed with frozen methanol and
stained with crystal violet (0.5% w/v, Sigma) for
1 minute.
Anchorage-Independent Growth Ability Assay
Me45 cells were digested with trypsin. 1000 cells
were suspended in 2 mL complete medium con
taining 0.3% agar (Sigma) and inoculated on the
top of the agar layer. The colonies were incubat-
ed at 37°C for 2 weeks until the colonies were
formed. After that, the colony was stained with
1% crystal violet and counted under a microscope
and photographed at magnification of 100. The
number of colonies with diameter exceeding 0.1
mm was calculated. Viable colonies >0.1 mm were
counted.
5-Bromodeoxyuridine (BrdU) Labeling and
Immunofluorescence
As previously reported,13 cells grown on cover-
slips (Fisher, Pittsburgh, Pennsylvania, USA)
were incubated with BrdU for 1 hour and fur-
ther stained with an anti-BrdU antibody (Upstate,
Temecula, California, USA) according to the man-
ufacturer’s instructions. Gray-level images were
acquired under a laser scanning microscope (FV-
1000, Olympus, Tokyo, Japan).
Statistical Analysis
Statistical analyses were conducted using Graph-
Pad Prism Software v. 6.01 (GraphPad, La Jolla,
California, USA). The difference between 2 groups
was analyzed by the analysis of Student’s t test,
and differences among 3 groups were analyzed by
ANOVA with Tukey’s multiple comparisons test,
respectively. Statistical significance was defined as
a value of p<0.05.
Results
miR-138-5p Expression Upregulated in Melanoma
Tissues and Cell Lines
To explore the potential effect of miR-138-5p on
melanoma progression, the expression of miR-
138-5p in different human melanoma cell lines
and clinical tissues was determined by qRT-PCR.
The results showed that the expression of miR-
138-5p in human melanoma cell lines was sig
nificantly upregulated as compared with NGM
cell line (Figure 1A). In addition, the expression
of miR-138-5p in melanoma tissues were assayed
to confirm its function during tissue tumor de-
velopment. The expression of miR-138-5p in mel
anoma tissues was significantly higher than that
in adjacent noncancer tissues (Figure 1B). Taken
together, the cell and tissue models of melano-
ma revealed that upregulation of miR-138-5p may
be involved in the development of malignant mel-
anoma.
MiR-138-5p Promoted the Proliferation of
Melanoma Cells
To investigate the role of miR-138-5p in human
melanoma, Me45 cells stably overexpressed miR-
138-5p or downregulation of miR-138-5p were
constructed. qRT-PCR analysis showed that these
2 kinds of cells had high transfection efficiency
Volume 41, Number 2/April 2019 41
Role of miR-138-5p in Human Melanoma

(Figures 2A and 3A). MTT and colony formation
assay indicated that the ectopic expression of
miR-138-5p drastically increased the growth rate
of ME45 cells as compared with normal cells (Fig-
ure 2B–C). In addition, anchorage-independent
growth ability assay suggested that the colony
formation of ME45 cells with stable miR-138-5p
overexpression was larger than that of the con-
trol group (Figure 2D). BrdU incorporation ex-
periment indicated that the level of DNA synthe-
sis was significantly increased in ME45 cells with
stable miR-138-5p overexpression, while the incor-
poration rate of BrdU in control cells was relative-
ly low (Figure 2E). Moreover, as compared with
the control group, the growth rate of Me45 cells
was significantly reduced by miR-138-5p inhibitor
(Figure 3B–C). Meanwhile, the number and size
of Me45 cell colonies in the miR-138-5p inhibitor
group were both lower than those of the control
group, as shown by the anchorage-independent
growth experiment (Figure 3D). BrdU incorpora-
tion experiment demonstrated that miR-138-5p
inhibitor could reduce the level of DNA synthe-
sis (Figure 3E). All of these results validated that
upregulation of miR-138-5p could promote the
proliferation and tumorigenesis of malignant mel
anoma cells in vitro.
MiR-138-5p Promoted the Proliferation of Me45
Cells by Downregulating hTERT-3’UTR
To clarify the molecular mechanism of how miR-
138-5p regulates proliferation of human melano-
ma cells, a public open algorithm (Target Scan,
http:/
/www.targetscan.org/) was used to predict
the potential binding sites of miR-138-5p in
hTERT’s mRNA 3’-UTR (Figure 4A). In M45 cells
transfected with miR-138-5p, the expression of
hTERT protein was decreased, while the expres-
sion of hTERT was increased by miR-138-5p in-
hibitor (Figure 4B). Subsequently, whether miR-
138-5p could bind to the 3’-UTR region of hTERT
was determined by luciferase reporter gene as-
say. The results revealed that overexpression of
miR-138-5p significantly decreased the luciferase
activity in the hTERT 3’-UTR region, while miR-
138-5p inhibitor increased the luciferase activity.
However, the luciferase activity was not affected
by overexpression or inhibition of miR-138-5p
(Figure 4C). Since miR-138-5p could promote cell
proliferation, further studies were conducted to
test the effects of miR-138-5p on proliferation-
related gene expression, including cyclin D1 and
c-myc. QPCR and Western blotting analysis re-
sults consistently indicated that the expression
of cyclin D1 and MYC was upregulated by miR-
138-5p in comparison with the normal control
transfected cells, while their expressions were
downregulated by miR-138-5p inhibitor (Figure
4D–E). These results suggested that miR-138-5p
might moderate cell growth through targeting
hTERT.
Downregulation of hTERT Counteracted the
Inhibition of Proliferation Induced by miR-138-5p
Inhibitor
To further verify the effect of hTERT on the pro
liferation of human melanoma cells, the expres-
sion of hTERT was detected in Me45 cells trans-
fected with miR-138-5p inhibitor and the specific
siRNA of hTERT (Figure 5A). Colony formation
assay and anchorage-independent growth assay
Ye et al
Figure 1 The expression of miR-138-5p in human melanoma
tissue samples and cell lines. (A) The expression of miR-138-5p
in human melanoma cell line and NGM cell line was analyzed
by qRT-PCR. (*p<0.05 vs. NGM group.) (B) The relative
expression level of miR-138-5p in 38 primary melanoma
carcinoma and adjacent noncancer tissues was determined by
qRT-PCR. (**p<0.01 vs. normal control group.)

showed that hTERT expression was inhibited in
Me45 cells transfected with miR-138-5p inhibitor,
which then could significantly promote the pro
liferation of Me45 cells (Figure 5B–C). In conclu-
sion, the results demonstrated that hTERT might
be an important functional target of miR-138-5p
and be involved in the regulation of miR-138-5p
on human melanoma cell proliferation.
Discussion
Malignant melanoma remains the most lethal kind
of skin cancer.14 The incidence of malignant mel-
anoma has dramatically increased over the years,
with a mortality rate which appears higher than
any other type of skin cancer.15 In this study it
was revealed that the expression of miR-138-5p
in human melanoma cell lines and clinical tissues
was significantly upregulated. miR-138-5p overex-
pression and inhibition was monitored to examine
the effects of miR-138-5p on melanoma cell pro-
liferation. It indicated that miR-138-5p could en-
hance the proliferation and tumorigenicity of hu-
man melanoma cells. In addition, the potential
mechanism of miR-138-5p inhibitor inducing cell
proliferation was determined, and it revealed that
hTERT was the direct target of miR-138-5p and
played a vital role in the positive effects of miR-
138-5p on human melanoma. To sum up, the pres-
ent results suggested that miR-138-5p plays a sig-
nificant role in the occurrence and progression of
human melanoma.
Recently, increasing attention has focused on
the regulatory role of miRNAs in the occurrence
and progression of various types of cancer.16,17
Some studies have shown that the imbalance of
miR-138-5p could promote tumorigenesis, includ-
ing pancreatic cancer, colorectal cancer, cervical
cancer, and bladder cancer.18-20 In addition, miR-
Figure 2
Overexpression of miR-138-5p
promoted the proliferation of
human melanoma cells.
(A) The expression level of
miR-138-5p after transfection
was verified by qRT-PCR.
(B) Overexpression of
miR-138-5p promoted the
proliferation of Me45 cell line
detected by MTT assay.
(C) Representative quantitative
analysis of cell colonies.
(D) miR-138-5p upregulation
promoted anchorage-
independent growth of Me45
cells. (E) Representative
BrdU-incorporated Me45 cells.
(*p<0.05 vs. NC group.)

138-5p overexpression could inhibit the invasion
and metastases of bladder cancer and squamous
cell carcinoma.21,22 A previous study showed that
miR-19 stimulated hTERT transcription through
direct targeting of PITX1 in melanoma cells.23
Chai et al reported that miR-497-5p, miR-195-
5p, and miR-455-3p functioned as tumor sup-
pressors by targeting hTERT in melanoma A375
cells.24 Numerous reports have focused on the
biological characteristics and progression of hu-
man melanoma. However, the biological function
and mechanism of miR-138-5p in the progres-
sion of human melanoma remain unclear. This
study provided convincing biological evidence
that the expression of miR-138-5p was signifi-
cantly upregulated in human melanoma tissues
and cell lines, suggesting that miR-138-5p func-
tioned as an oncogene in promoting the prolif
eration of human melanoma cells.
Conclusion
Based on all that has been discussed above, this
study revealed that the expression of miR-138-
5p was significantly upregulated in human mel-
anoma, and miR-138-5p promoted the prolifera-
tion of cancer cells. miR-138-5p inhibited hTERT
3’-UTR region and resulted in the imbalance of
cyclin D1 and c-myc, thus promoting the prolif-
eration of human melanoma cells. Our findings
provide new insights into the molecular mech-
anism and the intervention targets of human mel-
anoma.
References
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Figure 3
Downregulation of miR-138-
5p inhibited the proliferation
of human melanoma cells.
(A) The expression level of
miR-138-5p after transfection
with miR-138-5p-inhibitor was
verified by qRT-PCR. (B) MTT
assay showed that inhibition
of miR-138-5p inhibited the
growth of Me45 cell line.
(C) Representative quantitative
analysis of cell colonies.
(D) Downregulation of
miR-138-5p inhibited
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(E) Representative
BrdU-incorporated Me45 cells.
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Figure 4
miR-138-5p inhibited the
expression of hTERT by
directly binding to hTERT
3’-UTR in Me45 cells. (A) The
predicted binding sequence of
miR-138-5p (WT and mutant)
in hTERT 3’-UTR. (B) Western
blotting analysis of hTERT
expression in M45 cells
transfected with miR-138-5p
or miR-138-5p inhibitor.
(C) The luciferase activity
assay of the cells transfected
with pGL3-hTERT 3’-UTR
reporter gene and miR-138-5p,
miR-138-5p inhibitor, or
miR-138-5p-mut (*p<0.05).
(D) qRT-PCR analysis of cyclin
D1 and c-myc expression in
Me45 cells. (E) Western
blotting analysis of cyclin D1
and c-myc expression in Me45
cells. (*p<0.05 vs. NC group.)

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Figure 5
Downregulation of hTERT
counteracted the inhibition
of proliferation induced by
miR-138-5p inhibitor.
(A) Western blotting analysis
confirmed that hTERT
silencing effectively reduced
the expression of hTERT in
Me45 cells transfected with
miR-138-5p inhibitor.
(B) After transfection of hTERT-siRNA, transfection of miR-138-5p inhibitor promoted colony formation of Me45 cells. (C) After
transfection of hTERT-siRNA, transfection of miR-138-5p inhibitor promoted anchorage-independent growth of Me45 cells. (*p<0.05 vs.
NC group.)

miR-138-5p Promotes Proliferation of Human Melanoma Cells by Inhibiting hTERT Expression

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miR-138-5p Promotes Proliferation of Human Melanoma Cells by Inhibiting hTERT Expression