ORIGINAL PAPER

MicroRNA-205 suppresses proliferation and promotes apoptosisin laryngeal squamous cell carcinoma

Linli Tian • Jiarui Zhang • Jingchun Ge •

Hui Xiao • Jianguang Lu • Songbin Fu •

Ming Liu • Yanan Sun

Received: 15 October 2013 / Accepted: 22 November 2013 / Published online: 3 December 2013

� Springer Science+Business Media New York 2013

Abstract MicroRNAs were reported to be involved in the

modulation of tumor development. The aim of our study

was to investigate the effect of miR-205 on proliferation

and apoptosis of laryngeal squamous cell carcinoma

(LSCC) and seek associations between miR-205 and Bcl-2

using in vitro and in vivo methods. Real-time qPCR was

used to analyze the expression of miR-205 in LSCC sam-

ples and Hep-2 cell line. Apoptosis, cell cycle, and pro-

liferation (MTT) assays were performed to test the

apoptosis and proliferation of LSCC cells after miR-205

transfection. Bcl-2 expression in cells was assessed with

Western blotting. The tumorigenicity of LSCC cells was

evaluated in nude mice model. MiR-205 was significantly

down-regulated in LSCC tissues compared to adjacent

normal tissues. Lower expression of miR-205 was indi-

cated to be statistically related with advanced clinical stage

and T3–4 grades. We found that restoration of miR-205

down-regulated the proliferative markers of dihydrofolate

reductase and proliferating cell nuclear antigen and apop-

totic regulator of Bcl-2. The findings in vitro and in vivo

showed miR-205 could suppress cell proliferation and

induce cell apoptosis. In addition, Bcl-2 was identified as

one of the direct targets of miR-205 in LSCC cells. These

results suggest that miR-205 may play as a tumor sup-

pressor in LSCC, probably by targeting Bcl-2 and serve as

a potential target for therapeutic intervention.

Keywords Laryngeal squamous cell carcinoma �MiR-205 � Bcl-2 � Proliferation � Apoptosis

Introduction

Laryngeal squamous cell carcinoma (LSCC) is among the

most common cancers in incidence and mortality of head

and neck region [1]. Despite improvements in diagnostic

and therapeutic modalities, there has been no significant

improvement in laryngeal cancer survival over the past

20 years. Molecular mechanism underlying the invasion

and metastasis of LSCC has long been investigated.

Recently, we and others have focused on the implications

of the aberrant expression of microRNAs (miRNAs) in

squamous cell carcinoma of head and neck (SCCHN),

including LSCC [2–6].

MicroRNAs are small noncoding RNA molecules

encoded in the genome that are important for diverse cel-

lular processes, including development, differentiation, cell

cycle regulation, and apoptosis [7]. Since their first dis-

covery in the early 2000s, more than a thousand miRNA

molecules have been identified in the human genome,

exerting their regulatory functions by binding to specific

sequences usually in the 30-untranslated region (30UTR) to

degrade or translationally repress mRNA of the target

genes [8, 9]. It has been suggested that up to 30 % of

human protein coding genes may be regulated by miRNAs

[10]. The role of miRNAs has been well established in

Linli Tian and Jiarui Zhang are co-first authors. Ming Liu and Yanan

Sun are co-corresponding authors.

L. Tian � J. Zhang � J. Ge � H. Xiao � J. Lu � M. Liu (&) �Y. Sun (&)

Department of Otolaryngology, Head and Neck Surgery, The

Second Affiliated Hospital, Harbin Medical University,

Harbin 150081, China

e-mail: liuming002@outlook.com

Y. Sun

e-mail: syn2767@yahoo.com.cn

S. Fu

Laboratory of Medical Genetics, Harbin Medical University,

Harbin, China

123

Med Oncol (2014) 31:785

DOI 10.1007/s12032-013-0785-3

various human cancers [11], and aberrant miRNA expres-

sion is involved in the initiation and progression of cancer

[12, 13]. Moreover, the possible therapeutic use of miR-

NAs has been studied by using antagomirs to silence

miRNAs in mice [14] and non-human primates [15]. Up-

regulation of miR-21 and down-regulation of miR-206

have been detected in LSCC specimens [5, 6]. These

findings give the evidence that the miRNAs are actively

complicated in carcinogenesis of LSCC and merit further

studies to investigate different miRNAs’ functions in the

development of LSCC.

MiR-205 is a highly conserved miRNA among different

species and is down-regulated in many tumors and cancer

cell lines, such as breast [16], bladder [17], prostate [18,

19], and renal cancer [20]. Expression of miR-205 is

abundant in squamous cells of head and neck tissues

compared to other cell types in other tissues [3]. It has been

suggested that down-regulation of miR-205 may be

responsible for the epithelial–mesenchymal transition

(EMT) in head and neck carcinomas [21], and significantly

associate with loco-regional recurrence and poor prognosis

for HNSCC patients [4]. Nevertheless, the function of miR-

205 in LSCC has not yet been clearly investigated and need

to further elucidate.

In this study, we demonstrated that miR-205 was sig-

nificantly down-regulated in LSCC compared to adjacent

non-cancerous tissues, and lower expression of miR-205

was indicated to be statistically related with advanced

clinical stage and T3–4 grades. We found that the resto-

ration of miR-205 could down-regulate the proliferative

markers of dihydrofolate reductase (DHFR) and prolifer-

ating cell nuclear antigen (PCNA) and clearly inhibit the

cell proliferation. In addition, the expression of Bcl-2

protein, a key regulator of apoptotic pathway, was signif-

icantly down-regulated by the overexpression of miR-205.

To our knowledge, this was the first study regarding the

investigation of miR-205 function in tumor apoptosis by

testing Bcl-2 expression. Our data indicated that miR-205

may act as a tumor suppressor in LSCC to perform the

functions of suppressing proliferation and promoting

apoptosis by regulating Bcl-2, and the restoration of miR-

205 may become a potential novel strategy in therapeutic

intervention of LSCC.

Materials and methods

Samples

Pairs of tumor tissues and adjacent non-cancerous mat-

ched tissues in this study were obtained from 30 patients

who underwent partial or total laryngectomy between

December 2011 and December 2012 at the Department of

Otorhinolaryngology, the Second Affiliated Hospital of

Harbin Medical University, under an approved protocol of

Harbin Medical University. The patients had not received

any antineoplastic therapy before admission. After sur-

gery, the specimens were preserved in liquid nitrogen

within 5 min of excision, then transported frozen to the

laboratory and stored at -80 �C till next experiment

within 1 year. The human LSCC cell line (Hep-2 cell line)

was purchased from Shanghai Cell Bank of Chinese

Academy of Sciences.

Cell culture and transfection

Hep-2 cells were cultured in Dulbecco’s modified Eagle’s

medium (DMEM, GIBCO, Grand Island, NY, USA) con-

taining 10 % fetal bovine serum (FBS), 100 U/mL peni-

cillin, and 100 lg/mL streptomycin and incubated in a

humidified (37 �C, 5 % CO2) incubator. Transfection of

miR-205 plasmids that incorporated GFP as a reporter gene

was performed with Lipofectamine 2000 (Invitrogen) fol-

lowing the manufacturer’s instructions. Briefly, Hep-2 cells

were plated onto 6-well plates (4 9 105 cells/well) for a

day till the cells had reached 80–85 % confluency. The

plasmids and Lipofectamine 2000 were each diluted in

250 lL of serum-free OPTI-MEM (Gibco BRL) and incu-

bated for 5 min at room temperature. The diluted plasmids

and Lipofectamine 2000 were combined at a 1:2 ratio (3 lg

of plasmid with 6 lL of Lipofectamine 2000). This com-

bination was mixed gently and incubated for 20 min at

room temperature. A total of 500 lL of the combination

was added to each well in a final volume of 2 mL per well.

Quantitative real-time PCR

For LSCC samples and Hep-2 cells, total RNA was isolated

using Trizol reagent (Invitrogen) according to the manu-

facturer’s instruction. About 200 ng of total RNA was

reverse transcripted using All-in-OneTM miRNA q-PCR

Detection Kit (Genecopoeia, Germantown, MD, USA)

according to the manufacturer’s manual. To estimate the

expression of miR-205, the Ct values were normalized using

18S rRNA as internal control. The relative miRNA expres-

sion was calculated using the 2-44Ct. The primers for miR-

205 detection were 50-TTCATTCCACCGGAGTCTGAA

A-30. The hsRNA-U6 primer (TCGTGAAGCGTTCC

ATATTTTTAA) was used as an internal control for the

normalization in the Taqman microRNA assay. After

reverse transcription at 50 �C for 30 min and denaturation at

95 �C for 10 min, amplification and detection were per-

formed using the 7500 Fast Real-Time PCR System

(Applied Biosystems, Foster City, CA, USA), using 40

cycles of denaturation at 95 �C (15 s) and annealing/exten-

sion at 60 �C (60 s). Each sample was run in triplicate.

785 Page 2 of 10 Med Oncol (2014) 31:785

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MTT assay

After transduction of Hep-2 cells by miR-205 plasmid for

varying time periods—24, 48, and 72 h-20 lL of sterile

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoli-

um bromide) was added and incubated for another 4 h at

37 �C. Then, 150 lL of dimethyl sulfoxide was added to

each well and the plates were thoroughly mixed for 10 min.

Spectrometric absorbance at a wavelength of 492 nm was

measured on an enzyme immunoassay analyzer (model

680, Bio-Rad Laboratories, Hercules, CA, USA). The cell

growth rate was calculated using the following formula:

Cell growth rate (%) = (mean absorbance in six wells of

the treatment group/mean absorbance in six wells of the

cells control group) 9 100.

Apoptosis analysis

Annexin V/APC and propidium iodide (PI) apoptosis

detection kit I (BD Pharmingen, San Diego, CA, USA)

were used to identify apoptotic following the manufac-

turer’s instructions. The percentage of apoptotic cells was

calculated from the data originating from flow cytometry.

The cells were washed twice with ice-cold PBS and

resuspended in 19 binding buffer (BD Biosciences, San

Jose0, CA, USA) at a concentration of 1 9 106 cells/mL.

Cells were stained with Annexin V-PI and APC. Hep-2

cells without any treatment were used as an internal con-

trol, and the experiments were repeated at least three times.

Western blotting analysis

Hep-2 cells were collected and analyzed by Western blot-

ting to assess Bcl-2 and DHFR expressions according to the

standard methods using anti-bcl-2 and anti-DHFR anti-

bodies purchased from Boster (Wuhan, China). Western

blot of b-actin on the same membrane was used as a

loading control. The intensity of the respective signals in

these blots was determined with an image analysis using

Image J program.

Tumorigenicity assay in animal experiments

Eighteen BALB/c mice (5–6 weeks old), provided by

Beijing Charles River Laboratory Animal Technology

Company, were bred in the animal laboratory of the Harbin

Medical University in aseptic conditions and kept at a

constant humidity and temperature (25–28 �C) according

to the standard guidelines under a protocol approved by

Harbin Medical University. All mice were divided into

three groups by injecting different 100 lL Hep-2 cell

suspension (1 9 106) subcutaneously in the dorsal scapula

region: (1) Experimental group was treated with cells

transfected by miR-205 plasmids. (2) Negative control

group was treated with cells transfected by GFP plasmids.

(3) Blank control group was treated with blank Hep-2 cells.

Animals were killed on week 4 of treatment. The tumors

were harvested, calculated for weight, and prepared for

further analysis.

Transmission electron microscope examination (TEM)

Tumor samples were cut into 1 mm 9 1 mm 9 1 mm

sections, fixed in 3 % glutaral for 24 h at 4 �C and in 1 %

osmium tetroxide for 2 h, dehydrated through a grated

series of ethanol, and immersed with Epon 821 for 72 h at

60 �C. After the samples were prepared into ultrathin

section (70 lm) and stained with uranyl acetate and lead

citrate, they were observed by transmission electron

microscopy (H-600, HITACHI, Japan).

TUNEL assay

TUNEL assay was performed using the In Situ Cell Death

Detection Kit (R&D, USA) according to the manufac-

turer’s instruction. After routine deparaffinization, sections

were digested with proteinase K working solution for

25 min, followed by applying block solution for 15 min.

Then, the slices were incubated with 50 lL TUNEL

reaction mixture for 60 min. Alkaline phosphatase anti-

body was added for 20 min afterward. DAB was used as

chromogen, and the slices were counterstained with

hematoxylin, dehydrated, and mounted. All processes

above were conducted at 37 �C in a humidified atmo-

sphere. A positive control was prepared by treating the

samples without TdT. For quantitative analysis, the per-

centage of TUNEL-positive cells among 200 tumor cells in

ten fields per section that were selected randomly was

determined at 400-fold magnification using an microscope

(Olympus, Tokyo, Japan).

Immunohistochemistry

Series tumor sections of 4 lm thick were prepared for im-

munohistological staining. Tissue sections were quenched for

endogenous peroxidase with freshly prepared 3 % H2O2 with

0.1 % sodium azide and then placed in an antigen retrieval

solution (0.01 mol/L citrate buffer, pH 6.0) for 15 min in a

microwave oven at 100 �C and 600 W. After incubation in the

casein block, rabbit multiclonal anti-Bcl-2 antigen (1/50,

Zhongshan Goldenbridge Biological Technology, Ltd. Bei-

jing, China) was applied to the sections for 1 h at room tem-

perature, followed by incubation with biotinylated anti-rabbit

IgG as a second antibody (1/200, Zhongshan Goldenbridge

Biological Technology, Ltd. Beijing, China) and ExtrAvidin-

conjugated horseradish peroxidase (1/30, Maixin Bio Co.,

Med Oncol (2014) 31:785 Page 3 of 10 785

123

Fuzhou, China). The immune reaction was revealed with di-

aminobenzidine tetrachloride, and slides were counterstained

with hematoxylin, dehydrated, and mounted. Consistent

negative control was obtained by replacement of primary

antibody with PBS.

Statistical analysis

Data are expressed as mean ± SD of three independent

experiments, each performed in triplicate. Differences

between groups were assessed by unpaired, two-tailed

Student’s t test, and P \ 0.05 was considered significant.

Results

MiR-205 expression was down-regulated in LSCC

tissues and Hep-2 cells

Total RNA was isolated from Hep-2 cell line and 30

specimens of LSCC tissues and adjacent non-cancerous

tissues. MiR-205 expression was determined by real-time

qPCR. As shown in Fig. 1, miR-205 expression was sig-

nificantly higher (about 6.0-folds) in adjacent non-cancer-

ous tissues than that in LSCC tissues and Hep-2 cell line

(P \ 0.05), indicating that miR-205 was highly repressed

in either laryngeal cancer specimens or Hep-2 cell line. In

addition, lower expression of miR-205 was indicated to be

statistically related with advanced clinical stage and T3–4

grades (Table 1). No correlation was found with gender,

age, lymph node metastasis, and differentiation. The data

suggested that miR-205 may serve as a suppressor in the

developing process of LSCC.

MiR-205 expression was up-regulated by miR-205

plasmid in Hep-2 cells

Hsa-miR-205 plasmid vector system that incorporates GFP

as a reporter gene was used to up-regulate the expression of

miR-205 in order to further explore the functional roles of

miR-205 in LSCC. As shown in Fig. 2a, a high percentage

(more than 80 %) of fluorescent signals in the cytoplasm of

Hep-2 cells was detected at 48 h after transfected by either

the miR-205 plasmid (a, experimental group) or control

GFP transfection (b, negative control group), whereas no

signal was found for blank Hep-2 cells (c, blank control

group). Furthermore, quantitative real-time PCR revealed

that miR-205 was up-regulated in the Hep-2 cells of

experimental group (d), indicating that Hep-2 cell line was

an ideal model for the functional study of miR-205 and

miR-205 expression can be adjusted.

MiR-205 inhibited the proliferation of Hep-2 cells

Based on the restoration of miR-205 by hsa-miR-205

plasmid transfection, we next examined the ability of miR-

205 to influence the viability of Hep-2 cells in culture. As

shown in Fig. 3, cell proliferation of Hep-2 cells trans-

fected by miR-205 plasmid was evidently inhibited at 48

and 72 h. Further, we chose a proliferative marker of

DHFC to further reveal the effect of miR-205 in the pro-

liferation of tumor. We found that the level of DHFR was

markedly down-regulated in experimental group by

Fig. 1 MiR-205 expression in LSCC tissues, adjacent non-neoplastic

tissues, and Hep-2 cell line. The miR-205 level in both cancer tissues

and Hep-2 cell line was significantly lower than that in the adjacent

non-cancerous tissues. P \ 0.05

Table 1 Relationship between miR-205 expression and patients’

parameters

Patients’

characteristics

Number

(n = 30)

MiR-205 P value

Sex 0.218

Male 23 22.92 ± 4.41

Female 7 25.68 ± 6.51

Age 0.999

B64 24 23.62 ± 4.89

[64 6 23.63 ± 5.85

Differentiation 0.221

Well 24 22.84 ± 3.96

Moderately 3 27.96 ± 8.62

Poorly 3 24.85 ± 7.12

T classification 0.021

T1–2 12 27.40 ± 2.47

T3–4 18 21.58 ± 1.42

Lymph node metastasis 0.365

Negative 20 22.52 ± 3.05

Positive 10 24.26 ± 5.81

Clinical stage 0.021

I–II 12 27.40 ± 2.47

III–IV 18 21.58 ± 1.42

785 Page 4 of 10 Med Oncol (2014) 31:785

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Western blot analysis (Fig. 4). These findings suggested

that the restoration of miR-205 was highly specific for the

suppression of proliferation of Hep-2 cell line.

MiR-205 suppressed Bcl-2 expression and induced

the apoptosis of Hep-2 cells

We utilized flow cytometry to analyze the apoptotic per-

centage of transfected Hep-2 cells. At 72-h time point of

transfection, we observed the more significant increase of

apoptotic cells (48.42 ± 1.60 %) in miR-205 experimental

group (Fig. 5A) than those in the negative control group

(Fig. 5B, 1.67 ± 0.37 %) and blank control group

(Fig. 5C, 1.08 ± 0.12 %) (P \ 0.05). The finding of miR-

205 inducing apoptosis effect promoted us to investigate

the possible mechanism. On the basis of the literature, we

chose anti-apoptotic protein Bcl-2 to study as possible

regulator and detected the protein level of Bcl-2 in Hep-2

cells by Western blotting analysis. As shown in Fig. 5, Bcl-

2 expression was clearly suppressed in miR-205 experi-

mental group compared to the controls.

MiR-205 suppressed tumor growth in vivo

To confirm the findings shown in vitro, we established an

in vivo tumor model and found the growth of tumor was

Fig. 2 GFP expression and

expression of miR-205 in three

groups. A high GFP expression

at 48 h was detected after Hep-2

cells were transfected by hsa-

miR-205 plasmid (a) or control

GFP plasmid (b), whereas no

GFP expression in blank Hep-2

cells (c). Fluorescence

microscope images (9200). The

qRT PCR analysis (d) showed

miR-205 expression was

significantly up-regulated after

hsa-miR-205 plasmid

transfection.* P \ 0.05

Fig. 3 Curve of cell survival rate. After miR-205 plasmids’ trans-

fection, the survival rate of Hep-2 cells was evidently decreased at

different time points of 48 h and 72 h, respectively, compared with

the control group and blank group. P \ 0.05

Fig. 4 The protein level of DHFC in Hep-2 cells. MiR-205 down-

regulated DHFC expression in Hep-2 cells infected by miR-205

plasmids. a miR-205 plasmids infected Hep-2 cells, b blank Hep-2

cells, c plasmids without miR-205-infected Hep-2 cells. P \ 0.05

Med Oncol (2014) 31:785 Page 5 of 10 785

123

Fig. 5 MiR-205 induced

apoptosis of Hep-2 cells. Flow

cytometry analyzed the effect of

miR-205 on the cell cycle of

Hep-2 cells at 72-h time point of

transfection. Apoptotic cells in

miR-205 experimental group

(A) were dramatically higher

than those in negative control

group (B) and in blank control

group (C). MiR-205 clearly

suppressed the protein level of

Bcl-2 in experimental group by

Western blot analysis (D).

a miR-205 plasmids infected

Hep-2 cells, b blank Hep-2

cells, c: plasmids without miR-

205-infected Hep-2 cells.

P \ 0.05

Fig. 6 MiR-205 suppressed tumor growth in vivo. Tumor xenografts were dissected 28 days after treatment. a Experimental group, b negative

control group, c blank control group, d difference of tumor weight in the three groups. P \ 0.05

785 Page 6 of 10 Med Oncol (2014) 31:785

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markedly suppressed in mice of experimental group. As

shown in Fig. 6, a significant reduction in tumor weight

(1.1 ± 0.1 g) was detected in Hep-2 cells transfected with

miR-205 plasmids compared with that in either negative

control group (NC) (1.8 ± 0.3 g) or blank control group

(1.8 ± 0.2) (P \ 0.05). Meanwhile, PCNA, a widely rec-

ognized proliferative maker, was chosen to verify the effect

of miR-205. We detected that the PCNA protein expression

was down-regulated on the sections of the paraffin-

embedded xenograft tumor tissues through immunohisto-

chemistry (Fig. 7), indicating that miR-205 played an

important inhibitory role in the proliferation of LSCC.

MiR-205 promoted tumor apoptosis and down-

regulated Bcl-2 expression in vivo

To measure the discovery of miR-205-promoting apoptosis

in Hep-2 cells, we then performed apoptosis experiments in

the tumor model in vivo. The results from TUNEL assay

showed that apoptosis staining in miR-205 experimental

group (Fig. 8a) was dramatically stronger than that in

negative control group (Fig. 8b) and blank control group

(Fig. 8c), indicating that miR-205 induced significant

apoptosis in the experimental group. In addition, trans-

mission electron microscope revealed the typical signs of

apoptotic cells, such as cell shrinkage, chromatin conden-

sation, and aggregated under nuclear membrane (Fig. 8d),

whereas the tumor cells in the controls were lack of the

morphological features of apoptotic cells (Fig. 8e, f). To

further understand the relevance between miR-205 and

Bcl-2, we detected the Bcl-2 protein expression on the

sections of the paraffin-embedded xenograft tumor tissues

through immunohistochemistry. Immunolabeling of Bcl-2

was confined to the cytoplasm of the xenograft tumor cells.

Bcl-2 immunoreactivity was evaluated based on the per-

centage staining of the tumor cell population. As shown in

Fig. 8, the date of Bcl-2 in miR-205-treated group was

7.8 ± 1.4 % (Fig. 8g), whereas that in negative control

group or blank control group was 46.5 ± 2.2 % (Fig. 8h)

and 48.3 ± 3.4 % (Fig. 8i), respectively. The data

collected from the in vivo experiments further demon-

strated that the overexpression of miR-205 resulted in the

apoptosis-inducing effect on xenograft tumors probably by

regulating Bcl-2.

Discussion

Evidence has been accumulated that miRNAs may play an

important role in human carcinogenesis. A review of the

literature indicates that miR-205 expression is dysregulated

in different human cancer entities. It has been described

that miR-205 is down-regulated in breast cancer [16] and

bladder cancer [17] compared to matched adjacent normal

tissues. In contrast, miR-205 expression has been found to

be up-regulated in esophageal squamous cell carcinoma

[22]. These findings suggest that miR-205 can act as an

oncogene or a tumor suppressor, probably depending on

the cellular context of tumors. Moreover, the literature has

showed that miR-205 expression can be either down-reg-

ulated [4] or up-regulated [3] in head and neck cancer. Cao

et al. [23] identify that miR-205 overexpression is com-

pared with adjacent normal tissue, while our data indicate

the opposite result in LSCC. Thus, like many other miR-

NAs, miR-205 may exert a biphasic effect on human car-

cinogenesis, functioning either as oncogene that promotes

tumor development or as tumor suppressor that possesses

anti-oncogenic activity. There were several explanations

for the contradictory finding, partially due to different

histotypes [24], different subtypes [25], and microenvi-

ronment [26, 27] of tumor. Recently, it has been suggested

that molecular information of miRNAs can be carried by

tumor cell-derived microvesicles in relation to cancer

progression and promotion of metastasis [28, 29]. Here, our

results show that down-regulation of miR-205 is signifi-

cantly correlated with clinical stage and T grade, but not

with lymph node metastasis, suggesting that miR-205 may

play an anti-tumor role in the relative early stage of LSCC.

Identification of action consistent with a tumor suppressive

function of miR-205 expression in LSCC prompts us to

Fig. 7 MiR-205 down-regulated PCNA expression in tumor xeno-

grafts. a Tumors injected with miR-205 plasmids exhibited weak

PCNA protein staining (9400), b (tumors injected with GFP

plasmids) and c (tumors injected with bland Hep-2 cells) exhibited

strong protein staining (9400)

Med Oncol (2014) 31:785 Page 7 of 10 785

123

investigate its biological function. Our data show that cell

proliferation of Hep-2 cells transfected by miR-205 plas-

mids is evidently decreased contrast to the control Hep-2

cells. Overexpression of miR-205 dramatically down-reg-

ulates the level of DHFR as a proliferative marker in Hep-2

cells using Western blotting analysis. DHFR is a key

enzyme of the folate cycle, and the metabolism of one

carbon unit catalyzes the NADPH-dependent reduction of

dihydrofolate (DHF) to tetrahydrofolate (THF) [30] and an

S-phase-specific enzyme associated with cellular prolifer-

ation [31]. Therefore, the restoration of miR-205 is highly

specific for the suppression of proliferation of Hep-2 cell

line. Furthermore, our results demonstrate that overex-

pression of miR-205 may suppress tumor growth in BALB/

c mice and down-regulate the level of PCNA as another

proliferative marker in tumor xenografts. PCNA is an

essential protein in DNA replication associated with pro-

cesses such as chromatin remodeling, cell cycle control,

and DNA repair [32, 33] and has been demonstrated that

the expression level of PCNA is related to proliferation [34,

35]. The results implied our data showed that miR-205

played an important role as a proliferative suppressor in

LSCC.

According to the literature, several direct targets of miR-

205 are described. For example, ErbB3 and vascular

endothelial growth factor A (VEGF-A) are targeted by

miR-205 in breast cancer [16], and it has been shown that

the ERBB3 receptor has a central role in maintenance and

malignancy of lung cancer, often involving signaling

through the PI3 K/AKT pathway [36]. Another target of

miR-205 is PTEN that can affect the activity of Akt as a

phosphatase in squamous cell carcinoma, thus inhibiting

the tumor proliferation [37]. Recent studies have shown

that miR-205 inhibits tumor proliferation modulated by the

polycomb protein Mel-18, loss of which consistently

increased ZEB1 and ZEB2 expression and down-regulated

E-cadherin expression, leading to increased migration and

invasion in MCF-7 cells [38]. Furthermore, accumulating

Fig. 8 MiR-205 induced apoptosis of xenograft tumor cells. The

cellular apoptotic rate detected by TUNEL assay (9200) showed that

apoptosis staining in miR-205 experimental group (a) was dramat-

ically stronger than that in negative control group (b) and blank

control group (c). Apoptotic structure was detected in tumor cells of

miR-205 group (d) by transmission electron microscopy (912,000),

not in GFP control group (e) and in blank Hep-2 cell group (f). Bcl-2

protein staining tested by immunohistochemistry in experimental

group (g) was weaker than that in negative control group (h) or blank

control group (i)

785 Page 8 of 10 Med Oncol (2014) 31:785

123

data have shown that the importance of miRNAs as

potential prognostic indicators for cancer is underscored by

their functions in regulating fundamental cellular pro-

cesses, such as apoptosis [39]. The report of Jian Li found

that overexpression of miR-203 may decrease the anti-

apoptotic gene Bcl-xl expression in colon cancer [40]. In

addition, Qiu T and colleagues suggest that miR-503 can

modulate the resistance of non-small cell lung cancer cells

to cisplatin by targeting Bcl-2 [41]. Recent study has also

shown that miR-205, together with miR-125a and miR-

125b, functionally cooperates with entinostat, a synthetic

benzamide derivative class I HDACi, to down-regulate

erbB2/erbB3 receptors and induce apoptosis in breast

cancer cells [42]. Therefore, we speculate that Bcl-2 may

also be a possible target gene of miR-205, responsible for

the mechanism of regulating apoptosis of LSCC. Bcl-2, as

an anti-apoptotic membrane-associated molecule, resides

in the nuclear envelope and mitochondria, and is key reg-

ulatory protein of the apoptotic pathway [43]. It exerts its

anti-apoptotic functions by modulating the mitochondrial

release of cytochrome c and the interaction of apoptosis-

activating factors (Apaf-1) with caspase 9, Bax, and c-Myc

[44]. In this study, our data have indicated a strong link

between miR-205 and Bcl-2 in LSCC that restoration of

miR-205 may increase the apoptotic effects on Hep-2 cells

and decrease the expression of Bcl-2 both in vitro and

in vivo. Therefore, our results indicated that miR-205

participated in promoting apoptosis of LSCC cells proba-

bly by regulating the target gene of Bcl-2.

In summary, miR-205 was down-regulated in LSCC

tumor tissues. Moreover, we have unraveled the distinct

tumor suppressive properties of miR-205 in the relative

early stage of LSCC and verified miR-205 acting function

as a Bcl-2-responsive miRNA in LSCC. Consistent with

the date on the miR-205/Bcl-2 pathway, we propose that

miR-205 is a tumor suppressor that may inhibit cell pro-

liferation and induce apoptosis of LSCC by regulating Bcl-

2. On the basis of our studies, we evolve that miR-205 may

be a potentially attractive and promising target for thera-

peutic intervention in LSCC.

Acknowledgments The research was supported by grants from the

Heilongjiang Postdoctoral Fund (LBH-Z12157), the foundation of

Heilongjiang Educational Committee (12531343), the foundation of

Heilongjiang Health Bureau (2012-624), the Youth Foundation of the

Second Affiliated Hospital of Harbin Medical University (QN2011-01),

the National science Foundation of china (81241085, 81372902,

81272965), the key project of Natural Science Foundation of Hei-

longjiang Province of China (ZD201215/H1302), the Research Fund

for the Doctoral Program of Higher Education of China

(20102307110007), and the science and technology innovation talent

research funds of Harbin (2012RFXXS072).

Conflict of interest The authors declare that they have no conflict

of interest.

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