1 s2.0 S0753332217304390 Main PDF
1 s2.0 S0753332217304390 Main PDF
1 s2.0 S0753332217304390 Main PDF
Original article
A R T I C L E I N F O A B S T R A C T
Keywords: Recent studies have showed that microRNA-150 (miR-150) is up-regulated in various cancers including cervical
MiR-150 cancer. However, the specific mechanism of miR-150 in the regulation of cell proliferation, migration and in-
Cervical cancer vasion is still unclear. In this study, a total of 150 cervical cancer samples, including 50 cervical cancer tissues,
Proliferation 50 corresponding adjacent non-neoplastic tissues, and 50 serum samples were collected from cervical cancer
Invasion
patients. 50 matched normal tissues and 50 serum samples were collected from the control group. MiR-150 was
Migration
evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Programmed cell death protein 4
PDCD4
(PDCD4) was evaluated by qRT-PCR and western blot. Cell migration and invasion were assessed by transwell
assays. Proliferative abilities were determined by MTT assays. Luciferase reporter assay was employed to vali-
date the direct target of PDCD4 by miR-150. We found that miR-150 was increased in cervical cancer specimens.
In contrast, PDCD4 was decreased in cervical cancer tissues. MiR-150 promoted cell migration, invasion and
proliferation through targeting PDCD4. These results collectively indicated that miR-150 might be used as a
potential therapeutic biomarker in cervical cancer.
1. Background Recently, the PDCD4 gene has been identified as the target gene of
miR-150 [12]. PDCD4, a direct suppressor of NF-кB (p65), can also
Cervical cancer is one of the most prevalent cancers in the world prevent AKT activation and suppress the expression of matrix me-
and has a high mortality rate in women [1–3]. If we have a strong tallopeptidase 9 (MMP-9) [13]. In addition, PDCD4 was identified as
advantage in early detection, cervical cancer may be cured and has the upstream suppressor of MMP-9, which facilitates cancer cell mi-
available varieties of treatment options. However, the initial stages of gration through degrading the collagenous substrates in the sur-
cervical cancer are usually asymptomatic [2,4]. Therefore, a certain rounding extracellular matrix [14].
amount of specific and sensitive noninvasive biomarkers are urgently Therefore, there are two human cervical cancer cells which are
needed for predicting the prognosis of cervical cancer [5]. called HeLa and SiHa cells were used to clarify the role of miR-150 in
Recent studies have demonstrated that microRNAs (miRNAs) played cell migration and invasion in cervical cancer. In this study, we focused
crucial roles in different cancers such as breast cancer and cervical on whether miR-150 could determine the migration and invasion of
cancer [5–7]. MiRNAs are small RNA molecules of 19–25 nucleotides in cervical cancer cells through regulating PDCD4.
length that could bind the partial sequence homology to the 3′-un-
translated regions (3′-UTRs) of mRNA of its target genes, lead to the 2. Methods
repression of translation, and then play a vital role in regulating the
gene expression [6,8]. MiRNAs are also seen as new biomarkers in the 2.1. Patients and samples
development of many diseases [3,6]. MiR-150 is located at chromosome
19q13 and works as the primary oncomiR in a large amount of cancers A total of 50 cervical cancer tissues, 50 corresponding adjacent non-
such as leukemia and colorectal cancer [9,10]. Besides, the aberrant neoplastic tissues and 50 serum samples were collected from patients
expression of miR-150 is relevant to the development of different who underwent cervical surgical resection without preoperative systemic
cancers, predicting that it may be used as a diagnostic and prognostic therapy, while 50 matched normal tissues and 50 serum samples were
biomarker in cancer [11]. collected from patients with chronic cervicitis (control group) at the third
⁎
Corresponding author at: The Third Affiliated Hospital of Zhengzhou University, No. 7 Kangfu Front Street, Zhengzhou, Henan 450000, PR China.
E-mail address: zhangzhanzdsfy@126.com (Z. Zhang).
http://dx.doi.org/10.1016/j.biopha.2017.09.143
Received 27 January 2017; Received in revised form 25 September 2017; Accepted 26 September 2017
0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
Z. Zhang et al. Biomedicine & Pharmacotherapy 97 (2018) 511–517
HeLa and SiHa cells were procured from ATCC and were maintained
2.7. Western blot assay
in prescribed culture conditions. These cell lines were cultured in
RPMI1640 (Invitrogen, CA), supplemented with 1% penicillin/strep-
Total protein was lysed by a cell RIPA lysis buffer (Beyotime,
tomycin (Invitrogen, CA) and 10% fetal bovine serum (FBS). A humi-
China). The protein concentration of each sample was determined by
dified incubator with a 5% CO2 – 95% air atmosphere at 37 °C was
using a BCA protein assay kit (Beyotime, China) and equal amounts of
needed for culturing cells and cells were passaged every 2–3 days by
protein were separated by SDS–PAGE (10% acrylamide gel). The anti-
using trypsin-EDTA.
bodies used for rabbit anti-PDCD4 antibody (1:1000, Abcam, UK), and
mouse anti-β-actin antibody (1:5000, Santa Cruz, USA) were used for
2.3. MiR-150 mimics, miR-150 inhibitors and transfection
western blot.
HeLa and SiHa cells were transfected by using Lipofectamine 2000 For the invasion assay, the transwell chambers were coated with
(Invitrogen, Carlsbad, CA). PDCD4 cDNA without its 3′-UTR was in- matrigel (BD Biosciences, USA) to form a matrix barrier. Then,
serted into pcDNA3.1 (+) to produce the recombinant vector 1.0 × 105 cells in 200 μl of serum-free medium were placed into the
pcDNA3.1 (+)/PDCD4. We verified all constructions for sequence upper chamber. A 500 μl medium containing 15% FBS was added in the
correctness via direct sequencing. lower chamber. For the migration and invasion assays, HeLa and SiHa
cells were incubated for 24 h. The migrated or invaded cells that tra-
2.5. Luciferase reporter assay velled through the pores were counted under a microscope, and the
cells in three pores of each group were quantified.
The sequences of PDCD4 3′-UTR were amplified from normal human
genomic DNA and subcloned into the pmirGLO luciferase reporter vector 2.10. Statistics
(Promega, USA). HeLa and SiHa cells (1.0 × 105) were seeded in 24-well
plates and co-transfected with wild-type (WT) or mutant (MUT) 3′-UTR Statistical analyses were compared by using SPSS software 21.0.
vectors and miR-150 mimics or Mi-c by using Lipofectamine 2000. Then, Data were presented as the mean ± standard deviation. Student’s t-test
the Dual-Luciferase Reporter Assay System (Promega) was used to con- was performed to compare the significant differences between two
duct Luciferase reporter assay according to the manufacturer’s protocol. groups and ANOVA (one-way analysis of variance) was used to measure
The luciferase activities were normalized to Renilla luciferase activity. the difference between more than two groups. P < 0.05 was con-
All experiments were performed three times. sidered significantly different.
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3. Results Three databases (miRDB, TargetScan, and PicTar) were used to identify
whether PDCD4 is a candidate target of miR-150. It can clearly be seen
3.1. MicroRNA-150 was overexpressed in cervical cancer patients that the 3′-UTR of PDCD4 is the direct target of miR-150 (Fig. 2A). In
order to further confirm the targeting relationship between miR-150
The expression of miR-150 in cervical cancer tissues and serum and PDCD4 in cervical cancer, we transfected HeLa and SiHa cells with
samples was measured by qRT-PCR, and the expression of PDCD4 in miR-150 mimics or miR-150 inhibitors, and verified that more than
cervical cancer tissues was measured by qRT-PCR and western blot. As 90% of the cervical cancer cells were transfected successfully. The ex-
showed in Fig. 1A, the miR-150 expression determined by qRT-PCR was pression of miR-150 determined by qRT-PCR was significantly up-
significantly increased in the cervical cancer tissues compared with the regulated or down-regulated after transfection with miR-150 mimics or
matched adjacent non-neoplastic tissues and the normal controls miR-150 inhibitors, separately (Fig. 2B). Furthermore, qRT-PCR
(P < 0.05). As showed in Fig. 1C, miR-150 determined by qRT-PCR showed that the elevated levels of miR-150 in HeLa and SiHa cells were
was significantly increased in serum samples in cervical cancer patients concurrent with the lower level of PDCD4 mRNA (Fig. 2C). Moreover,
compared with the normal controls (P < 0.05). As showed in Fig. 1B, the protein levels of PDCD4 measured by western blot also declined
D, the expression of PDCD4, measured by using qRT-PCR and western following the overexpression of miR-150 in cervical cancer cells
blot, was notably decreased in the cervical cancer tissues compared (Fig. 2D). In order to better understand the interaction between miR-
with the normal control (P < 0.05). As showed in Fig. 1A, B, D, there 150 and PDCD4, luciferase reporter assays were then conducted. The
was not any significant difference in miR-150 and PDCD4 detected in luciferase activities were decreased when co-transfected with WT-
the adjacent non-neoplastic tissues from cervical cancer patients and PDCD4-3′-UTR plasmid and miR-150 mimics in HeLa and SiHa cells, in
the normal controls (all P﹥0.05). contrast, there was no obvious differences when co-transfected with
MUT-PDCD4-3′-UTR plasmid and miR-150 mimics. These results clar-
ified that miR-150-5p target PDCD4 through binding to its 3′-UTR.
3.2. PDCD4 is one of the target genes of miR-150 in HeLa and SiHa cells
Since miRNAs mainly regulate protein expression through directly 3.3. MiR-150 promoted the proliferation of HeLa and SiHa cells
binding RNA, we had a strong interest in the underlying target genes
related to the oncogenetic functions of miR-150 in cervical cancer. Cell growth rate in the Mi-c group was lower than that in the blank
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control and miR-150 mimics group (all P < 0.05), while cell growth in migration trend (all P < 0.05) (Fig. 4B, D). These findings suggested that
the miR-150 inhibitor group was obviously inhibited when compared with miR-150 promote the migration and invasion of the cervical cancer cells.
the blank control and In-c group (all P < 0.05); While not significant
differences in cell growth were found between the blank control and the
Mi-c group and In-c group (all P > 0.05). There was not any significant 3.5. Overexpression of PDCD4 attenuated the positive effects of miR-150
differences detected in cell growth between the miR-150 mimics group
and the blank control (P > 0.05). These data indicated that miR-150 To further clarify whether the positive effects of miR-150 on cell
could promote the proliferation of cervical cancer cells (Fig. 3). proliferation, migration and invasion of cervical cancer cells were
mediated by the suppression of PDCD4, we rescued PDCD4 expression
in miR-150-overexpressing HeLa and SiHa cells by transfecting with
3.4. MiR-150 increased the migration and invasion of HeLa and SiHa cells pcDNA3.1/PDCD4 plasmid. After transfection, western blot showed
that the decreased PDCD4 level was upregulated remarkably
To understand the function of miR-150 on cell mobility, transwell (Fig. 4A–B). MTT assasy, cell migration and invasion assays were then
assays were performed. As shown in Fig. 4A, C, the migrative abilities of conducted to determine the cell proliferation, migration and invasion,
cervical cancer cells were significantly up-regulated when transfected with respectively. As showed in Fig. 4C–D, the cell proliferation was sig-
miR-150 mimics, while were down-regulated when transfected with miR- nificantly decreased in the miR–150 + pcDNA3.1/PDCD4 group, when
150 inhibitors (all P < 0.05). Besides, the effect of miR-150 on the cell compared with the miR-150 group. The cell migration and invasion
invasion of HeLa and SiHa cells was measured by a Matrigel Transwell were also lower in the miR–150 + pcDNA3.1/PDCD4 group, when
assay. The invasive abilities of HeLa and SiHa cells exhibited a similar cell compared with the miR-150 group (Fig. 4E–H).
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Fig. 3. MiR‐150 promoted the proliferation, migration and invasion of HeLa and SiHa cells. HeLa and SiHa cells were transfected with miR-150 mimics, Mi-c, miR-150 inhibitor and In-c,
respectively.
A–F, Cell proliferation were measured by MTT assay, cell migration and invasion were measured by transwell assay and Matrigel transwell analysis,*P < 0.05.
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Fig. 4. Overexpression of PDCD4 attenuates the positive effects of miR-150 on the proliferation, migration and invasion of HeLa and SiHa cells. HeLa and SiHa cells were transfected with
miR-150 mimics control (Mi-c), miR-150 mimics (miR-150), or co-transfected with miR-150 mimics and pcDNA3.1/PDCD4, respectively.
A–B, PDCD4 were measured by western blot for each group of transfected HeLa and SiHa cells.
C–H, Cell proliferation by MTT assays (C,D), migration capacity by transwell assays(E,G), invasion capacity by Matrigel transwell assays(F,H),*P < 0.05.
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