MicroRNA-214-3p inhibits proliferation and cell cycle progression by targeting MELK in hepatocellular carcinoma and correlates cancer prognosis
Li et al. Cancer Cell Int
MicroRNA-214-3p inhibits proliferation and cell cycle progression by targeting MELK in hepatocellular carcinoma and correlates cancer prognosis
Yue Li 1
You Li 0
Yao Chen 1
Qian Xie 1
Ningning Dong 1
Yanjun Gao 1
Huan Deng 1
Chunhua Lu 0
Suihai Wang 1
0 Department of Biotechnology, College of Life Science and Technology, Guangxi University , No. 100, Daxue Road, Nanning 530004, Guangxi Province , China
1 Institute of Antibody Engineering, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University , No. 1838, Guangzhou Avenue North, Guangzhou 510515 , China
Background: MicroRNAs are considered as potential regulators in various biological pathways and contribute to the diagnosis and prognosis of cancers. MicroRNA-214-3p (miR-214-3p) was proved to be correlated with various cancers in recent studies. However, the biological functions of miR-214-3p in hepatocellular carcinoma (HCC) and its association with the prognosis of HCC after liver transplantation are still unevaluated. Here we intended to elucidate the functional implication of miR-214-3p in regulation of cell proliferation and apoptosis and its potential prediction of clinical prognosis of HCC patients. Methods: Expressions of miR-214-3p in 98 HCC patients and three HCC cell lines were detected by quantitative reverse transcription PCR (qRT-PCR) to explore the association of miR-214-3p expression and clinicopathological characteristics. The effects of miR-214-3p on cell proliferation and apoptosis were examined by proliferation and flow cytometry assay, respectively. The direct target gene of miR-214-3p was also detected by luciferase reporter assay. Results: The effects of miR-214-3p on cell proliferation and apoptosis were examined by proliferation and flow cytometry assay, respectively. The direct target gene of miR-214-3p was also detected by luciferase reporter assay. The results showed that miR-214-3p expression was downregulated in primary HCC samples compared with normal liver tissues, and was decreased in HCC recurrence species compared with non-recurrence controls (P = 0.001). Low miR214-3p level was associated with poor overall survival (OS) (Log rank P = 0.003) and recurrence-free survival (RFS) (Log rank P = 0.007). Moreover, miR-214-3p precursor transfection resulted in decreased cell proliferation, cell cycle arrest at G1 phase, and enhanced cell apoptosis in HepG2 and HUH-7 cells. Further investigation showed that miR-214-3p could regulate its target gene maternal embryonic leucine zipper kinase (MELK) by directly binding to MELK-3′-UTR. Conclusions: miR-214-3p suppresses HCC progression by directly down-regulating MELK expression, indicating a potential therapeutic target for the treatment and prognosis of HCC patients.
microRNA-214-3p; MELK; Hepatocellular carcinoma; Recurrence; Proliferation; Apoptosis; Cell cycle arrest
Hepatocellular carcinoma (HCC) is the third leading
cause of cancer mortality worldwide with poor survival
and unsatisfied prognosis [
]. Over decades, the
incidence of HCC has been dramatically increased
especially in hepatitis B or C virus (HBV or HCV) infection
induced phenotype [
]. Until now, surgical hepatic
resection and liver transplantation are still the main curative
treatment for HCC patients. Although great advances
have been made in treatment and diagnosis, the
prognosis of HCC remains limited, with its survival rates
under 20% at 5 years [
]. Therefore, it’s an urgent need
to understand the molecular mechanisms responsible for
the pathogenesis of HCC and to identify effective
treatment strategies. However, tumor recurrence rates remain
a major concern for the exhibition of active hepatitis or
cirrhosis in surrounding non-tumor liver tissues, even in
patients who have received curative treatments [
better understanding of the molecular mechanisms that
can distinguish progressive from non-progressive HCC
is indispensable for exploring novel prognostic markers
and therapeutic targets which may guide the surveillance
after liver transplantation.
Recent evidences support that microRNAs (miRNAs)
serve as potential indicators for diagnosis and
prognosis of cancers [
]. miRNAs are small noncoding RNAs,
which contain 20 ~ 23 nucleotides, processed from
primiRNAs and contribute to post-transcriptional
regulation of target gene expression through binding directly
to the specific sequences of target genes’ 3′-UTRs [
Researches show that miRNAs have effects on cell
proliferation, migration, and apoptosis via making a
difference in the stability or translation of target mRNA.
Additionally, miRNAs are considered as crucial
participators in tumor progression through influencing
multiple biological functions and pathways [
Previous study has demonstrated that miR-214-3p is
correlated with tumor onset and progression [
]. It has been
reported that miR-214-3p is downregulated in HCC
tissues and closely related to fibrotic stages [
however, the biological functions of miR-214-3p in HCC
and its position in HCC prognosis after transplantation
In our current study, the expression of miR-214-3p
in the formalin-fixed paraffin-embedded (FFPE) tumor
tissues from HCC patients was detected, and the
correlation of miR-214-3p expression with lymph node
metastasis, recurrence, pathological T stage, and age
was analyzed as well. Moreover, a further insight into the
function of miR-214-3p in regulating HCC cell
proliferation, cell cycle, and apoptosis was gained by
overexpressing miR-214-3p in human HCC cells.
Patients and tissue samples
A cohort of 98 patients undergoing liver transplantation
for HCC was obtained with their follow-up data from the
Nanfang Hospital of Southern Medical University from
January 2006 to November 2011. All the patients were
followed until December 2011. The median
recurrencefree period was 12 months for patients with HCC
recurrence compared to 65 months for patients without HCC
recurrence. All of these 98 patients enrolled in this study
met the transplantation criteria for HCC [
samples were from the paraffin embedded archival tissue
blocks and the normal liver tissues were from the liver
hemangioma resection. The clinicopathological
parameters of patients with HCC were summarized in Table 1.
Informed consents from all patients were provided
according to the protocols approved by the Institutional
Review Boards of the Nanfang Hospital of Southern
Cell culture and transfection
HepG2, HUH-7, SNU398 and L-O2 cell lines used in this
study were purchased from the ATCC (Manassas, VA).
All cells then were cultured in EMEM and supplemented
with 10% (v/v) heat-inactivated fetal bovine serum (FBS,
Gemini Bio-Products, Sacramento, CA) and antibiotics
(98 U/ml penicillin and 98 μg/ml streptomycin) at 37 °C
in a humidified atmosphere of 5% CO2. Double-stranded
RNAs that mimic endogenous precursor miR-214-3p
(Invitrogen-Life Technologies, Carlsbad, CA) as well as
negative oligonucleotide control was transfected into
cells using Oligofectamine (Thermo Scientific, Waltham,
MA) according to the manufacturer’s instruction.
RNA isolation and Taqman real‑time PCR
Total RNA was isolated and then reverse transcribed to
cDNA with the stem-loop RT primer for miR-214-3p.
miR-214-3p expression was normalized and quantificated
using U6 small RNA as an internal control. For
miR214-3p analysis, primers were 5′-GCATCCTGCCTCC
ACATGCAT-3′ and 5′-GCGCTGAGGAATAATAG
AGTATGTAT-3′. PCR primers for the internal control
U6 were 5′-TGACTTCCAAG TACCATCGCCA-3′ and
relative expression levels were calculated using the 2−ΔΔCt
method. All the experiments were run in triplicate.
Cell proliferation assay
Cell proliferation assay was carried out using cell Titer 96
Aqueous one Solution Cell Proliferation Assay (Promega,
Madison, WI) follow in the manufacturer’s protocol.
Three independent experiments were done.
CA). Finally, DNA histograms were analyzed with
modified software. Three independent experiments were done.
A total of 5 × 105 cells were harvested and centrifuged
for apoptotic evaluation. Propidium iodide (BD
Bioscience) and the fluorescein isothiocyanate-conjugated
(FITC) anti-human Annexin V Apoptosis Detection Kit
I (BD Pharmingen) was used to characterize cells
according to the manufacturer’s instructions. Labeled cells were
detected using the fluorescence activated cell sorting
(FACS) Aria II Cell Sorter System (BD Biosciences),
followed by data analysis using the Diva program (BD
Biosciences). Three independent experiments were done.
Luciferase activity assay
For luciferase reporter assay, HEK293T cells were
cultured in 48-well plates and then cotransfected with 10 ng
pGL3 cm-MELK-3′ UTR-Wt or pGL3 cm-MELK-3′
UTR-Mut, 30 pmol of miR-214-3p precursor or NC
oligonucleotides, and 2 ng of pRL-TK (Ruibo, Guangzhou,
China). After transfection for 72 h, cells were collected
separately and then analyzed following the
Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI).
The data were presented as relative luciferase activity.
Three independent experiments were done.
Immunohistochemical (IHC) staining
Briefly, before antigen retrieval in citrate buffer, tissue
sections were dewaxed and subsequently rehydrated in
graded series of ethanols. After that, the sections were
incubated overnight with antibody against MELK
(Epitomics, Burlingame, USA; 1:200) at 4 °C, followed by
incubation with an HRP-conjugated secondary antibody and
DAB (Dako, Carpenteria, CA). DAB was used for color
development, compared with dark brown staining was
Western blot analysis
Cells were harvested and lysed in lysis buffer
supplemented with proteinase inhibitor cocktail on ice for
20 min. Cell lysates were resolved by SDS-PAGE and
transferred to PVDF membranes (Millipore). The
membranes were blocked for 1 h in 5% non-fat dry milk and
incubated with primary MELK (Epitomics, Burlingame,
USA or GAPDH (Cell signaling technology, Danvers,
USA) antibodies at 4 °C overnight. Then the membranes
were incubated with HRP-conjugated secondary
antibodies and detected with ECL Plus (Millipore).
* Unpaired student t-test; #Chi square test; ∆ Fisher’s exact test
Cell cycle analysis
HepG2 and HUH-7 cells were collected in the log phase
of growth and incubated for 24 h. Then the cells were
trypsinized, washed with PBS twice, and fixed overnight
in cold 75% ethanol at 4 °C. After that, the fixed cells
were stained with propidium iodide, follow by
examination using flow cytometer (BD Biosciences, San Jose,
The SPSS version 17.0 (SPSS Inc. Chicago, IL) was used
for statistical analysis in this study. Comparisons between
two groups were performed using Student’s t test.
Correlations between clinicopathologic characteristics and
immunohistochemical variables were analyzed using
Chi square test or Fisher’s exact test. The Kaplan–Meier
method was selected to graph survival curves. Log-rank
statistic was applied to calculate the differences between
the groups. The impact of prognostic factors on RFS and
OS were analyzed by Cox proportional hazard models. A
two-sided P value less than 0.05 was considered
Downregulation of miR‑214‑3p in primary HCC tissues
We first determined miR-214-3p expression in HCC
cell lines (HepG2, HUH-7 and SNU398) and a
normal hepatic cell line (L-O2). Our results showed that
miR214-3p exhibited decreased expressions in HCC cell lines
compared with L-O2 cells (Fig. 1a). To explore the clinical
significance of miR-214-3p in HCC, the expression of
miR214-3p was examined in HCC tissues. The results showed
that miR-214-3p expression in HCC tissues (n = 98) was
dramatically lower in tumor recurrence patients than in
non-recurrence patients (P = 0.020; Fig. 1b). Similarly,
a significant reduction of miR-214-3p expression was
observed in liver transplant recipients who died compared
with that in the survived patients (P = 0.003; Fig. 1c).
Association of miR‑214‑3p expression with clinicopathological variables
In this study, we found that the decreased
expression of miR-214-3p in HCC was closely link to various
pathologic parameters, including tumor size (P = 0.016),
multinodular HCC (P = 0.044), and vascular invasion
(P = 0.025, Table 1).
Correlation of miR‑214‑3p in HCC with tumor recurrence and poor prognosis
Kaplan–Meier analysis showed that decreased
miR214-3p expression was connected with shorter OS
(median survival 27 vs. 63 months, P = 0.003) and
RFS (median survival 32 vs. 53 months P = 0.007) of
liver transplant recipients (Fig. 2a and b). In
multivariate analysis using a continuous variable Cox
proportional hazards model, miR-214-3p expression was
found to be predictive of outcomes independent of
gender, age, and tumor stages (OS, P = 0.004; RFS,
P = 0.018).
Effects of Pre214‑3p transfection on cell proliferation
To uncover whether overexpression of miR-214-3p had
an effect on cell proliferation, HepG2 and HUH-7 cells
were transfected with miR-214-3p precursor
(Pre2143p) (Fig. 3a and b). Cell proliferation assay showed
that the proliferation rate was decreased in HepG2 and
HUH-7 cells with the inhibitory efficiencies were 42.5
and 36.2% after Pre214-3p transfection, respectively
(Fig. 3c). Cell cycle analysis showed that compared
with the control group, the percentages of G1-phase
cells were increased in Pre214-3p-transfected HepG2
(P = 0.005) and HUH-7 cells (P = 0.012), while the
percentages of S-phase cells were decreased both cell
lines (P = 0.007 and P = 0.018) (Fig. 3d and e).
Nevertheless, no statistical significance was found in the
percentages of G2/M phase cells in HepG2 or HUH-7 cells
compared with the control group (Fig. 3d and e). These
results suggested that overexpression of miR-214-3p
blocks cell cycle progression by inducing cell cycle
arrest at G1 phase.
Effects of Pre214‑3p transfection on apoptosis
Flow cytometry analysis was conducted to investigate
whether miR-214-3p could induce apoptosis of HCC
cells. Increasing apoptotic rates were observed in
Pre2143p-transfected HUH-7 (P = 0.012) and HepG cells
(P = 0.008), compared with the control group (Fig. 4 a
MiR‑214‑3p downregulates MELK expression by directly
targeting its 3′‑UTR
Moreover, potential targeted genes of miR-214-3p were
predicted using online databases, i.e. TargetScan, PicTar,
and miRanda. MELK was chosen for further
experimental validation due to its frequent overexpression detected
by the three databases and its unknown anti-apoptotic
function. Dual-luciferase reporter analysis showed that
enforced expression of miR-214-3p remarkably reduced
the luciferase activity of the reporter gene with the WT
construct rather than with the mutant MELK 3′-UTR
construct (Fig. 5a and b). Moreover, overexpression of
miR-214-3p in HepG2 and HUH-7 resulted in a reduced
MELK expression (Fig. 5c). Similarly, HCC sample with
decreased miR-214-3p showed higher MELK expression
(Fig. 5d). Together, these results suggest that miR-214-3p
downregulates MELK expression by targeting MELK
In recent decades, miRNAs have been considered as
contributive regulators involved in transcriptional
regulation, cell differentiation, tumorigenesis, and other
biological processes [
]. Globally aberrant miRNA
expression profiles of tumors have provided valuable
insights into the molecular pathways of oncogenesis
]. Nowadays, more than 2000 human miRNAs have
been reported as regulational factors in cell proliferation,
migration, and invasion of tumors [
]. Newly papers
indicated that upregulation of miR-214-3p was found
in breast cancer patients with osteolytic bone
metastasis, and a knock-in miR-214-3p remarkably increased
bone resorption by straightly targeting Traf3 to promote
osteoclast activity and bone-resorbing activity [
However, other studies demonstrated that miR-214-3p
was significantly downregulated in two esophageal
squamous cancer cell lines compared with esophageal
epithelial cells . Further investigations pointed out that
downregulation of miR-214-3p suppressed
chemoresistance in esophageal cancer cells by targeting both
survivin and CUG-BP1 [
]. The anti-apoptotic Bcl-2 family
member MELK/A1 is one of these PRDI-BF1/Blimp-1
target genes. MELK as a target of miR-214-3p provides
a novel perspective on the mechanisms underlying HCC
proliferation and resistance to apoptosis [
MELK expression was detected in the bone marrow
as well as in some other tissues [
]. Specifically, a
connection was found in clinical samples between the
MELK expression and the progression of
stomach carcinoma .
Our studies showed that miR-214-3p expression was
decreased in both HCC tissues and HCC cell lines, which
were consistent with previous results [
]. It has been
reported that miR-214-3p expression is strongly related
to with fibrotic stages [
]. Interestingly, in this study, we
found that miR-214-3p expression is also closely
associated with recurrence and living status of liver transplant
patients. Moreover, downregulation of miR-214-3p was
associated with poor survival and tumor recurrence
in HCC patients. Moreover, miR-214-3p restoration
inhibited cell cycle progression and accelerated
apoptosis in vitro. To our best knowledge, this is the first study
showing that miR-214-3p regulates cellular proliferation in
HCC cells and links to the prognosis of HCC. In all, this
study shown that miR-214-3p was decreased in HCC
tissues and the expression level of miR-214-3p might be a
significant prognostic marker for HCC patients. Based on
gain-of-function approach, it is suggested that miR-214-3p
could remarkably block HCC cell proliferation and induce
apoptosis in vitro by directly targeting MELK 3′-UTR.
Collectively, our data not only demonstrates novel
insights regarding miR-214-3p function and the potential
mechanisms of HCC cell proliferation, but also indicates
a possible regulation pathway for MELK and a potential
therapeutic strategy for HCC treatment.
HCC: hepatocellular carcinoma; miR-214-3p: microRNA-214-3p; qRT-PCR:
quantitative reverse transcription PCR; OS: overall survival; RFS: recurrence-free
survival; MELK: maternal embryonic leucine zipper kinase; FITC: fluorescein
isothiocyanate-conjugated; FACS: fluorescence activated cell sorting.
LY carried out the experiments, and also revised the manuscript. LY and CY
carried out the experiments and drafted the manuscript. XQ, DNN, DH and GYJ
carried out the experiments. LCH participated in the design of the study and
performed the statistical analysis. WSH conceived of the study, and
participated in its design and coordination and helped to draft the manuscript. All
authors read and approved the final manuscript.
The authors thank statistical assistance provided by the Department of
Laboratory Medicine, The Second Hospital of Hebei Medical University.
The authors declare that they have no competing interests.
Availability of data and materials
Please contact author for data requests.
Consent for publication
Informed consent was obtained from all individual participants included in
Ethics approval and consent to participate
Ethical approval for this study was obtained from the institutional review
board of the Nanfang Hospital of Southern Medical University.
This study was supported by the National Natural Science Foundation of
China (No. 81201663) and the National Natural Science Foundation of China
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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