- Email: [email protected]
miR-613 inhibits the growth and invasiveness of human hepatocellular carcinoma via targeting DCLK1
Accepted Manuscript miR-613 inhibits the growth and invasiveness of human hepatocellular carcinoma via targeting DCLK1 Wenyao Wang, Hongfei Zhang, Lichao Wang, Shaojun Zhang, Miao Tang PII:
S0006-291X(16)30488-0
DOI:
10.1016/j.bbrc.2016.04.003
Reference:
YBBRC 35598
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 30 March 2016 Accepted Date: 1 April 2016
Please cite this article as: W. Wang, H. Zhang, L. Wang, S. Zhang, M. Tang, miR-613 inhibits the growth and invasiveness of human hepatocellular carcinoma via targeting DCLK1, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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miR-613
inhibits
the
growth
and
invasiveness
of
human
hepatocellular carcinoma via targeting DCLK1
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Wenyao Wang1,*, Hongfei Zhang1, Lichao Wang1, Shaojun Zhang1, and Miao Tang1
Department of General Surgery, The Second Hospital of Hebei Medical University,
*
Corresponding author.
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Shijiazhuang, China
Department of General Surgery, The Second Hospital of Hebei Medical university, Shijiazhuang 050000, China
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Tel.: +86-0311-66007330 Fax: +86-0311-66007334
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Email: [email protected]
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Abstract
microRNAs (miRNAs) play key regulatory roles in various biological processes. In
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this study, we aimed to determine the expression and biological roles of miR-613 in hepatocellular carcinoma (HCC). Compared with non-cancerous liver tissues, miR-613 was significantly downregulated in HCC tissues. Ectopic expression of
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miR-613 significantly suppressed the proliferation and invasion of Hep3B and
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SMMC-7721 HCC cells. Bioinformatic and luciferase reporter analysis identified doublecortin-like kinase 1 (DCLK1) as a direct target of miR-613. Overexpression of miR-613 inhibited the expression of DCLK1 in HCC cells. There was a significant inverse correlation between miR-613 and DCLK1 protein expression in HCC samples.
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Small interfering RNA-mediated silencing of DCLK1 phenocopied the suppressive effects of miR-613 in HCC cells. Rescue experiments demonstrated that co-transfection of DCLK1 lacking the 3'-untranslated region partially prevented
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miR-613-induced suppression of HCC cell proliferation and invasion. In vivo studies
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confirmed that miR-613 overexpression retarded the growth of Hep3B xenograft tumors in nude mice, coupled with a reduction in the percentage of Ki67-positive tumor cells and DCLK1 protein expression. In conclusion, we provide first evidence for the suppressive activity of miR-613 in HCC, which is causally linked to targeting of DCLK1. Restoration of miR-613 may provide a potential therapeutic strategy for HCC. Key words: dysregulation; growth; invasiveness; microRNA; target.
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Introduction
Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies
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worldwide [1]. Despite many efforts to develop novel agents and modalities [2,3], the long-term survival of HCC patients is still dismal. Understanding of the molecular
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therapeutic strategies for this disease.
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pathogenesis of HCC tumorigenesis is of significance in establishing effective
Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated protein that has been identified as a tumor stem cell marker [4,5]. DCLK1 is upregulated in many solid tumors such as pancreatic cancer [6], colorectal cancer [7], and HCC [8]. This protein
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plays an important role in tumorigenesis [9,10]. It has been documented that DCLK1 promotes intestinal tumorigenesis in Apc mutant intestinal tumors [9]. Inhibition of DCLK1 accounts for XMD8-92-mediated suppression of pancreatic tumor xenograft
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growth [10]. Targeting DCLK1 can also impair the growth of HCC xenograft tumors
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[8]. These studies suggest DCLK1 as a promising target for anticancer treatment.
microRNAs (miRNAs) are small, endogenous, non-coding RNA molecules, and play important roles in numerous biological processes, such as proliferation, differentiation, migration, apoptosis, and transformation [11]. Some miRNAs have been found to be involved in tumor development and progression by acting as potential oncogenes or tumor suppressors [12,13]. For instance, miR-1180 overexpression can render HCC
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ACCEPTED MANUSCRIPT cells resistant to apoptosis via activation of nuclear factor-κB signaling pathway [12]. miR-149 can repress metastasis of HCC by targeting actin-regulatory proteins [13]. Recent studies have shown that miR-613 suppresses the proliferation and invasion of
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ovarian cancer cells [14] and prostate cancer cells [15], suggesting that this miRNA may represent a novel tumor suppressor. However, the expression and biological roles
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of miR-613 in HCC are poorly investigated.
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In this study, we examined the expression of miR-613 in HCC and matched normal liver tissues, as well as a panel of HCC cells. The functions of miR-613 in HCC cell proliferation, invasion, and tumorigenesis are determined. The potential target(s) of
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miR-613 in HCC are also identified.
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Materials and methods
Cells and tissues
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Human HCC cell lines (HepG2, Hep3B, Sk-Hep1, and SMMC-7721) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal liver epithelial cells THLE-3 were purchased from the American Type Cell Culture
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Collection (ATCC, Rockville, MD, USA). All cells were cultured in Dulbecco’s
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modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2.
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A total of 38 pairs of HCC and adjacent normal liver tissues were collected from HCC patients who underwent tumor resection in the Second Hospital of Hebei Medical University (Shijiazhuang, China) between December 2013 and September 2014. The
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patients did not receive any anticancer treatment before surgery. All tissues were
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pathologically confirmed. Fresh tissue samples were snap-frozen in liquid nitrogen and stored at -80°C until RNA or protein extraction. Written informed consent for research purposes was obtained from each patient. The study protocol was approved by the Ethical Committee of the Second Hospital of Hebei Medical University.
Plasmids, small interfering RNAs (siRNAs), and transfections A DNA fragment containing human miR-613 precursor was amplified by PCR and
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ACCEPTED MANUSCRIPT cloned into pmR-ZsGreen1 vector (Clontech, Mountain View, CA, USA), which can coexpress a fluorescent protein and an miRNA sequence. The DCLK1 open reading frame without the 3'-UTR region was amplified by PCR with human DCLK1 cDNA
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(OriGene, Rockville, MD, USA) as a template and inserted into pcDNA3.1(+) expression vector (Invitrogen). Wild-type DCLK1 3'-UTR were obtained by PCR and cloned into the pmirGLO vector (Promega, Madison, WI, USA) immediately of
the
firefly
luciferase
gene,
yielding
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downstream
the
plasmid
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pmirGLO-DCLK1-3'-UTR-wt. The mutant construct of DCLK1 3'-UTR was generated using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) and also
inserted
into
the
pMIR-reporter
vector,
yielding
the
plasmid
pmirGLO-DCLK1-3'-UTR-mut. All of the constructs were verified by DNA
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sequencing. siRNA duplexes targeting DCLK1 and negative control siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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Hep3B and SMMC-7721 cells were seeded onto 12-well plates and allowed to grow
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to about 75% confluence. Cells were transfected with pmR-ZsGreen1-miR-613 or empty vector using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Transfected cells were selected in the presence of 800 µg/mL of G418 (Invitrogen) for 2 weeks before assessment of tumorigenesis and invasiveness. For downregulation of DCLK1, cells were transiently transfected with DCLK1 or control siRNA (50 nM for each). During rescue experiments, cells were co-transfected with 50 nM miR-613 mimic or negative control miRNA (Applied Biosystems, Foster City,
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Luciferase reporter assay
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HEK293 cells were seeded onto 24-well plates and allowed to grow to about 80% confluence. Cells were co-transfected with pmirGLO-DCLK1-3'-UTR-wt or pmirGLO-DCLK1-3'-UTR-mut along with miR-613 mimic or negative control
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miRNA using Lipofectamine 2000. A renilla luciferase-expressing plasmid pRL-TK
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(Promega) was co-transfected and used as an internal control. At 24 h posttransfection, cells were harvested and luciferase activity was determined using the Dual Luciferase Reporter Assay Kit (Promega). The results are expressed as relative luciferase activity
qRT-PCR analysis
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(firefly luciferase/renilla luciferase).
Total RNA was isolated from tissues or cells using TRIzol reagent (Invitrogen)
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according to the manufacturer’s instructions. For detection of mature miR-613, total
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RNA was subjected to reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR analysis of miR-613 expression was carried out using TaqMan MicroRNA assay kits (Applied Biosystems). Results were normalized to U6 snRNA using the comparative threshold cycle (Ct) method [16]. For measurement of DCLK1 mRNA, reverse transcription was done using the Prime-Script RT reagent kit (TaKaRa, Otsu, Japan). qRT-PCR was performed using SYBR Green Master Mix (Solarbio, Beijing, China), with the specific primers:
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ACCEPTED MANUSCRIPT DCLK1
forward
5'-TGAAGGGTACGCTCCTCAGT-3', β-actin
5'-GCTACACTCTGACCGCATGA-3'; 5'-CTTAGTTGCGTTACACCCTTTCTTG-3',
reverse forward reverse
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5'-CTGTCACCTTCACCGTTCCAGTTT-3'. Relative DCLK1 mRNA levels were determined using the comparative Ct method after normalization to β-actin.
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Western blot analysis
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Cell or tissue lysates were prepared with RIPA Lysis buffer (Beyotime, Haimen, China) supplemented with the protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Protein samples were separated
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by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to to a nitrocellulose membrane. After blocking with 5% fat-free milk, the membrane was probed with primary anti-DCLK1 (dilution 1:300; Sigma, St. Louis, MO, USA) and
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anti-β-actin (dilution 1:2,000; Santa Cruz Biotechnology) antibody. After washing, the
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membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The signal was visualized using the ECL detection system (Amersham Pharmacia Biotech, Duebendorf, Switzerland) and quantified by densitometry using Quantity One software (Bio-Rad, Hercules, CA, USA).
Cell proliferation assay Cells transfected with indicated constructs were seeded in sextuplicate onto 96-well
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brief, cells were incubated with MTT at a final concentration of 0.5 mg/mL (Sigma) for 4 h. The supernatant was discarded, and the precipitated formazan was dissolved
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in dimethyl sulfoxide. Absorbance was measured at 570 nm with a microplate reader.
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Transwell invasion assay
Cells transfected with indicated constructs in serum-free medium (1 × 105 cells) were plated onto the 24-well upper chamber with a membrane that was pre-coated with Matrigel (100 µg per well; BD Biosciences, San Jose, CA, USA). In the lower
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chamber, fresh DMEM with 10% FBS was added. After incubation for 24 h at 37°C, the cells in the upper chamber were carefully removed with a cotton swab. Invaded cells were fixed with 4% formaldehyde, stained with 0.5% crystal violet, and counted
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under a microscope.
Tumorigenesis in vivo
Four-week old, male BALB/c (nu/nu) mice purchased from the Experimental Animal Center of Hebei Medical University were kept under controlled temperature and lighting conditions (a 12-h light/dark cycle) and were given food and water ad libitum. Hep3B cells stably transfected with miR-613-expressing plasmid or vector (2 × 106 cells per mouse) were implanted subcutaneously into the right flank of mice (4 mice
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Western blot analysis.
Immunohistochemistry for Ki67
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Paraffin-embedded tissue sections (5 µm) were deparaffinized, rehydrated, and
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microwave-heated in an antigen retrieval buffer. After inactivation of endogenous peroxidase, sections were incubated with anti-Ki67 antibody (dilution 1:600; Beckman Coulter, Fullerton, CA, USA). After washing, sections were incubated with a secondary biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) and
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then with Avidin-Biotin Complex (Vector Laboratories). 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) was then added for development and the sections were counterstained with hematoxylin (Sigma). The percentage of Ki67-positive cells
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was estimated after counting at least 800 tumor cells under a microscope.
Statistical analysis
Continuous data are expressed as the mean ± standard error (S.E.) and analyzed by the Student’s t test or one way analysis of variance followed by the Tukey's post hoc test. Correlation analysis was performed using the Spearman method. Statistical analysis was determined with the SPSS software, version 19.0 (SPSS, Chicago, IL, USA). Differences were considered to be statistically significant when P < 0.05.
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Results
miR-613 inhibits the aggressive phenotypes of HCC cells
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The expression of miR-613 in HCC and adjacent non-cancerous liver tissues was examined using qRT-PCR analysis. As shown in Fig. 1A, HCC tissues had significantly (P = 0.012) lower levels of miR-613, compared to adjacent liver tissues.
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In accordance with clinical data, the expression level of miR-613 was significantly
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reduced in all the HCC cell lines tested, relative to normal cells (Fig. 1B).
To determine the biological relevance of miR-613 downregulation in HCC, HCC cells were transfected with miR-613-expressing plasmid and tested for cell proliferation
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and invasion. MTT assay revealed that miR-613-overexpressing HCC cells had significantly (P < 0.05) lower proliferation capacity than control cells (Fig. 1C). Transwell invasion assay demonstrated that ectopic expression of miR-613
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significantly (P < 0.05) inhibited the invasion of Hep3B and SMCC-7721 cells
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through a matrigel-coated chamber (Fig. 1D). These results indicate that miR-613 suppresses the aggressive phenotypes of HCC cells.
DCLK1 is a direct target of miR-613 in HCC Next, we attempted to identify the target genes of miR-613 that may be involved in HCC growth and progression. Bioinformatic analysis suggested that DCLK1 mRNA harbored a putative miR-613 binding site in its 3'-UTR (Fig. 2A). To verify whether
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ACCEPTED MANUSCRIPT miR-613 directly regulates DCLK1 expression via interaction with its 3'-UTR, we co-transfected
wild-type
or
mutant
DCLK1
3'-UTR
reporter
along
with
miR-613-expressing plasmid or vector. The luciferase activity of wild-type reporter
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was significantly inhibited in the presence of miR-613 (Fig. 2B). However, miR-613-mediated repression of the reporter expression was compromised by
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mutation of the miR-613 binding site in the DCLK1 3'-UTR.
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To confirm that miR-613 modulates endogenous DCLK1 expression in HCC cells, we measured the protein levels of DCLK1 in HCC cells expressing ectopic miR-613. The results showed that the DCLK1 protein level in Hep3B and SMMC-7721 cells was reduced by 78% and 56%, respectively, upon overexpression of miR-613 (Fig. 2C).
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Analysis of DCLK1 protein in primary human HCCs showed a significant increase (P = 0.006) in tumors relative to surrounding liver tissues (Fig. 2D). Moreover, there was a significant negative correlation between miR-613 and DCLK1 protein expression in
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HCC samples (r = -0.432, P = 0.002; Fig. 2D). These results suggest that miR-613
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directly targets DCLK1 expression in HCC.
miR-613 suppresses the proliferation and invasion of HCC cells via repression of DCLK1 expression
Next, we checked whether miR-613 modulates the aggressive phenotypes of HCC cells through downregulation of DCLK1. To this end, we knocked down DCLK1 or co-transfected DCLK1-expressing plasmid lacking the 3'-UTR together with miR-613.
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ACCEPTED MANUSCRIPT The delivery of DCLK1 siRNA led to an effective downregulation of endogenous DCLK1 in HCC cells, as determined by qRT-PCR analysis (Fig. 3A). Knockdown of DCLK1 significantly (P < 0.05) reduced the proliferation (Fig. 3B) and invasion (Fig.
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3C) of HCC cells, compared to the transfection of control siRNA. Rescue experiments demonstrated that co-transfection of DCLK1 lacking 3'-UTR significantly abolished miR-613-induced suppression of HCC cell proliferation (Fig. 3B) and invasion (Fig.
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3C). Collectively, these data indicate that miR-613 exerts its suppressive effects on
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HCC cells via targeting of DCLK1.
miR-613 impairs HCC tumorigenesis in vivo
To further confirm the biological role of miR-613 in vivo, a subcutaneous exnograft
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tumor model with miR-613-overexpressing and control Hep3B cells was generated in nude mice. From 2 weeks after cell implantation, mean tumor volume was significantly (P < 0.05) lower in the miR-613-overexpressing group than that in the
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control group (Fig. 4A). At the end of the experiment (4 weeks after cell implantation),
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xenograft tumors were excised and weighed. miR-613-overexpressing xenograft tumors had ~45% lower weights than control tumors (Fig. 4B). Immunohistochemical analysis with Ki67 staining demonstrated that the percentage of Ki67-positive proliferating cells was ~50% lower in miR-613 tumors than that in control tumors (Fig. 4C). Additionally, Western blot analysis confirmed the downregulation of DCLK1 in miR-613 tumors (Fig. 4D). These results demonstrate that miR-613 hampers subcutaneous HCC tumor growth.
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Discussion
Although thousands of miRNAs have been detected in humans [17], only a minority
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of them have been functionally characterized. miR-613 were originally reported to be implicated in lipid metabolism in HepG2 cells [18] and macrophages [19]. Recent evidence indicates a link between miR-613 and tumorigenesis [14,15,20].
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Dysregulation of miR-613 has been found in several types of cancers such as
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non-small cell lung cancer [20] and prostate cancer [15]. Downregulation of miR-613 was also detected in HCC tissues, compared to adjacent non-cancerous tissues. Li et al [20] reported that miR-613 induces a cell cycle arrest in non-small cell lung cancer cells. Consistently, we showed that enforced expression of miR-613 hampered the
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proliferation of HCC cells. Moreover, ectopic expression of miR-613 was found to impair the invasiveness of HCC cells. Altogether, these results provide first evidence
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that miR-613 acts as a tumor suppressor in HCC.
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To further explore the therapeutic potential of overexpression of miR-613 in HCC, we examined the role of miR-613 in the growth of HCC subcutaneous xenograft tumors in nude mice. In accordance with in vitro data, miR-613 overexpression retarded the growth of Hep3B xenograft tumors, which was accompanied by a significant decline in the percentage of Ki67-positive tumor cells. Similarly, the delivery of miR-613 mimic inhibited in vivo tumor growth in a xenograft model of non-small cell lung cancer [20]. These findings highlight the therapeutic significance of restoration of
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ACCEPTED MANUSCRIPT miR-613 expression in the treatment of cancers.
Several direct targets of miR-613 have been identified [19,20]. For instance, miR-613
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has been reported to modulate cholesterol efflux via targeting LXRα and ABCA1 in THP-1 macrophages [19]. Another study documented that miR-613 exerts its anticancer effects against non-small cell lung cancer by targeting CDK4 [20]. In this
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study, we identified a novel direct target of miR-613, DCLK1. In silico and
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experimental studies revealed that miR-613 had the ability to negatively regulate the expression of DCLK1, which was causally linked to its interaction with a site in the DCLK1 3'-UTR. Mutation of this site impaired miR-613-mediated repression of DCLK1 3'-UTR reporter expression. DCLK1 is frequently upregulated in HCC [8]
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and contributes to tumorigenesis [9,10,21]. Targeting DCLK1 has been shown to suppress the growth of HCC xenograft tumors [8]. The studies, combined with our findings, suggest that DCLK1 may be a functional target of miR-613 in HCC.
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Interestingly, knockdown of DCLK1 yielded similar suppressive effects on HCC cells
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to those obtained with overexpression of miR-613. Most importantly, co-transfection of DCLK1 lacking 3'-UTR significantly reversed the suppression of HCC cell proliferation and invasion by miR-613 overexpression. Additionally, there was a negative correlation between miR-613 and DCLK1 expression in HCC tissues. Taken together, targeting DCLK1 accounts for the suppressive activity of miR-613 in HCC. However, the fact that restoration of DCLK1 could not completely reverse the phenotypes induced by miR-613 suggested the presence of other functional mediators
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ACCEPTED MANUSCRIPT of miR-613 in the regulation of HCC cell phenotypes.
In this study, we demonstrated for the first time that miR-613 suppresses HCC growth
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and invasiveness, at least partially through targeting DCLK1. Restoration of miR-613
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may represent a potential approach to prevent the growth and progression of HCC.
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Figure legends
Fig. 1. miR-613 inhibits the proliferation and invasion of HCC cells. (A) qRT-PCR
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analysis of miR-613 in 38 pairs of HCC and non-cancerous liver tissues. (B) Measurement of miR-613 in a panel of HCC cell lines and THLE-3 normal liver epithelial cells by qRT-PCR analysis. *P < 0.05 vs. THLE-3 cells. (C) MTT assay was
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done to measure the viability of HCC cells transfected with control miRNA or
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miR-613-expressing plasmid after incubation for indicated times. (D) HCC cells transfected with control miRNA or miR-613-expressing plasmid were tested for invasiveness in Matrigel-coated transwells. Bar graphs represent the results from three
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independent experiments. *P < 0.05 vs. control.
Fig. 2. DCLK1 is a direct target of miR-613 in HCC. (A) A potential target site for miR-613 in the 3′-UTR of human DCLK1 mRNA, as predicted by the program
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Targetscan. To disrupt the interaction between miR-613 and DCLK1 mRNA, the
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target site was mutated. (B) Luciferase reporter assays performed in HEK293 cells co-transfected with miR-613 or control miRNA (Control) and wild-type or mutant DCLK1 3′-UTR constructs. (C) HCC cells were transfected with miR-613 or control miRNA, and tested for the protein expression of DCLK1. *P < 0.05 vs. control. (D) Spearman correlation analysis of the relationship between DCLK1 protein and miR-613 expression in 38 HCC specimens.
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ACCEPTED MANUSCRIPT Fig. 3. miR-613 suppresses the proliferation and invasion of HCC cells via repression of DCLK1 expression. (A) qRT-PCR analysis of DCLK1 mRNA levels in HCC cells transfected with indicated constructs. C-siRNA: control siRNA. (B) MTT assay was
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done to measure the viability of HCC cells transfected with different constructs after incubation for indicated times. (C) HCC cells transfected with indicated constructs were tested for invasiveness in Matrigel-coated transwells. Bar graphs represent the
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cells co-transfected with miR-613 and empty vector.
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results from three independent experiments. *P < 0.05 vs. C-siRNA; #P < 0.05 vs.
Fig. 4. miR-613 impairs HCC tumorigenesis in vivo. Hep3B cells stably transfected with miR-613 or control miRNA were implanted into nude mice and tumor growth
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and gene expression were assessed. (A) Tumor volume was measured every week after cell injection. (B) At the end of the experiment, animals were sacrificed and tumors were excised and weighted. Inserts show a representative gross morphology of
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tumors. (C) Tumor samples were subjected to Ki67 staining. Representative images
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are shown in top panels. Scale bar = 100 µm. The percentage of Ki67-positive tumor cells was determined. (D) Western blot analysis of DCLK1 protein in tumors. Bar graphs represent the results from three independent experiments. *P < 0.05 vs. control.
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Highlights
miR-613 inhibits the aggressive phenotypes of HCC cells.
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miR-613 impairs HCC tumorigenesis in vivo.
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DCLK1 is a direct target of miR-613 in HCC
S0006-291X(16)30488-0
DOI:
10.1016/j.bbrc.2016.04.003
Reference:
YBBRC 35598
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 30 March 2016 Accepted Date: 1 April 2016
Please cite this article as: W. Wang, H. Zhang, L. Wang, S. Zhang, M. Tang, miR-613 inhibits the growth and invasiveness of human hepatocellular carcinoma via targeting DCLK1, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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miR-613
inhibits
the
growth
and
invasiveness
of
human
hepatocellular carcinoma via targeting DCLK1
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Wenyao Wang1,*, Hongfei Zhang1, Lichao Wang1, Shaojun Zhang1, and Miao Tang1
Department of General Surgery, The Second Hospital of Hebei Medical University,
*
Corresponding author.
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Shijiazhuang, China
Department of General Surgery, The Second Hospital of Hebei Medical university, Shijiazhuang 050000, China
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Tel.: +86-0311-66007330 Fax: +86-0311-66007334
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Email: [email protected]
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Abstract
microRNAs (miRNAs) play key regulatory roles in various biological processes. In
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this study, we aimed to determine the expression and biological roles of miR-613 in hepatocellular carcinoma (HCC). Compared with non-cancerous liver tissues, miR-613 was significantly downregulated in HCC tissues. Ectopic expression of
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miR-613 significantly suppressed the proliferation and invasion of Hep3B and
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SMMC-7721 HCC cells. Bioinformatic and luciferase reporter analysis identified doublecortin-like kinase 1 (DCLK1) as a direct target of miR-613. Overexpression of miR-613 inhibited the expression of DCLK1 in HCC cells. There was a significant inverse correlation between miR-613 and DCLK1 protein expression in HCC samples.
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Small interfering RNA-mediated silencing of DCLK1 phenocopied the suppressive effects of miR-613 in HCC cells. Rescue experiments demonstrated that co-transfection of DCLK1 lacking the 3'-untranslated region partially prevented
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miR-613-induced suppression of HCC cell proliferation and invasion. In vivo studies
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confirmed that miR-613 overexpression retarded the growth of Hep3B xenograft tumors in nude mice, coupled with a reduction in the percentage of Ki67-positive tumor cells and DCLK1 protein expression. In conclusion, we provide first evidence for the suppressive activity of miR-613 in HCC, which is causally linked to targeting of DCLK1. Restoration of miR-613 may provide a potential therapeutic strategy for HCC. Key words: dysregulation; growth; invasiveness; microRNA; target.
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Introduction
Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies
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worldwide [1]. Despite many efforts to develop novel agents and modalities [2,3], the long-term survival of HCC patients is still dismal. Understanding of the molecular
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therapeutic strategies for this disease.
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pathogenesis of HCC tumorigenesis is of significance in establishing effective
Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated protein that has been identified as a tumor stem cell marker [4,5]. DCLK1 is upregulated in many solid tumors such as pancreatic cancer [6], colorectal cancer [7], and HCC [8]. This protein
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plays an important role in tumorigenesis [9,10]. It has been documented that DCLK1 promotes intestinal tumorigenesis in Apc mutant intestinal tumors [9]. Inhibition of DCLK1 accounts for XMD8-92-mediated suppression of pancreatic tumor xenograft
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growth [10]. Targeting DCLK1 can also impair the growth of HCC xenograft tumors
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[8]. These studies suggest DCLK1 as a promising target for anticancer treatment.
microRNAs (miRNAs) are small, endogenous, non-coding RNA molecules, and play important roles in numerous biological processes, such as proliferation, differentiation, migration, apoptosis, and transformation [11]. Some miRNAs have been found to be involved in tumor development and progression by acting as potential oncogenes or tumor suppressors [12,13]. For instance, miR-1180 overexpression can render HCC
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ovarian cancer cells [14] and prostate cancer cells [15], suggesting that this miRNA may represent a novel tumor suppressor. However, the expression and biological roles
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of miR-613 in HCC are poorly investigated.
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In this study, we examined the expression of miR-613 in HCC and matched normal liver tissues, as well as a panel of HCC cells. The functions of miR-613 in HCC cell proliferation, invasion, and tumorigenesis are determined. The potential target(s) of
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miR-613 in HCC are also identified.
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Materials and methods
Cells and tissues
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Human HCC cell lines (HepG2, Hep3B, Sk-Hep1, and SMMC-7721) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal liver epithelial cells THLE-3 were purchased from the American Type Cell Culture
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Collection (ATCC, Rockville, MD, USA). All cells were cultured in Dulbecco’s
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modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2.
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A total of 38 pairs of HCC and adjacent normal liver tissues were collected from HCC patients who underwent tumor resection in the Second Hospital of Hebei Medical University (Shijiazhuang, China) between December 2013 and September 2014. The
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patients did not receive any anticancer treatment before surgery. All tissues were
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pathologically confirmed. Fresh tissue samples were snap-frozen in liquid nitrogen and stored at -80°C until RNA or protein extraction. Written informed consent for research purposes was obtained from each patient. The study protocol was approved by the Ethical Committee of the Second Hospital of Hebei Medical University.
Plasmids, small interfering RNAs (siRNAs), and transfections A DNA fragment containing human miR-613 precursor was amplified by PCR and
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ACCEPTED MANUSCRIPT cloned into pmR-ZsGreen1 vector (Clontech, Mountain View, CA, USA), which can coexpress a fluorescent protein and an miRNA sequence. The DCLK1 open reading frame without the 3'-UTR region was amplified by PCR with human DCLK1 cDNA
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(OriGene, Rockville, MD, USA) as a template and inserted into pcDNA3.1(+) expression vector (Invitrogen). Wild-type DCLK1 3'-UTR were obtained by PCR and cloned into the pmirGLO vector (Promega, Madison, WI, USA) immediately of
the
firefly
luciferase
gene,
yielding
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downstream
the
plasmid
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pmirGLO-DCLK1-3'-UTR-wt. The mutant construct of DCLK1 3'-UTR was generated using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) and also
inserted
into
the
pMIR-reporter
vector,
yielding
the
plasmid
pmirGLO-DCLK1-3'-UTR-mut. All of the constructs were verified by DNA
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sequencing. siRNA duplexes targeting DCLK1 and negative control siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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Hep3B and SMMC-7721 cells were seeded onto 12-well plates and allowed to grow
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to about 75% confluence. Cells were transfected with pmR-ZsGreen1-miR-613 or empty vector using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Transfected cells were selected in the presence of 800 µg/mL of G418 (Invitrogen) for 2 weeks before assessment of tumorigenesis and invasiveness. For downregulation of DCLK1, cells were transiently transfected with DCLK1 or control siRNA (50 nM for each). During rescue experiments, cells were co-transfected with 50 nM miR-613 mimic or negative control miRNA (Applied Biosystems, Foster City,
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Luciferase reporter assay
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HEK293 cells were seeded onto 24-well plates and allowed to grow to about 80% confluence. Cells were co-transfected with pmirGLO-DCLK1-3'-UTR-wt or pmirGLO-DCLK1-3'-UTR-mut along with miR-613 mimic or negative control
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miRNA using Lipofectamine 2000. A renilla luciferase-expressing plasmid pRL-TK
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(Promega) was co-transfected and used as an internal control. At 24 h posttransfection, cells were harvested and luciferase activity was determined using the Dual Luciferase Reporter Assay Kit (Promega). The results are expressed as relative luciferase activity
qRT-PCR analysis
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(firefly luciferase/renilla luciferase).
Total RNA was isolated from tissues or cells using TRIzol reagent (Invitrogen)
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according to the manufacturer’s instructions. For detection of mature miR-613, total
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RNA was subjected to reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR analysis of miR-613 expression was carried out using TaqMan MicroRNA assay kits (Applied Biosystems). Results were normalized to U6 snRNA using the comparative threshold cycle (Ct) method [16]. For measurement of DCLK1 mRNA, reverse transcription was done using the Prime-Script RT reagent kit (TaKaRa, Otsu, Japan). qRT-PCR was performed using SYBR Green Master Mix (Solarbio, Beijing, China), with the specific primers:
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forward
5'-TGAAGGGTACGCTCCTCAGT-3', β-actin
5'-GCTACACTCTGACCGCATGA-3'; 5'-CTTAGTTGCGTTACACCCTTTCTTG-3',
reverse forward reverse
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5'-CTGTCACCTTCACCGTTCCAGTTT-3'. Relative DCLK1 mRNA levels were determined using the comparative Ct method after normalization to β-actin.
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Western blot analysis
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Cell or tissue lysates were prepared with RIPA Lysis buffer (Beyotime, Haimen, China) supplemented with the protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Protein samples were separated
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by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to to a nitrocellulose membrane. After blocking with 5% fat-free milk, the membrane was probed with primary anti-DCLK1 (dilution 1:300; Sigma, St. Louis, MO, USA) and
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anti-β-actin (dilution 1:2,000; Santa Cruz Biotechnology) antibody. After washing, the
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membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The signal was visualized using the ECL detection system (Amersham Pharmacia Biotech, Duebendorf, Switzerland) and quantified by densitometry using Quantity One software (Bio-Rad, Hercules, CA, USA).
Cell proliferation assay Cells transfected with indicated constructs were seeded in sextuplicate onto 96-well
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brief, cells were incubated with MTT at a final concentration of 0.5 mg/mL (Sigma) for 4 h. The supernatant was discarded, and the precipitated formazan was dissolved
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in dimethyl sulfoxide. Absorbance was measured at 570 nm with a microplate reader.
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Transwell invasion assay
Cells transfected with indicated constructs in serum-free medium (1 × 105 cells) were plated onto the 24-well upper chamber with a membrane that was pre-coated with Matrigel (100 µg per well; BD Biosciences, San Jose, CA, USA). In the lower
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chamber, fresh DMEM with 10% FBS was added. After incubation for 24 h at 37°C, the cells in the upper chamber were carefully removed with a cotton swab. Invaded cells were fixed with 4% formaldehyde, stained with 0.5% crystal violet, and counted
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under a microscope.
Tumorigenesis in vivo
Four-week old, male BALB/c (nu/nu) mice purchased from the Experimental Animal Center of Hebei Medical University were kept under controlled temperature and lighting conditions (a 12-h light/dark cycle) and were given food and water ad libitum. Hep3B cells stably transfected with miR-613-expressing plasmid or vector (2 × 106 cells per mouse) were implanted subcutaneously into the right flank of mice (4 mice
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Western blot analysis.
Immunohistochemistry for Ki67
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Paraffin-embedded tissue sections (5 µm) were deparaffinized, rehydrated, and
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microwave-heated in an antigen retrieval buffer. After inactivation of endogenous peroxidase, sections were incubated with anti-Ki67 antibody (dilution 1:600; Beckman Coulter, Fullerton, CA, USA). After washing, sections were incubated with a secondary biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) and
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then with Avidin-Biotin Complex (Vector Laboratories). 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) was then added for development and the sections were counterstained with hematoxylin (Sigma). The percentage of Ki67-positive cells
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was estimated after counting at least 800 tumor cells under a microscope.
Statistical analysis
Continuous data are expressed as the mean ± standard error (S.E.) and analyzed by the Student’s t test or one way analysis of variance followed by the Tukey's post hoc test. Correlation analysis was performed using the Spearman method. Statistical analysis was determined with the SPSS software, version 19.0 (SPSS, Chicago, IL, USA). Differences were considered to be statistically significant when P < 0.05.
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Results
miR-613 inhibits the aggressive phenotypes of HCC cells
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The expression of miR-613 in HCC and adjacent non-cancerous liver tissues was examined using qRT-PCR analysis. As shown in Fig. 1A, HCC tissues had significantly (P = 0.012) lower levels of miR-613, compared to adjacent liver tissues.
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In accordance with clinical data, the expression level of miR-613 was significantly
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reduced in all the HCC cell lines tested, relative to normal cells (Fig. 1B).
To determine the biological relevance of miR-613 downregulation in HCC, HCC cells were transfected with miR-613-expressing plasmid and tested for cell proliferation
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and invasion. MTT assay revealed that miR-613-overexpressing HCC cells had significantly (P < 0.05) lower proliferation capacity than control cells (Fig. 1C). Transwell invasion assay demonstrated that ectopic expression of miR-613
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significantly (P < 0.05) inhibited the invasion of Hep3B and SMCC-7721 cells
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through a matrigel-coated chamber (Fig. 1D). These results indicate that miR-613 suppresses the aggressive phenotypes of HCC cells.
DCLK1 is a direct target of miR-613 in HCC Next, we attempted to identify the target genes of miR-613 that may be involved in HCC growth and progression. Bioinformatic analysis suggested that DCLK1 mRNA harbored a putative miR-613 binding site in its 3'-UTR (Fig. 2A). To verify whether
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ACCEPTED MANUSCRIPT miR-613 directly regulates DCLK1 expression via interaction with its 3'-UTR, we co-transfected
wild-type
or
mutant
DCLK1
3'-UTR
reporter
along
with
miR-613-expressing plasmid or vector. The luciferase activity of wild-type reporter
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was significantly inhibited in the presence of miR-613 (Fig. 2B). However, miR-613-mediated repression of the reporter expression was compromised by
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mutation of the miR-613 binding site in the DCLK1 3'-UTR.
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To confirm that miR-613 modulates endogenous DCLK1 expression in HCC cells, we measured the protein levels of DCLK1 in HCC cells expressing ectopic miR-613. The results showed that the DCLK1 protein level in Hep3B and SMMC-7721 cells was reduced by 78% and 56%, respectively, upon overexpression of miR-613 (Fig. 2C).
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Analysis of DCLK1 protein in primary human HCCs showed a significant increase (P = 0.006) in tumors relative to surrounding liver tissues (Fig. 2D). Moreover, there was a significant negative correlation between miR-613 and DCLK1 protein expression in
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HCC samples (r = -0.432, P = 0.002; Fig. 2D). These results suggest that miR-613
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directly targets DCLK1 expression in HCC.
miR-613 suppresses the proliferation and invasion of HCC cells via repression of DCLK1 expression
Next, we checked whether miR-613 modulates the aggressive phenotypes of HCC cells through downregulation of DCLK1. To this end, we knocked down DCLK1 or co-transfected DCLK1-expressing plasmid lacking the 3'-UTR together with miR-613.
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3C) of HCC cells, compared to the transfection of control siRNA. Rescue experiments demonstrated that co-transfection of DCLK1 lacking 3'-UTR significantly abolished miR-613-induced suppression of HCC cell proliferation (Fig. 3B) and invasion (Fig.
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3C). Collectively, these data indicate that miR-613 exerts its suppressive effects on
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HCC cells via targeting of DCLK1.
miR-613 impairs HCC tumorigenesis in vivo
To further confirm the biological role of miR-613 in vivo, a subcutaneous exnograft
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tumor model with miR-613-overexpressing and control Hep3B cells was generated in nude mice. From 2 weeks after cell implantation, mean tumor volume was significantly (P < 0.05) lower in the miR-613-overexpressing group than that in the
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control group (Fig. 4A). At the end of the experiment (4 weeks after cell implantation),
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xenograft tumors were excised and weighed. miR-613-overexpressing xenograft tumors had ~45% lower weights than control tumors (Fig. 4B). Immunohistochemical analysis with Ki67 staining demonstrated that the percentage of Ki67-positive proliferating cells was ~50% lower in miR-613 tumors than that in control tumors (Fig. 4C). Additionally, Western blot analysis confirmed the downregulation of DCLK1 in miR-613 tumors (Fig. 4D). These results demonstrate that miR-613 hampers subcutaneous HCC tumor growth.
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Discussion
Although thousands of miRNAs have been detected in humans [17], only a minority
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of them have been functionally characterized. miR-613 were originally reported to be implicated in lipid metabolism in HepG2 cells [18] and macrophages [19]. Recent evidence indicates a link between miR-613 and tumorigenesis [14,15,20].
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Dysregulation of miR-613 has been found in several types of cancers such as
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non-small cell lung cancer [20] and prostate cancer [15]. Downregulation of miR-613 was also detected in HCC tissues, compared to adjacent non-cancerous tissues. Li et al [20] reported that miR-613 induces a cell cycle arrest in non-small cell lung cancer cells. Consistently, we showed that enforced expression of miR-613 hampered the
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proliferation of HCC cells. Moreover, ectopic expression of miR-613 was found to impair the invasiveness of HCC cells. Altogether, these results provide first evidence
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that miR-613 acts as a tumor suppressor in HCC.
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To further explore the therapeutic potential of overexpression of miR-613 in HCC, we examined the role of miR-613 in the growth of HCC subcutaneous xenograft tumors in nude mice. In accordance with in vitro data, miR-613 overexpression retarded the growth of Hep3B xenograft tumors, which was accompanied by a significant decline in the percentage of Ki67-positive tumor cells. Similarly, the delivery of miR-613 mimic inhibited in vivo tumor growth in a xenograft model of non-small cell lung cancer [20]. These findings highlight the therapeutic significance of restoration of
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Several direct targets of miR-613 have been identified [19,20]. For instance, miR-613
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has been reported to modulate cholesterol efflux via targeting LXRα and ABCA1 in THP-1 macrophages [19]. Another study documented that miR-613 exerts its anticancer effects against non-small cell lung cancer by targeting CDK4 [20]. In this
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study, we identified a novel direct target of miR-613, DCLK1. In silico and
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experimental studies revealed that miR-613 had the ability to negatively regulate the expression of DCLK1, which was causally linked to its interaction with a site in the DCLK1 3'-UTR. Mutation of this site impaired miR-613-mediated repression of DCLK1 3'-UTR reporter expression. DCLK1 is frequently upregulated in HCC [8]
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and contributes to tumorigenesis [9,10,21]. Targeting DCLK1 has been shown to suppress the growth of HCC xenograft tumors [8]. The studies, combined with our findings, suggest that DCLK1 may be a functional target of miR-613 in HCC.
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Interestingly, knockdown of DCLK1 yielded similar suppressive effects on HCC cells
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to those obtained with overexpression of miR-613. Most importantly, co-transfection of DCLK1 lacking 3'-UTR significantly reversed the suppression of HCC cell proliferation and invasion by miR-613 overexpression. Additionally, there was a negative correlation between miR-613 and DCLK1 expression in HCC tissues. Taken together, targeting DCLK1 accounts for the suppressive activity of miR-613 in HCC. However, the fact that restoration of DCLK1 could not completely reverse the phenotypes induced by miR-613 suggested the presence of other functional mediators
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In this study, we demonstrated for the first time that miR-613 suppresses HCC growth
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and invasiveness, at least partially through targeting DCLK1. Restoration of miR-613
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may represent a potential approach to prevent the growth and progression of HCC.
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[18]. D. Zhong, Y. Zhang, Y.J. Zeng, M. Gao, G.Z. Wu, C.J. Hu, G. Huang, F.T. He, MicroRNA-613 represses lipogenesis in HepG2 cells by downregulating LXRα, Lipids Health Dis. 12(2013):32. [19]. R. Zhao, J. Feng, G. He, miR-613 regulates cholesterol efflux by targeting LXRα and ABCA1 in PPARγ activated THP-1 macrophages, Biochem. Biophys. Res. Commun. 448(2014):329-334. [20]. D. Li, D.Q. Li, D. Liu, X.J. Tang, MiR-613 induces cell cycle arrest by targeting
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ACCEPTED MANUSCRIPT CDK4 in non-small cell lung cancer. Cell Oncol (Dordr), 2016 Jan 7. [21]. N. Weygant, D. Qu, W.L. Berry, R. May, P. Chandrakesan, D.B. Owen, S.M. Sureban, N. Ali, R. Janknecht, C.W. Houchen, Small molecule kinase inhibitor
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through inhibition of doublecortin-like kinase 1, Mol. Cancer. 13(2014):103.
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Figure legends
Fig. 1. miR-613 inhibits the proliferation and invasion of HCC cells. (A) qRT-PCR
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analysis of miR-613 in 38 pairs of HCC and non-cancerous liver tissues. (B) Measurement of miR-613 in a panel of HCC cell lines and THLE-3 normal liver epithelial cells by qRT-PCR analysis. *P < 0.05 vs. THLE-3 cells. (C) MTT assay was
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miR-613-expressing plasmid after incubation for indicated times. (D) HCC cells transfected with control miRNA or miR-613-expressing plasmid were tested for invasiveness in Matrigel-coated transwells. Bar graphs represent the results from three
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independent experiments. *P < 0.05 vs. control.
Fig. 2. DCLK1 is a direct target of miR-613 in HCC. (A) A potential target site for miR-613 in the 3′-UTR of human DCLK1 mRNA, as predicted by the program
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Targetscan. To disrupt the interaction between miR-613 and DCLK1 mRNA, the
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target site was mutated. (B) Luciferase reporter assays performed in HEK293 cells co-transfected with miR-613 or control miRNA (Control) and wild-type or mutant DCLK1 3′-UTR constructs. (C) HCC cells were transfected with miR-613 or control miRNA, and tested for the protein expression of DCLK1. *P < 0.05 vs. control. (D) Spearman correlation analysis of the relationship between DCLK1 protein and miR-613 expression in 38 HCC specimens.
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ACCEPTED MANUSCRIPT Fig. 3. miR-613 suppresses the proliferation and invasion of HCC cells via repression of DCLK1 expression. (A) qRT-PCR analysis of DCLK1 mRNA levels in HCC cells transfected with indicated constructs. C-siRNA: control siRNA. (B) MTT assay was
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done to measure the viability of HCC cells transfected with different constructs after incubation for indicated times. (C) HCC cells transfected with indicated constructs were tested for invasiveness in Matrigel-coated transwells. Bar graphs represent the
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cells co-transfected with miR-613 and empty vector.
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results from three independent experiments. *P < 0.05 vs. C-siRNA; #P < 0.05 vs.
Fig. 4. miR-613 impairs HCC tumorigenesis in vivo. Hep3B cells stably transfected with miR-613 or control miRNA were implanted into nude mice and tumor growth
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and gene expression were assessed. (A) Tumor volume was measured every week after cell injection. (B) At the end of the experiment, animals were sacrificed and tumors were excised and weighted. Inserts show a representative gross morphology of
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tumors. (C) Tumor samples were subjected to Ki67 staining. Representative images
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are shown in top panels. Scale bar = 100 µm. The percentage of Ki67-positive tumor cells was determined. (D) Western blot analysis of DCLK1 protein in tumors. Bar graphs represent the results from three independent experiments. *P < 0.05 vs. control.
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Highlights
miR-613 inhibits the aggressive phenotypes of HCC cells.
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miR-613 impairs HCC tumorigenesis in vivo.
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DCLK1 is a direct target of miR-613 in HCC