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Accepted Manuscript
MicroRNA-374b reduces the proliferation and invasion of colon
cancer cells by regulation of LRH-1/Wnt signaling
Rongfeng Qu, Shuhong Hao, Xianmei Jin, Guang Shi, Qiong Yu,
Xianshuang Tong, Dongrui Guo
PII:
DOI:
Reference:
S0378-1119(17)30962-9
doi:10.1016/j.gene.2017.11.019
GENE 42327
To appear in:
Gene
Received date:
Revised date:
Accepted date:
14 September 2017
28 October 2017
7 November 2017
Please cite this article as: Rongfeng Qu, Shuhong Hao, Xianmei Jin, Guang Shi, Qiong
Yu, Xianshuang Tong, Dongrui Guo , MicroRNA-374b reduces the proliferation and
invasion of colon cancer cells by regulation of LRH-1/Wnt signaling. The address for
the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Gene(2017), doi:10.1016/j.gene.2017.11.019
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ACCEPTED MANUSCRIPT
MicroRNA-374b reduces the proliferation and invasion of colon cancer cells by regulation of
LRH-1/Wnt signaling
Rongfeng Qu1, Shuhong Hao1, Xianmei Jin2, Guang Shi1, Qiong Yu1, Xianshuang Tong1, Dongrui
Guo1,*
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Department of Hematology and Oncology, The Second Hospital of Jilin University, Changchun, Jilin
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130022, China
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Department of Pediatric Oncology, The First Hospital of Jilin University, Changchun, Jilin 130021,
China
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*Corresponding author: Dongrui Guo
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Correspondence to Dongrui Guo; Department of Hematology and Oncology, The Second Hospital of
Jilin University, No. 218 Ziqiagn Street, Changchun, Jilin 130022, China
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Email: guo_ruidong@163.com
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Abstract
Deregulation of microRNA (miRNA) has been suggested as a critical event in colon cancer
development and progression. Recent studies have suggested that miR-374b is a novel cancer-related
miRNA involved in several cancer types. Thus far, very li ttle is known about the role of miR-374b in
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colon cancer; therefore, the goal of this study was to investigate the potential role of miR-374b in colon
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cancer. Here, we showed that miR-374b expression was significantly downregulated in colon cancer
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tissues and cell lines. Overexpression of miR-374b inhibited the proliferation and invasion of colon
cancer cells, while miR-374b suppression promoted colon cancer cell proliferation and invasion. Liver
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receptor homolog-1 (LRH-1) was identified as a target of miR-374b in colon cancer cells. Both the
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mRNA and protein expression of LRH-1 were regulated by miR-374b. In addition, an inverse
correlation between LRH-1 mRNA and miR-374b expression was evidenced in colon cancer specimens.
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Notably, overexpression of miR-374b also downregulated the Wnt signaling in colon cancer cells.
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Furthermore, restoration of LRH-1 expression significantly abolished the antitumor effect of miR-374b
in colon cancer cells. These findings suggest that miR-374b inhibits colon cancer cell proliferation and
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invasion through downregulation of LRH-1 expression. Inhibiting LRH-1 by miR-374b may represent
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a novel therapeutic strategy for the treatment of colon cancer.
Keywords
Colon cancer; LRH-1; miR-374b; Wnt.
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1. Introduction
Colon cancer represents one of the most prevalent cancers, with morbidity increasing worldwide
(Siegel et al., 2016). Multiple factors, including genetic mutation, inflammation, dietary habit, alcohol
consumption and smoking contribute to the pathogenesis of colon cancer; however, the precise
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underlying molecular mechanisms remain elusive (Calvert and Frucht, 2002; Vasen et al., 2015).
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Although the 5-year survival rate of colon cancer has been significantly improved in recent years, colon
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cancer is still a major health concern causing tremendous financial burden (Zafar et al., 2015).
Therefore, it is of great importance to develop novel and effective therapies to improve the therapeutic
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efficacy for colon cancer.
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MicroRNAs (miRNAs) are a set of endogenous, small, non-coding RNAs that are involved in
physiological and pathological processes (Kloosterman and Plasterk, 2006). Essentially, miRNAs
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control gene expression through targeting the 3’-untranslated region (UTR) of target mRNAs in a
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base-pairing manner (Winter et al., 2009). miRNAs play critical roles in various biological processes,
including cell differentiation, proliferation, apoptosis, migration and invasion (Kim, 2005). The
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deregulation of miRNAs has been shown to be related to the carcinogenesis of human cancers
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(Esquela-Kerscher and Slack, 2006). Various studies have revealed that aberrantly expressed miRNAs
are found in colon cancer (Bandres et al., 2009; Hamfjord et al., 2012). Certain miRNAs have been
suggested to be potential biomarkers for prognosis and therapeutic targets for colon cancer (Ng et al.,
2009; Jafri et al., 2015; Mohammadi et al., 2016). Therefore, a better understanding of the molecular
mechanism of miRNAs in the pathogenesis of colon cancer may help development of novel
miRNA-based therapy for colon cancer.
Liver receptor homolog-1 (LRH-1; also called nuclear receptor subfamily 5 group A member 2,
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NR5A2) is a member of the orphan nuclear receptors and plays an important role in steroidogenesis,
bile acid synthesis, cholesterol metabolism and development (Fayard et al., 2004). Accumulating
evidence has suggested that LRH-1 is a novel oncogene that participates in the pathogenesis of
numerous cancers, including gastric (Wang et al., 2008), g hepatocellular (Wang et al., 2005),
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pancreatic (Benod et al., 2011) and breast (Chand et al., 2012) cancers. Importantly, LRH-1 has been
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identified as a coactivator of Wnt signaling (Botrugno et al., 2004; Nadolny and Dong, 2015). The Wnt
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signal has been suggested to be a classic oncogenic signal involved in the development and progression
of many cancers (Moon et al., 2004). Aberrantly activated Wnt signaling occurs in approximately 80%
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of colon cancer (Kolligs et al., 2002). Activated mutations in the adenomatous polyposis coli (APC)
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gene and β-catenin gene contribute to the abnormal activation of Wnt signaling in colon cancer
(Segditsas and Tomlinson, 2006; Schneikert and Behrens, 2007). However, other regulators, such as
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Wnt ligands or inhibitors, are also involved in the development of colon cancer (Bafico et al., 2004). It
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has been reported that LRH-1 interacts with β-catenin and T-cell factor 4 to promote the expression of
c-myc and cyclin D1 (Botrugno et al., 2004). Recent studies also have shown that LRH-1 is involved in
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tumorigenesis of colon cancer (Schoonjans et al., 2005; Bayrer et al., 2015). Therefore, LRH-1 may
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serve as a potential target for treatment of colon cancer.
Recent studies have shown that miR-374b is a novel cancer-related miRNA involved in
osteosarcoma (Liao et al., 2016), pancreatic cancer (Schreiber et al., 2016) and gastric cancer (Xie et al.,
2014). In a recent study, Wu et al. reported that miR-374b was significantly lower in colorectal cancer,
representing a putative biomarker for colorectal cancer (Wu et al., 2015). However, little is known
about the precise function of miR-374b in colon cancer. In this study, we aimed to investigate the
precise role and underlying mechanism of miR-374b in colon cancer. Our results showed that the
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miR-374b expression was significantly downregulated in colon cancer tissues and cell lines.
Overexpression of miR-374b inhibited the proliferation and invasion of colon cancer cells. LRH-1 was
identified as a target of miR-374b in colon cancer cells. Notably, overexpression of miR-374b
downregulated the Wnt signaling in colon cancer cells. These findings suggest that miR-374b inhibits
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colon cancer cell proliferation and invasion through the targeting of LRH-1/Wnt signaling, providing a
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potential therapeutic target for treatment of colon cancer.
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2. Materials and methods
2.1. Tissue specimens
Fifteen colon cancer specimens and 15 paired adjacent noncancerous specimens were obtained from
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patients undergoing surgical resection in The Second Hospital of Jilin University. The dissected tissue
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specimens were immediately frozen in liquid nitrogen and stored at -80 °C. All participants agreed to
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tissue donation and provided written informed consent. This study was reviewed and approved by the
Institutional Human Experiment and Ethic Committee of The Second Hospital of Jilin University. The
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experimental procedures were performed in line with the Declaration of Helsinki.
2.2. Cell lines
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The human colon cancer cell lines HT29, HCT116, SW480 and SW620 were obtained from the Type
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Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The human normal colon
mucosal epithelial (NCM460) cells were obtained from Incell Corporation (San Antonio, TX, USA).
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The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Rockville, MD, USA),
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supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher
Scientific, Rockford, IL, USA). Cells were cultured in a humidified incubator containing 5% CO 2 at
37 °C.
2.3. Cell transfection
The miR-374b mimics, miR-374b inhibitor and negative controls (NC) were purchased from
Shanghai GenePharma Co., Ltd (Shanghai, China). The open reading frame of LRH-1 was cloned into
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the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). The miR-374b mimics/inhibitor or vector were
transfected into cells using a Lipofectamine 2000 system (Invitrogen) according to the manufacturer’s
instructions. The transfection rate was confirmed by real-time quantitative PCR and western blot
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analysis.
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2.4. Real-time quantitative PCR
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Total RNA was isolated using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA) per the
manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using a PrimeScript 1st
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Strand cDNA Synthesis Kit (Takara, Dalian, China). Real-time quantitative PCR was performed with
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Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using an ABI 7500
Sequence Detection System (Applied Biosystems) with the following amplification procedure: 40
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cycles of 95 °C for 15 s and 60 °C for 1 min. The primer sequences used for real-time quantitative PCR
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were as follows: miR-374b (forward, 5’-TCAGCGGATATAATACAACCTGC-3’ and reverse,
5’-TATCGTTGTTCTCCACTCCTTCAC-3’); LRH-1 (forward, 5’-CTGATAGTGGAACTTTTGAA-3’
reverse,
5’-CTTCATTTGGTCATCAACCTT-3’);
c-myc
(forward,
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and
D1
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5’-AGAGAAGCTGGCCTCCTACC-3’ and reverse, 5’-CGTCGAGGAGAGCAGAGAAT-3’); cyclin
(forward,
5’-GTTCGTGGCCTCTAAGATGAAG-3’
and
reverse,
5’-GTGTTTGCGGATGATCTGTTTG-3’); U6 (forward, 5’-ATTGGAACGATACAGAGAAGATT-3’
and
reverse,
5’-GGAACGCTTCACGAATTTG-3’;
GAPDH
(forward,
5’-GAAATCCCATCACCATCTTCCAGG-3’ and reverse, 5’-GAGCCCCAGCCTTCTCCATG-3’). U6
served as the internal control for miR-374b expression, and GAPDH served as the internal control for
mRNA expression. Relative expression was calculated using the 2-∆∆Ct method.
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2.5. Cell counting kit-8 (CCK-8) assay
Cells were seeded into a 96-well plate at a density of 1 × 104 cells/well and cultured overnight. Cells
were transfected with miR-374b mimics or the miR-374b inhibitor for 48 h. Then, 10 µL of CCK-8
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solution (Sigma, St. Louis, MO, USA) was added to each well. After incubation for 1 h at 37 °C, the
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optical density (OD) at 450 nm was detected by an enzyme-linked immunoassay analyzer (BioTek
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Instruments, Inc. Winooski, VT, USA).
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2.6. Colony formation assay
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Cells were transfected with miR-374b mimics or the miR-374b inhibitor for 48 h and then
re-suspended in DMEM containing 10% FBS and 0.3% soft agar. Cells (1 × 103 cells/well) were then
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seeded onto 6-well plates containing 0.6% solidified agar in DMEM containing 10% FBS. After culture
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for approximately 2 weeks, cells were stained with 0.1% crystal violet (Sigma), and the colonies were
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counted. The average colony number was presented as a histogram.
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2.7. Transwell cell invasion assay
Cells were plated on a transwell 24-well chamber precoated with Matrigel Basement Membrane
Matrix (BD Science, San Jose, CA, USA) according to the manufacturer’s instructions. The upper
chamber was filled with 200 μL of serum-free DMEM containing (1 × 104 cells), and 500 μL of
DMEM containing 10% FBS wad added to the lower chamber. After culture for 24 h, the cells on the
upper side of the membrane were removed, and the cells on the lower side of the membrane were fixed
with methanol and stained with 0.1% crystal violet (Sigma). The cell numbers were counted under a
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microscope.
2.8. Dual-luciferase assay
The 3’-UTR of fragment of LRH-1 cDNA containing the predicted miR-374b binding site was
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cloned into the pmirGLO vector (Promega, Madison, WI, USA). Site-directed mutagenesis of the
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miR-374b binding site in the LRH-1 3’-UTR was performed using the Site-Directed Mutagenesis Kit
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(Stratagene, San Diego, CA, USA). The constructed vector was cotransfected with miR-374b mimics
or the miR-374b inhibitor using a Lipofectamine 2000 system (Invitrogen) and incubated for 48 h. To
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detect the activity of Wnt signaling, cells were cotransfected with miR-374b mimics or the miR-374b
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inhibitor, the TOPFlash firefly luciferase vector and the phRL-TK renilla luciferase vector (Promega)
and incubated for 48 h. The relative luciferase activity was detected by the Dual-Glo Luciferase Assay
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Kit (Promega) according to the manufacturer’s instructions.
2.9. Western blot
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Total protein was extracted using cell lysis buffer for western blots, and the protein concentration
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was quantitated using the BCA protein assay kit (Beyotime Biotechnology, Haimen, China). Equivalent
amounts of proteins (50 μg) from each sample were separated by 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Billerica,
MA, USA). The membrane was blocked with 3% skim milk and then immuno-blotted with primary
antibodies for LRH-1 and GAPDH (Abcam, Cambridge, UK). After incubation overnight at 4 °C, the
membrane was probed with horseradish peroxidase-linked secondary antibodies (Bioss Antibodies,
Beijing, China). The protein bands were visualized using Immobilon Western Chemiluminescent HRP
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substrate (Millipore). The band intensities were then quantified by Image-Pro Plus 6.0 software.
2.10. Statistical analysis
The data were presented as the means ± standard deviation. All statistical analyses were performed
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using SPSS 18.0 statistical software (SPSS Inc., Chicago, IL, USA). Student’s t-test was used to
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analyze the differences between two groups. One-way analysis of variance was used to analyze the
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differences among multiple groups followed by a Bonferroni post-hoc test. Correlation analysis was
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performed by Spearman’s correlation test. A p-value less than 0.05 indicates statistical significance.
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3. Results
3.1.The expression of miR-374b is downregulated in colon cancer specimens and cell lines
To investigate the potential role of miR-374b in colon cancer, the expression of miR-374b was
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assessed in colon cancer tissues by real-time quantitative PCR. The results showed that miR-374b
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expression was significantly downregulated in colon cancer tissues compared with adjacent
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noncancerous tissues (Fig. 1A). Furthermore, we measured the expression pattern of miR-374b in
several human colon cancer cell lines by real-time quantitative PCR. As shown in Fig. 1B, miR-374b
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levels were frequently lower in colon cancer cell lines compared with normal colon mucosal epithelial
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cells. Taken together, these results indicate that miR-374b is downregulated in colon cancer, suggesting
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a tumor-suppressive role of miR-374b in colon cancer.
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3.2. miR-374b suppresses cell proliferation and invasion of colon cancer cells
To investigate the potential antitumor effects of miR-374b in colon cancer, we carried out
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gain-of-function and loss-of-function experiments by transiently transfecting miR-374b mimics or a
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miR-374b inhibitor into HCT116 and SW480 cells. The expression of miR-374b was confirmed by
real-time quantitative PCR (Fig. 2A). The CCK-8 assay showed that miR-374b overexpression
significantly inhibited cell proliferation of colon cancer cells (Fig. 2B). Furthermore, the colony
formation capability of colon cancer cells was also markedly attenuated by miR-374b overexpression
(Fig. 2C). In addition, we detected a tumor-suppressive effect of miR-374b on cell invasion using a
Transwell invasion assay. The results showed that miR-374b overexpression significantly inhibited cell
invasion of colon cancer cells (Fig. 2D). In contrast, suppression of miR-374b expression showed the
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opposite effects (Fig. 2B–D). These results imply that miR-374b has strong antitumor effects on cell
proliferation and invasion of colon cancer cells.
3.3. LRH-1 is a direct target gene of miR-374b
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To investigate the underlying mechanism responsible for the antitumor effect of miR-374b, we
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predicted the potential target genes of miR-374b by bioinformatics analysis using online databases
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including TargetScan-Prediction of microRNA targets and microRNA.org-Targets and Expression.
Among these targets, LRH-1, a potential therapeutic target for cancer (Nadolny and Dong, 2015),
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attracts our attention. The 3’-UTR of LRH-1 contained a putative binding site for miR-374b (Fig. 3A).
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LRH-1 has been suggested as a novel oncogene in colon cancer (Schoonjans et al., 2005; Bayrer et al.,
2015). Also, studies have shown that LRH-1 expression is epigenetically regulated by miRNAs (Liang
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et al., 2015; Jiang et al., 2016). Herein, we investigated whether LRH-1 is a direct target gene of
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miR-374b in colon cancer using dual-luciferase reporter assay. The 3’-UTR of LRH-1 contained a
putative binding site for miR-374b was cloned into the pmirGLO reporter gene vector. The
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dual-luciferase reporter assay showed that the relative luciferase activity was significantly reduced by
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miR-374b overexpression or increased by miR-374b suppression (Fig. 3B and C). However, neither
miR-374b overexpression nor miR-374b suppression showed significant effects on the mutant reporter
(Fig. 3B and C). Furthermore, real-time quantitative PCR and western blot analysis were used to
investigate whether miR-374b regulates LRH-1 expression. The results showed that both the mRNA
and protein expression were downregulated by miR-374b overexpression, whereas miR-374b
suppression promoted LRH-1 mRNA and protein expression (Fig. 4A and B). In addition, we found
that LRH-1 mRNA expression was significantly upregulated in colon cancer tissues (Fig. 5A).
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Correlation analysis showed that LRH-1 mRNA expression was inversely correlated with miR-374b
expression in colon cancer tissues (Fig. 5B). Taken together, these results suggest that LRH-1 is a direct
target gene in colon cancer.
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3.4. miR-374b suppresses Wnt signaling by LRH-1 in colon cancer cells
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Given that LRH-1 is a critical coactivator of Wnt signaling (Botrugno et al., 2004; Nadolny and
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Dong, 2015), we analyzed the effect of miR-374b on Wnt signaling in colon cancer cells. The results
showed that miR-374b overexpression significantly downregulated Wnt signaling (Fig. 6A).
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Furthermore, the transcription of c-myc (Fig. 6B) and cyclin D1 (Fig. 6C) was also markedly
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suppressed by miR-374b overexpression. In contrast, suppression of miR-374b significantly activated
Wnt signaling (Fig. 6A–C). These data suggest that miR-374b regulates Wnt signaling possibly by
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targeting LRH-1.
3.5. Restoration of LRH-1 expression abolishes the antitumor effect of miR-374b
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To validate whether miR-374b inhibits colon cancer cell proliferation and invasion by targeting
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LRH-1, we restored the expression of LRH-1 in miR-374b mimic-transfected cells by introducing the
pcDNA3.1/LRH-1 vector (without the 3’-UTR of LRH-1). The results showed that transfection of the
pcDNA3.1/LRH-1 vector significantly restored the reduced LRH-1 protein expression in miR-374b
mimic-transfected cells (Fig. 7A). The promoting effect of miR-374b on Wnt signaling was
significantly reversed by LRH-1 restoration (Fig. 7B). Furthermore, restoration of LRH-1 expression
markedly abolished the suppressive effect of miR-374b on colon cancer proliferation (Fig. 7C) and
invasion (Fig. 7D). These results indicate that miR-374 suppresses the proliferation and invasion of
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colon cancer cells through downregulation of LRH-1.
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Discussion
In this study, we demonstrated a tumor-suppressive role of miR-374b in colon cancer. We showed
that miR-374b was significantly decreased in colon cancer tissues and cell lines. Overexpression of
miR-374b inhibited the proliferation and invasion of colon cancer cells. Interestingly, we identified
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LRH-1 as a novel target gene of miR-374b. The overexpression of miR-374b also downregulated the
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activation of Wnt signaling. However, restoration of LRH-1 expression markedly abolished the
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antitumor effects of miR-374b. Our study indicates that miR-374b may inhibit colon cancer
development through downregulation of LRH-1.
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Recent studies have suggested miR-374b is a novel cancer-related miRNA involved in several types
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of human cancers (He et al., 2013). Global analysis of miRNA expression in prostate cancer revealed
that miR-374b is downregulated in prostate cancer tissues, serving as an independent predictor for
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prognosis (He et al., 2013). High expression of miR-374b predicted a favorable outcome of breast
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cancer (Liu et al., 2015). Overexpression of miR-374b inhibited the proliferation and invasion of breast
cancer cells (Liu et al., 2015), and the overexpression of miR-374b overcomes the cisplatin resistance
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of pancreatic cancer cells (Schreiber et al., 2016). In human osteosarcoma, miR-374b inhibited
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angiogenesis and metastasis by targeting the vascular endothelial growth factor (Liao et al., 2016).
Moreover, a recent study demonstrated that miR-374b is frequently decreased in T-cell lymphoblastic
lymphoma and that overexpression of miR-374b suppresses cell growth and promotes cell apoptosis in
vitro and in vivo through targeting AKT1 and Wnt-16 (Qian et al., 2015). These studies revealed a
tumor-suppressive role of miR-374b. However, an oncogenic function of miR-374b has been found in
gastric cancer (Xie et al., 2014). Xie et al. reported that miR-374b was increased in gastric cancer,
which promoted the metastasis and invasion of gastric cancer by inhibiting reversion-inducing
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cysteine-rich proteins with the Kazal motif (RECK) (Xie et al., 2014). In this study, we found that
miR-374b was decreased in colon cancer tissues as well as cell lines. Our findings are consistent with
those of a recent study that reported miR-374b is significantly decreased in colorectal cancer (Wu et al.,
2015). In our study, we further investigated the biological function of miR-374b in colon cancer. We
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showed that overexpression of miR-374b inhibits the proliferation and invasion of colon cancer cells.
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Our results suggest a tumor-suppressive role of miR-374b in colon cancer. Consistent with our findings,
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a recent study reports that miR-374b induced by p53 promotes the apoptosis of colon cancer cells by
targeting and inhibiting Akt1 in response to bleomycin–induced DNA damage (Gong et al., 2017) ,
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supporting a tumor-suppressive function of miR-374b in colon cancer.
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LRH-1 is emerging as a novel target for cancer therapy (Lazarus et al., 2012; Nadolny and Dong,
2015). LRH-1 controls the vital transcriptional programs of the proliferation and invasion of breast
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cancer cells (Chand et al., 2010; Chand et al., 2012; Bianco et al., 2014; Bianco et al., 2015). LRH-1 is
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highly expressed in pancreatic cancer cells, and knockdown of LRH-1 suppresses the growth and
proliferation of pancreatic cancer cells (Benod et al., 2011). Inhibition of LRH-1 also blocks the
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epithelial-mesenchymal transition of pancreatic cancer stem cells (Luo et al., 2016). LRH-1 is
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abundantly expressed in gastric cancer tissues and promotes gastric cancer proliferation via induction
of cyclin E1 (Wang et al., 2008). In a genetic model of colon cancer, haploinsufficiency of LRH-1
abates colon cancer formation through affecting the cell cycle and inflammation (Schoonjans et al.,
2005). The silencing of LRH-1 in colon cancer cells inhibited cell proliferation (Bayrer et al., 2015).
Moreover, a recent study reported that LRH-1 promotes colon cancer cell proliferation by inhibiting the
expression of the cell cycle inhibitor p21 (Kramer et al., 2016). These studies suggest that LRH-1 is a
potential target for colon cancer. In this study, we identified that LRH-1 is a direct target gene of
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miR-374b in colon cancer. We showed that LRH-1 expression is regulated by miR-374b, suggesting
that miR-374b is a novel upstream regulatory factor for LRH-1. We also showed an inverse correlation
between miR-374b and LRH-1 expression in colon cancer specimens. The decreased miR-374b
expression may contribute to the promotion of LRH-1-mediated tumorigenesis of colon cancer.
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Therefore, inhibition of LRH-1 by miR-374b may be a promising therapeutic strategy for colon cancer.
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Aberrant activation of Wnt signaling occurs in approximately 80% of colon cancer (Kolligs et al.,
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2002). LRH-1 has been reported to be a critical regulator of Wnt signaling that can synergize with
β-catenin and T-cell factor 4 to stimulate the transcription of c-myc and cyclin D1 (Botrugno et al.,
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2004; Nadolny and Dong, 2015). LRH-1 has also been reported to activate Wnt signaling in gastric
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cancer (Wang et al., 2008; Li et al., 2016), osteosarcoma (Li et al., 2015), lung cancer (Jiang et al.,
2016) and colon cancer (Bayrer et al., 2015). In this study, we have shown that inhibiting LRH-1 by
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for modulating Wnt signaling.
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miR-374b significantly downregualted the Wnt signaling in colon cancer, suggesting a novel approach
Several chemical inhibitors for LRH-1 have been developed that show promising effects on the
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inhibition of cancer cell proliferation (Benod et al., 2013; Corzo et al., 2015). Interestingly, miRNAs
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are emerging as novel inhibitors of LRH-1. An increasing number of studies have shown that various
miRNAs, including miR-451 (Li et al., 2015), miR-27b-3p (Zhu et al., 2016), miR-381 (Zhang et al.,
2016) and miR-376c (Jiang et al., 2016), can directly target the 3’-UTR of LRH-1, inhibiting LRH-1
expression. Intriguingly, miR-381 is reported to control the proliferation and invasion of colon cancer
cells through targeting of LRH-1 (Liang et al., 2015). More recently, LRH-1 is reported to be targeted
and regulated by miR-136 in colon cancer cells and inhibition of LRH-1 by miR-136 impedes the
proliferation and invasion of colon cancer cells (Yuan et al., 2017). Overall, these findings suggest that
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the dysregulation of LRH-1 in colon cancer development may be controlled by various miRNAs. In
this study, we identified miR-374b as a novel miRNA for targeting LRH-1. Our study suggests that
miR-374b may serve as a novel target for development of miRNA-based inhibitor of LRH-1.
In conclusion, our results for the first time demonstrated that miR-374b is a novel tumor-suppressive
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miRNA in colon cancer that can inhibit colon cancer proliferation and invasion through targeting of
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LRH-1. Our study provides novel insights into the function of miR-374b/LRH-1 in the development of
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colon cancer. miR-374b/LRH-1 may represent a potential and promising target for therapeutic
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applications in colon cancer treatment.
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Conflict of interest
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The authors declare that they have no conflict of interest.
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Figure legends
Fig. 1. miR-374b is repressed in colon cancer. (A) Relative expression of miR-374b in colon cancer
specimens and adjacent noncancerous specimens was detected by real-time quantitative PCR. * p <
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0.05. (B) Relative expression of miR-374b in colon cancer cell lines (HT29, HCT116, SW480 and
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SW620) and normal colon mucosal epithelial cells NCM460 was measured by real-time quantitative
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PCR. * p < 0.05 vs. NCM460.
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Fig. 2. miR-374b inhibits colon cancer cell proliferation and invasion. (A) Expression of miR-374b in
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colon cancer cells was detected by real-time quantitative PCR. The miR-374b mimics or the miR-374b
inhibitor were transfected into HCT116 and SW480 cells and incubated for 48 h. (B) Cell proliferation
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was detected by the CCK-8 assay in HCT116 and SW480 cells transfected with miR-374b mimics or
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the miR-374b inhibitor for 48 h. (C) Transfected cells were seeded into a 6-well plate, and colony
formation was detected after 2 weeks by staining with 0.1% crystal violet. The number of colonies was
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counted. (D) The effect of miR-374b on invasion was assessed by the Transwell invasion assay.
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Transfected cells were cultured in a transwell 24-well chamber and cultured for 24 h. The number of
invaded cells was counted. * p < 0.05 vs. NC.
Fig. 3. miR-374b directly targets the 3’-UTR of LRH-1. (A) The putative miR-374b binding site in the
3’-UTR of LRH-1 mRNA. Mutations were generated at the complementary site for the seed region of
miR-374b. The dual-luciferase reporter assay was used in HCT116 (B) and SW480 (C) cells. An
LRH-1 3’-UTR fragment containing the wild-type or mutant miR-374b binding site was cloned into the
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pmirGLO reporter gene vector. Cells were cotransfected with the reporter gene construct vector and
miR-374b mimics or the miR-374b inhibitor for 48 h. * p < 0.05 vs. NC.
Fig. 4. miR-374b represses LRH-1 expression in colon cancer cells. HCT116 and SW480 cells were
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transfected with miR-374b mimics or the miR-374b inhibitor for 48 h. (A) Relative mRNA expression
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of LRH-1 was detected by real-time quantitative PCR. (B) Relative protein expression of LRH-1 was
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detected by western blot. * p < 0.05 vs. NC.
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Fig. 5. LRH-1 is inversely correlated with miR-374b expression in colon cancer specimens. (A)
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Relative mRNA expression of LRH-1 in colon cancer specimens and adjacent noncancerous specimens
was detected by real-time quantitative PCR. (B) Correlation between LRH-1 mRNA and miR-374b
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expression was assessed by Spearman’s correlation test. r = -0.824; p < 0.001.
Fig. 6. miR-374b suppresses Wnt signaling in colon cancer cells. SW480 and HCT116 cells were
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cotransfected with the TOPFlash firefly luciferase reporter vector, phRL-TK renilla luciferase vector,
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miR-374b mimics or the miR-374b inhibitor and cultured for 48 h. (A) Relative luciferase activity was
detected by the Dual-Glo luciferase system. Relative mRNA expression of c-myc (B) and cyclin D1 (C)
was detected by real-time quantitative PCR. * p < 0. 05 vs. NC.
Fig. 7. Restoration of LRH-1 expression abolishes the antitumor effects of miR-374b. SW480 and
HCT116 cells were cotransfected with miR-374b mimics and the pcDNA3.1/LRH-1 expression vector
(without the 3’-UTR) and cultured for 48 h. (A) Protein expression of LRH-1 was detected by western
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blot. (B) Wnt signaling was assessed using the Tcf-dependent TOPFlash reporter assay by transfecting
the TOPFlash firefly luciferase reporter vector and the phRL-TK renilla luciferase vector. (C) Cell
proliferation was detected by the CCK-8 assay. (D) Cell invasion was detected by the Transwell
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Abbreviations
miRNA, microRNA; LRH-1, liver receptor homolog-1; FBS, fetal bovine serum; CCK-8, cell counting
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kit-8; UTR, untranslated region.
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Highlights
miR-374b is decreased in colon cancer.
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miR-374b represses the progression of colon cancer
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miR-374b targets and regulates LRH-1.
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miR-374b inhibits colon cancer progression by inhibiting LRH-1.
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