Background
Colorectal cancer (CRC) is one of the most common malignancies in the world, with high rates of incidence and disease-related mortality and morbidity [
1]. Approximately 5-10% of colorectal cancers are caused by heritable mutations. Scientists think that roughly 50% of the remaining cases are caused by sporadic mutations [
2]. However, the precise mechanisms involved in CRC tumorigenesis are still not well understood. MicroRNAs (miRNAs) are small noncoding RNA molecules that modulate the expression of target mRNAs at a post-transcriptional level [
3]. They regulate critical aspects of the oncogenic phenotype through the disruption of protein translation by selective binding and degradation of target mRNAs [
4]. An increasing number of studies have shown that dysregulation of miRNA expression plays an important role in cancer initiation, progression, and prognosis, as well as acquired resistance toanticancer agents [
5‐
7]. Several miRNAs have been found to participate in the pathogenesis of CRC, including miR-21, miR-451, miR-499-5p, miR-375, and miR-142-5p [
8]. However, there have been few studies examining miRNAs that regulate the oncogenic mutations in CRC.
The AP-1 (activator protein 1) transcription factor is a dimeric complex that comprises members of the JUN, FOS, ATF (activating transcription factor), and MAF (musculoaponeurotic fibrosarcoma) protein families [
9]. AP-1 factors regulate diverse cellular processes, including differentiation, proliferation, and cell survival, and are also critically involved in the development of various cancers [
10‐
13]. They contain a basic DNA binding domain and a leucine zipper dimerization domain, and dimers preferentially bind to consensus TGA(C/G)TCA (TRE) or TGACGTCA (CRE) DNA motifs [
14]. Several studies have reported that functional loss or downregulation of AP-1, or post-transcriptional control of JunD by miRNA can effectively suppress the proliferation of cancer cells [
15,
16]. However, post-transcriptional regulation of AP-1 in CRC cells was rarely mentioned as in other cancers. Basic leucine zipper transcription factor ATF-like (BATF), BATF2, and BATF3 belong to the AP-1 family of transcription factors that regulate numerous cellular processes. Initially, BATF family members were thought to function only as inhibitors of AP-1-driven transcription [
17‐
19]; however, recent studies have uncovered that these factors have unique, non-redundant, and positive transcriptional activities in dendritic cells [
20], B cells, and T cells [
21‐
23]. Given their functional redundancy [
24], this suggests BATF3 might also has oncogenic potential.
In the present study, we performed an integrated analysis and identified miRNAs with altered expressionin CRC cells using miRNA sequencing data from The Cancer Genome Atlas (TCGA,
http://tcga-data.nci.nih.gov/docs/publications/coadread_2012). We identified miR-760 as a clinically noteworthy miRNA in CRC and confirmed that miR-760 expression was lower in CRC patients with poor survival. We further found that ectopic expression of miR-760 suppresses proliferation and tumorigenicity in CRC cells. Moreover, our data indicated that the full-length 3′-untranslated region (3′-UTR) of human BATF3 mRNA was a direct target of miR-760. Thus, miR-760 plays an essential role during the regulation of BATF3 in CRC cells in vitro and in vivo. Our present study suggests that miR-760 suppresses cell proliferation and tumorigenicity in CRC cells by targeting BATF3 mRNA and suppressing the expression of BATF3 and downstream cyclin D1.
Methods
Cell line culture and morphological observation
The human CRC cell lines SW620, DLD1, and HCT116, and colorectal mucosa cell line FHC were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 50 mg/ml streptomycin. All cells were incubated at 37 °C with a humidified atmosphere of 5% CO2. The morphology of the CRC cells was observed using phasemicroscopy (Leica,Wetzlar, Germany).
Patients, specimens, and follow-up
In this study, CRC samples used in the immunohistochemistry (IHC) assay were obtained randomly from patients who underwent radical resection of pathologically confirmed tumors between 2013 and 2015 in the First Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China). None of the patients received any preoperative anticancer treatment. The present research was approved by the research ethics committee of the First Affiliated Hospital of Zhengzhou University. The group of patient population used in different experiments was listed in Additional file
1: Table S1. A total of 80 cases were used in this study to examine BATF3 expression, among which 45 samples were obtained from patients between 2014 and 2015 that tested positive for miR-760 expression, and the other 35 samples were obtained from patients in 2013 from the Department of Pathology at the First Affiliated Hospital of Zhengzhou University. All patients included were monitored until 2017, with a median observation time of approximately 33 months. All patients provided written informed consent to participate in this study.
Vectors, lentiviral infection, and transfection of miR-760 mimic, inhibitor, and siRNA of target gene
The miR-760 gene was PCR-amplified from genomic DNA and cloned into a psicoR-GFP lentiviral vector. Psico-miR-760 was cotransfected with the psPAX2 and pMD2.G packaging plasmid in HEK293 T cells using Lipo3000 (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. At 48 h after the cotransfection, supernatants were collected and incubated with CRC cells for a 24-h infectionperiod in the presence of polybrene (5 μg/ml). After infection, GFP-positive cells were sorted by MoFloXDP (Beckman, USA) to obtain miR-760 overexpression cells. The 3′-UTR of BATF3 was amplified and cloned downstream to the luciferase gene in a modified pmirGLO control vector (received as a gift from Professor Guoqiang Zhao, Zhengzhou University). Hsa-miR-760 mimic, the negative control (NC) mimic, hsa-miR-760 inhibitor, and the NC inhibitor were purchased from Genepharma Company (Shanghai, China).
RNA extraction and quantitative reverse transcription PCR (qRT-PCR)
As described in our previous study, total miRNA from cultured cells and CRC tissues were extracted using Trizol solution (Invitrogen Life Technologies) [
25]. Independently, RNA from each sample was reverse-transcribed using PrimeScript RT reagent Kit (Takara Bio, Otsu, Shiga, Japan). Subsequently, expression levels of miR-760 were quantified by qRT-PCR using SYBR Premix ExTaqII (TaKaRa, Japan) in Agilent Mx3005P. Expression levels of genes were normalized to that of the housekeeping gene GAPDH as the control. miRNA expression was defined based on Ct values, and relative expression levels were normalized according to the expression of small nuclear RNA U6. We used melting curves to monitor non-specific amplifications. The 2
-ΔΔCt method was used to calculate relative expression changes.
The following primers were used:
miR-760 forward, 5′-CGGCTCTGGGTCTGTGGGGA-3′;
BATF3 forward, 5′-AGAGAGATCGGGAAGCTGACA-3′;
P21 forward, 5′-GAGCGATGGAACTTCGACTT-3′;
P27 forward, 5′-GCACTGCAGAGACATGGAAG-3′;
Cyclin D1 forward, 5′-CCCTCGGTGTCCTACTTCAA-3′;
and U6 forward: 5′-CTCGCTTCGGCAGCACA-3′.
Cell proliferation assay
CRC cells (2 × 103) were seeded into 96-well plates. Cell counting kit-8 (CCK-8; DOJINDO, Japan) assay was performed following the manufacturer’s instructions. Absorbance at 450 nm was measured 1 h after the addition of 10 μl of CCK-8 reagent per well to calculate the number of viable cells every 24 h over a 96 h period. All experiments were performed in triplicate. CRC cells were plated on a 6-well plate (500 cells per well) and cultured for 10 days. The colonies were stained with 1.0% crystal violet for 5 min after fixation with 10% formaldehyde for 15 min.
CFSE staining
CRC cells were washed and resuspended with RPMI1640 at a final concentration of 1 × 106 cells/ml. CRC cells were labeled with 5 Mmcarboxy-fluorescein succinimidyl ester (CFSE, Invitrogen) for 10 min at 37 °C. The labeling reaction was quenched by addition of cold RPMI-1640 with 10% FBS and incubation on ice for 10 min. CFSE-labeled cells were washed with culture medium twice and seeded into 24-well plates. Labeled cells were harvested at indicated time points and cell proliferation was determined by FACS.
Dual-luciferase reporter assay
293T cells were seeded in a 24-well plate and co-transfected with 0.5 μg pmirGLO vector, 80 nM of miR-760-mimic and 1 μl of Lipo3000 (Invitrogen) in 50 μl of Opti-MEM Reduced-Serum Medium (Invitrogen). NC mimic was used as the control. To verify the activation of the cyclin D1 promoter, TRE-containing sequence (5′-AAAATGAGTCAGAA-3′) was cloned into pGL4.27 vector to produce the plasmid pGL4.27-Cyclin D1-wild-type (WT). miR-760 overexpressed in SW620 or HCT116 cells, and control psico-transducted cells were seeded separately in 24-well plates and co-transfected with 0.25 μg pGL4.27-cyclinD1-WT plasmids, 0.25 μg pRL-TK plasmids (Promega), and 1 μl Lipo3000 (Invitrogen) in 50 μl Opti-MEM Reduced-Serum Medium following the manufacturer’s instructions. Twenty-four hours following transfection, the activities of Firefly and Renilla luciferases in cell lysates were measured using the Dual-Glo®Luciferase Assay System (Promega) and the Fluoroskan Ascent FL (Thermo Fischer Scientific). Firefly luciferase activity was normalized to Renilla luciferase activity. All transfection experiments were conducted in triplicate.
BALB/c athymic nude mice (female, 4–6 weeks old and 16–20 g) were purchased from Beijing Vital River Laboratory (Beijing, China). All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Zhengzhou University. To establish the CRC cancer xenograft model, the mice were randomly divided into two groups (n = 6 each). SW620-psico or SW620-miR-760 cells (5 × 106) were suspended in 150 μl PBS and inoculated subcutaneously into the flanks of the nude mice. Tumor dimension was measured by length (L) and width (W) with a caliper twice a week, and the volumes were calculated using the formula (L × W2)/2. Mice were sacrificed by cervical dislocation after being anaesthetized with 10% chloral hydrate at day 31, and the tumors were excised and snap-frozen for protein and RNA extraction. IHC of the tumor tissues was performed as described below.
IHC
Formalin-fixed, paraffin-embedded sections (3 mm) were deparaffinized in xylene, rehydrated with an alcohol gradient, and washed briefly in tap water. Endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide for 30 min. To retrieve antigenicity, sections were boiled in 10 mM citrate buffer (pH 5.8) for 30 min in a microwave (800 W). Next, sections were incubated with goat serum diluted in PBS (pH 7.4) for 30 min at 22 °C. Subsequently, sections were incubated at 4 °C overnight with primary antibodies specific for BATF3 diluted at 1:200 (Abcam, Cambridge, UK). The following day, sections were rinsed with fresh PBS and incubated with horseradish peroxidase-linked secondary antibodies at room temperature for 30 min. Finally, sections were stained with 3,30-diaminobenzidine (DAB) substrate (Dako, Carpinteria, CA, USA) and counterstained with Mayer’s hematoxylin. Images were recorded using a microscope (Leica, Wetzlar, Germany).
Statistical analysis
Data analysis was performed using SPSS 19.0 statistical software or GraphPad Prism 7 software. Data were expressed as the mean ± standard deviation(SD) of at least 3 replicated experiments. Student’s t-test was performed to analyze the differences between two groups with normally distributed continuous variables. Otherwise, the Mann-Whitney U test would be used. Pearson’s coefficient correlation or linear regression analysis was used to analyze the relationships between the expression levels of specific genes. The Kaplan-Meier method was used to establish survival curves, and the survival differences were compared using the log-rank test. In all cases, a two-tailed P value< 0.05 was considered statistically significant.
Discussion
In the present study, we found that miR-760 is dramatically downregulated in human CRC tissues compared with normal colorectal tissues. Moreover, upregulation of miR-760 suppresses proliferation of colorectal cancer cells by targeting BATF3 3′-UTR. The negative regulation of BATF3 by miR-760 leads to downregulation of cyclinD1 in CRC cells. Collectively, miR-760 suppresses tumor cell growth in vitro and tumorigenesis in vivo. Our study may serve as a rational for targeting the miR-760/BATF3 interaction in a novel therapeutic application to treat CRC patients.
miRNAs are a class of non-coding RNA molecules that play a vital role in cell differentiation, proliferation, and survival by binding to complementary target mRNAs, resulting in mRNA translational inhibition or degradation [
29]. Thus, miRNA mimics and miRNA inhibitors (antimiRs) have shown promise in preclinical development [
30]. It has been reported that miR-760 is downregulated in CRC plasma, and has an 80.0% sensitivity and 72.4% specificity (AUC = 0.788) for the early detection of CRC, suggesting it can be used for early CRC diagnosis [
31]. In the present study, we found that miR-760 expression in tumor tissues was significantly reduced compared with healthy cells, and low miR-760 expression was associated with advanced Dukes stage and lymph node metastasis (Table
1). Moreover, low miR-760 expression can predict poorer outcomes in CRC patients. Taken together, the expression of miR-760 in tumors might serve as a prognostic marker for CRC patients.
miRNAs are known to play an important role in human tumorigenesis by altering the expression of multiple genes, therefore, elucidating the molecular mechanism by which miRNAs function in tumor development may provide valuable diagnostic and therapeutic strategies for malignancy [
32‐
34]. It has been shown that miR-760 was downregulated in chemoresistant breast cancer tissues and might mediate breast cancer chemoresistance via the regulation of p-gp expression [
35]. Moreover, miR-760 was found to reduce the cancer stem cell population and inhibit breast cancer cell proliferation and metastasis via inactivation of NANOG transcription factor [
36]. However, there is also evidence to suggest that miR-760 acts as an onco-miR in certain cancer types. For example, it is reported that miR-760 was strongly overexpressed in ovarian cancer and miR-760 upregulation drastically promoted ovarian cancer cell proliferation by inhibiting the expression of PHLPP2 [
37]. Herein, we found that miR-760 was downregulated in CRC tissues and that miR-760 overexpression suppressed CRC cell proliferation by repressing BATF3 expression. As there are few reports that have explored the function of miR-760 in cancer and the precise role of miR-760 in cancer pathogenesis and progression remains unclear and even controversial, more studies are needed to determine the exact role and mechanism of miR-760 in tumor development.
In normal cells, BATF3 (also known as JDP1 and p21SNFT) is expressed in T helper type 1 cells and plays a major role in the development and function of conventional dendritic cells [
20]. Thanks to its leucine zipper motif sequence, BATF3 was recognized as belonging to a class of proteins that heterodimerize with JUN proteins [
38,
39]. The leucine zipper motif of BATF3 has been shown to be involved in the dimerization of the bZIP proteins to generate composite transcription factors that recognize palindromic TPA response elements (TREs) in their target genes [
40,
41]. Although BATF3 molecules lack transcriptional activation domains, unlike the AP-1 factors FOS and JUN, it has been reported that BATF3 can exert unique positive transcriptional specificity through interacting with members of the interferon regulatory factor (IRF) family [
24]. Recently, it has been reported that BATF3 forms AP-1 complexes with JUN in the MYC promoter and controls MYC expression in cHL (classical Hodgkin lymphoma) and ALCL (anaplastic large cell lymphoma) cell lines, critically supporting their proliferation and survival [
28]. However, there are a few conflicting reports showing that BATF3 might play a positive regulatory role in other tumor cells. Here, we observed miR-760 directly targeted BATF3 and ectopic miR-760 resulted in a reduction of BATF3 and cyclinD1.
It has been previously shown that the JNK-mediated phosphorylation of AP-1 (c-Jun) upregulates cyclin D1 to drive proliferation in liver cell regeneration and mouse epidermal cell transformation [
42,
43], which supported our data that miR-760 inhibited cyclinD1 via AP-1 in CRC cells. Experimentally, we verified that cyclin D1 mRNA 3′-UTR had AP-1(c-Jun)-binding sites in both SW620 and HCT116 CRC cells. Interestingly, we also found that loss of miR-760 accelerated the expression of BATF3, c-Jun (Additional file
2: Figure S4), cyclin D1 (Fig.
4f) and vice versa. Therefore, we propose that BATF3 forms AP-1 complexes with c-Jun in the cyclin D1 promoter and controls cyclin D1 expression in CRC cell lines, consequently supporting CRC cell proliferation and survival. As for the mechanism how BATF3 dimers with c-Jun and the complicated network between BATF3 and other AP1 family members like c-Jun, we will further explore the complex members of AP1 and their function or clinical significance in CRC in the future. Therefore, cyclin D1 may be tightly regulated by miR-760 in CRC, and miR-760 restoration could target BATF3/AP-1/cyclin D1 pathway to suppress CRC progression. Furthermore, it is likely that other molecules or signaling pathways will be discovered that are also targeted by miR-760 in CRC. Future work will focus on revealing additional functions of miR-760 in CRC carcinogenesis and progression.