Background
Colorectal cancer (CRC), a common digestive tract tumor, is considered as the third leading cause of cancer-related deaths all over the worldwide[
1]. Although several kinds of treatments have been developed recently for the patients with CRC, poor prognosis continues to be in patients with advanced CRC[
2]. Metastasis of CRC cells to vital organs is responsible for the majority of cancer deaths. Despite years of studies, the molecular underpinnings of CRC metastasis remain incompletely understood.
microRNAs (miRNAs) are a cluster of non-coding RNA molecules whose size ranges 19 to 24 nucleotides, and suppress target gene expression by initiating mRNA degradation and/or blocking translation[
3]. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation, apoptosis, and carcinogenesis and metastasis[
4‐
6]. Researches have found that miR-182 plays an important role in regulating mammalian circadian rhythm, T-cell development and DNA repair[
7,
8]. Several studies have illustrated that miR-182 is abnormally expressed in various tumors[
9,
10] and directly involves in human cancer processes, such tumorigenesis, migration and metastasis[
11‐
15]. It has been identified that the expression of miR-182 is up-regulated in CRC[
10,
16,
17]. This up-regulated expression of miR-182 can be correlated with the poor prognosis of patients with CRC[
10]. However, the role and mechanism of miR-182 in CRC have not been completely understood till date. In this study, we explored the role of miR-182 in CRC and found that the up-regulation of miR-182 promoted the proliferation, invasion, and metastasis of tumor cell by suppressing SATB2 in CRC. In addition, we explored the underlying mechanism of miR-182 functions in CRC. Therefore, our data demonstrated that miR-182 was indeed a metastasis promoter in CRC, and the study also shed light on the molecular mechanisms of its anti-metastasis function in CRC patients.
Methods
Tissue specimens and cell culture
Fresh and formalin-fixed, paraffin-embedded, CRC specimens were obtained from patients who were diagnosed with primary CRC and then underwent elective surgery in Nanfang Hospital, Southern Medical University (Guangzhou, China). The tissues that have been used for conducting this study have been approved by the ethics committee of Nanfang Hospital, Southern Medical University. In total, 31 cases of fresh CRC tissues were freshly frozen in liquid nitrogen and stored at -80°C until further use.
The human embryonic kidney cells 293T and the human CRC cell lines DLD-1, HCT116, SW480, SW620, and Lovo were obtained from a cell bank at the Chinese Academy of Sciences (Shanghai, China). A subclone named M5 with enhanced metastatic abilities in liver and a subclone named SCP 51 with enhanced metastatic abilities in liver and lymph node were isolated by
in vivo selection of SW480 cells through a process described in our previous studies[
18‐
20]. All CRC cell lines were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum (HyClone, Logan, USA) and 100 U/ml penicillin/streptomycin (Gibco). They were maintained in a humidified chamber with 5% CO
2 at 37°C. 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with 10% FBS.
RNA isolation and quantitative real-time PCR
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized using the PrimeScript RT reagent Kit (Promega, Madison, WI, USA). A stem-loop quantitative RT-PCR was carried out to detect the expression of mature miR-182 using ABI TaqMan® MicroRNA Assay kit (Applied Biosystems, Foster City, USA) and gene-specific primers (ABI) using an ABI 7500 Real-Time PCR system. mRNA levels of SATB2 gene were measured as previously described[
18,
21].
Vector preparation
To generate pLV-miR-182 expression vector, a 110-bp DNA fragment corresponding to pre-miR-182 was amplified and cloned into pLVTHM lentiviral vector. pLVTHM lentiviral vector encodes enhanced green fluorescent protein (EGFP) which has been optimized for brighter fluorescence and greater expression in mammalian cells. The previously described vector pCAG-SATB2[
18] was used to upregulate SATB2 expression.
A 2771-bp fragment of 3' untranslated region (3’UTR) was amplified in accordance with a previously described procedure[
21] to generate pGL3-SATB2-3’UTR. The putative miR-182 binding sites at SATB2 3’UTR were site-directed and mutated using GeneTailor Site-Directed Mutagenesis System (Invitrogen). All plasmids were verified by sequencing.
Lentivirus production and transduction
Virus particles were harvested 48 hours after transfecting pLV-miR-182 with the envelope plasmid pMD2.G and the packaging vector psPAX2 into 293T cells using lipofectamine 2000 reagent (Invitrogen). The lentivirus generated from the empty pLVTHM vector was used as a control (miR-con). These cells were infected with the recombinant lentivirus- transducing units and 8 mg/ml Polybrene (Sigma, St Louis, Missouri, USA) to generate four stable cell lines: two stable over-expression miR-182 cell line (SW480/miR-182 and DLD-1/miR-182) and two control cell line (SW480/miR-con and DLD-1/miR-con).
Oligonucleotide transfection
miR-182 mimics and antisense inhibitors containing 2’-OMe (O-methyl) modifications were synthesized by GenePharma (Shanghai, China). Oligonucleotide transfection was performed with Lipofectamine 2000 reagent (Invitrogen).
Luciferase reporter assay
In the presence of either miR-182 or miR-con, the firefly luciferase construct was cotransfected into SW480 cells with a control Renilla luciferase vector pRL-CMV (Promega). A dual luciferase assay (Promega) was performed 48 hours after transfection. These experiments were performed independently in triplicate.
Cell proliferation assay and colony formation assay
Cells were seeded in 96-well plates at 2 × 10
3 per well. Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8, Dojindo, Rockville, USA) according to the manufacturer's instructions. For performing colony formation assay, the cells were plated in 6-well plates at 2 × 10
2 per well and maintained in RPMI1640 containing 10% FBS for 2 weeks. After 2 weeks, the cells were washed twice with PBS, fixed with methanol and stained with 0.5% crystal violet. The number of colonies were counted under a microscope[
22].
Wound healing and invasion assays
Cell migration was assessed by measuring the movement of cells in a scraped, acellular area that was created using a 200 μL pipette tube. The spread of wound closure was observed after 0 and 48 hours, respectively. Photographs were taken to assess the level of migration in each group of transfected cells. Migration was quantified by counting the total number of cells that migrated toward the original wound field. For performing invasion assay, matrigel-coated chambers (BD Biosciences, San José, CA, USA) containing 8 μm pores were used. Cells were seeded into the upper chambers (coated in matrigel) by maintaining a concentration of 2 × 105 in serum-free medium. The lower chamber of the transwell was filled with culture media containing 10% FBS as a chemo-attractant. After the chambers were incubated at 37°C for 48 hours, non-invaded cells present on the top of the transwell were scraped off with a cotton swab. The successfully translocated cells were fixed with 10% formalin. Then, they were stained using 0.1% crystal violet for 30 minutes and counted under a light microscope.
In vivo functions assays
4–6 weeks old Balb/C-nu/nu athymic nude mice were obtained from the Laboratory Animal Centre of Southern Medical University. Mice were housed under pathogen free conditions in a 12 hours dark/light cycle and ad libitum access to food and filtered water. For performing tumor growth assay, a total of 2 × 106 cells of SW480 with stable over-expression of miR-182 were injected subcutaneously in upper and lower limbs of right flank of mice, while the control cells were injected into upper and lower limbs of left flank of mice (n = 6 per group). The tumor size was measured using digital calipers every three days. After monitoring for 30 days, mice were sacrificed by cervical dislocation and the tumors were dissected. Tumor volume was calculated as follows: Volume = D × d2 ÷ 2, where D meant the longest diameter and d meant the shortest diameter.
For developing the metastatic model, mouse cecum was exteriorized by laparotomy under sodium pentobarbital anesthesia. The subcutaneous tumors were diced into 1 mm3 cubes and implanted into the mesentery at the cecum terminus of mice. Then, the gut was returned to the abdominal cavity and closed with surgical drapes. After performing the surgery, the health of these animals was monitored on a daily basis. Body weight of mice was also recorded every three days. Humane endpoints had been planned at the end of experiment and as a means to relive pain or distress. At the end of the experiment (8 weeks), the still alive mice were sacrificed by cervical dislocation. The organs were removed and fixed using 10% neutral buffered formalin. Subsequently, consecutive tissue sections were obtained and stained with haematoxylin-eosin (H&E) to observe the metastatic nodules of organs under microscope. All experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. All necessary steps were taken to minimize the suffering and distress to the mice.
Western blot analysis
Protein lysates were separated using 10% SDS-PAGE gel electrophoresis and transferred to PVDF membrane (Amersham Pharmacia Biotech, NJ, USA). The membrane was probed with the following antibodies: anti-SATB2 (Abcam, Cambridge, UK), anti-Snail (Abcam, Cambridge, UK), anti-E-cadherin (Cell Signaling Technology, Inc), and anti-Vimentin (Cell Signaling Technology). Finally, the membrane was probed with HRP (horseradish peroxidase)-labeled goat-antimouse IgG (Santa Cruz Biotechnology, USA) and detected by chemiluminescence. A polyclonal anti-β-actin or anti-Tublin antibody was used as a protein-loading control. The intensity of protein fragments was quantified using the Quantity One software (4.5.0 basic, Bio-Rad).
Statistical analysis
All statistical analyses were performed using the SPSS 16.0 statistical software package. In at least three independent experiments, the data were presented in terms of mean ± SD. The differences between variables were assessed by the following three statistical tests: χ2 test, Fisher’s exact test, or One-way ANOVA. A p value less than 0.05 was considered to be statistically significant.
Discussion
miR-182 belongs to the miR-183 family which is comprised of miR-96, miR-182 and miR-183. They are coordinately expressed from a single genetic locus that is located at human chromosome 7q32.2. In recent times, miR-182 has emerged as an important regulator of various physiological and pathological processes[
8,
11,
23]. However, the functional analyses of miR-182 in tumorigenesis have yielded inconsistent results in different cancer models[
7,
9,
12]. In this study, we extend the study of miR-182 in tumor. We observed that the upregulation of miR-182 frequently occursred in CRC tissues, and that miR-182 high-expressed in CRC patients with metastases compared with those of patients without metastasis. In functional studies, overexpression of miR-182 dramatically promoted CRC cells proliferation, invasion and migration
in vitro and
in vivo. Moreover, miR-182 could suppress SATB2 via directly targeting its 3’-UTRs, and subsequently regulate EMT signaling. Therefore, these results firmly illustrated that miR-182 acts as a pivotal mediator in the development and progression of CRC.
In the previous study, it was revealed that miR-182 plays an oncogenic role in breast cancer[
13]. It was shown that miR-182 is associated with poor survival of breast cancer patients and promotes breast cancer metastasis[
12]. In melanoma, an over-expression of miR-182 promoted cell migration, and sustained miR-182 suppression that prevented liver metastasis of melanoma cell in mice[
14,
15]. On the other hand, miR-182 suppresses tumorigenesis of lung adenocarcinoma[
7]. A recent systematic meta-analysis revealed that miR-182 is deregulated in colon or colorectal cancer[
24]. The up-regulation of miR-182 in CRC was determined by miRNA microarray or quantitative RT-PCR analysis was consistent across three studies[
16,
17,
25]. Our results indicate that miR-182 was remarkably up-regulated in CRC tissues compared with adjacent non-neoplastic tissues. This is consistent with the previous findings of CRC studies[
16,
17,
25], suggesting that a positive role for miR-182 in tumorigenesis.
To reveal the unambiguous role of miR-182 in CRC cells, we established two stable miR-182-overexpression cell lines to test the effect of miR-182 on the behaviors of tumor cells. We conducted our search for determining the effects of miR-182 on CRC cells growth. The results indicated that miR-182 promoted cell growth and colony formation and accelerated tumorigenesis in murine model of CRC xenograft. Thus, the confirming results helped us in deducing that miR-182 is an oncogenic miRNA for CRC. In this context, we also observed that the expression level of miR-182 in patients with lymph node metastases was higher than that in patients without metastases. The expression level of miR-182 in the metastasizing cells was higher than with little metastatic powers. This indicated that the up-regulation of miR-182 can be considered as a predicted factor of metastasis for CRC patients. Indeed, the functional assays in vitro revealed that miR-182 can facilitate tumor invasion and migration. In animal models, the over-expression of miR-182 was sufficient to promote the metastasis of CRC cells to lung, liver, lymph node and organs in the peritoneal cavity. This correlation indicates that miR-182 might be playing a pivotal role in CRC metastasis.
In general, each miRNA regulates hundreds of target genes, influencing on almost every biological pathway. A number of mRNAs involve in distinct pathways are targeted by miR-182. In bladder cancer, miR-182 promotes the cell proliferation, migration and invasion by suppressing Smad4 and RECK[
26]. miR-182 can target tumor suppressor gene FOXO1 and functions as oncogene in endometrial cancer and breast cancer[
13,
27]. miR-182 also suppresses cylindromatosis (CYLD) to sustain NF-κB activation in glioma[
28]. A recent study revealed that miR-182 represses the expression of MIM (missing in metastasis gene) and promotes breast cancer metastasis[
12]. These results indicate that miR-182 exerts its own functions depending on its specific target genes. To further explore the mechanism through which miR-182 regulates CRC invasion and metastasis, we analyzed miR-182 targets. Among these predicted targets, SATB2 was of particular interest as our previous studies have reported the correlation of its silencing with CRC metastasis. 3’UTR of SATB2 mRNA contains a complementary site for the binding region of miR-182. We conducted luciferase activity assays to validate the targeting of SATB2 by miR-182. The activities of SATB2 3’UTR luciferase reporter were responsive to miR-182 over-expression. Endogenous SATB2 expression in both mRNA and protein decreased in miR-182 over-expression CRC cells. In this study, we have clearly illustrated that miR-182 might directly regulate SATB2 expression by inducing mRNA degradation and translational suppression.
SATB2 is a member of nuclear matrix-attachment proteins family that recognizes AT-rich DNA sequences at the base of looped-out chromatin. SATB2 is involved in various important genetic processes, such as chromatin condensation, interaction with other chromatin remodeling complexes, and regulation of transcriptionc[
29‐
31]. SATB1 is found to be a switch to control tumor growth and metastasis of breast cancer[
32] and CRC[
33]. In previous studies, we observed that the expression of SATB2 mRNA and protein was lower in metastatic CRC tissues and cell lines[
18]. Furthermore, we and other group have demonstrated that the down-regulated expression of SATB2 is associated with metastasis and poor prognosis in CRC[
18,
34]. SATB2 is a target gene of miR-31[
21]. However, the function of SATB2 has never been documented in CRC. Here, we assessed the functions of SATB2 in CRC cells growth and metastasis with an approach of over-expression. The results indicated that SATB2 suppressed proliferation, migration and invasion of CRC cells. Furthermore, the most important effect exerted by miR-182 on cell proliferation, invasion, and migration is partially reversed after transfecting with a SATB2 expression vector. Both miR-182 and SATB2 affect migration and invasion of CRC cells but in an opposite direction. These findings indicate that SATB2 might be an important target of miR-182, wherein it regulated invasion and metastasis in CRC.
In addition, our study revealed the functional mechanism of miR-182 in CRC metastasis. Epithelial-mesenchymal transition (EMT) appears to be a key event in tumor invasion and metastasis. Epithelial cells lose their epithelial adherence and tight junction proteins, lose their polarity and cell-cell contact, and undergo remarkable remodeling of the cytoskeleton resulted in facilitating cell motility and invasion. The reintroduction of miR-182 into CRC cells induced EMT, indicating the down-expression of epithelial markers and up-expression of the mesenchymal markers. However, Qu et al. illustrated the miR-182 induced mesenchymal to epithelial transition (MET) via repressing SNAIL2 in prostate cells[
35]. These controversial results indicate that the role of miR-182 is possibly tumor-specific and highly dependent on its targets in different cancer cells. Indeed, the tissue- and time-dependent expression of miRNAs influences protein translation during distinct cellular processes. The aberrant expression of their target genes affected different biological pathways having diverse function[
36]. One of the limitations of our study was that it did not exhibit the direct evidence of EMT-related molecules that were regulated by miR-182/SATB2 in our studies. It was demonstrated that SATB2 forms a complex with histone deacetylase 1 (HDAC 1)
in vitro and
in vivo. SATB2/HDAC1 complex can regulate gene expression through recruiting chromatin remodelers[
31]. Furthermore, histone deacetylase inhibitor treatment enhances Snail acetylation and induces epithelial-mesenchymal transition via inducing Snail transcription[
37]. Here, we put forward a hypothesis that SATB2 might recruit HDAC1 to remodel chromatin of Snail, and further regulate the expression of Snail, a key factor in EMT, which needs to be confirmed in further studies.
Competing interests
The authors declared that they do not have any competing interests.
Authors’ contributions
SW designed research. MY, JY, DJ and WL contributed in the experimental procedures. MY and SW analyzed the data. SW and YD supervised all the work. All the authors have read and approved the final manuscript.