Background
Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer deaths globally, accounting for approximately 1.2 million new cases and 600,000 deaths each year [
1]. There is an urgent need to clarify the mechanisms underlying the pathogenesis of CRC and to develop novel and effective methods for its diagnosis and treatment [
2,
3]. The identification of tissue-specific biomarkers with prognostic and therapeutic significance is, therefore, an important strategy [
3,
4].
MicroRNAs (miRNAs) are small noncoding RNAs that induce degradation or translational repression of target gene mRNA. Recent evidence suggests that miRNAs are often aberrantly expressed in various cancers, and are correlated with prognosis and therapeutic outcomes in patients. In CRC, a number of miRNAs have been identified as regulators of cell proliferation and invasion, including miR-200a [
5], miR-214 [
6] and miR-221 [
7]. Therefore, more extensive investigations are required to identify additional relevant miRNAs and to clarify the roles of these molecules in CRC.
MiR-217 has been reported to play an important role in carcinogenesis. In pancreatic cancer [
8], hepatocellular carcinoma [
9], renal cell carcinoma [
10] and chronic myelogenous leukemia [
11], miR-217 is downregulated and functions as a tumor suppressor, while it overexpressed and acts as an oncogene in B-cell lymphomas [
12]. Moreover, miR-217 was demonstrated to modulate epithelia cell senescence in metabolic disorders [
13]. However, the role of miR-217 in CRC remains to be elucidated. In 2009, Stuckenholz et al. [
14] identified a number of novel genes, including miR-217, involved in mammalian gastrointestinal development, which were implicated as potential targets for therapeutic intervention in the management of gastrointestinal disease and cancer. Based on these findings, we hypothesized that miR-217 plays a role in human CRC. The present study compared the expression of miR-217 in CRC tissues and normal colorectal (CRN) tissues. Furthermore, the correlations between miR-217 and the clinical characteristics of CRC were analyzed.
Prediction software (TargetScan, microRNA and miRDB) analysis indicated that astrocyte-elevated gene-1 (AEG-1) is a potential target of miR-217. AEG-1, also known as metadherin (MTDH) or LYRIC, is induced in primary human fetal astrocytes infected with HIV-1 or treated with a recombinant HIV-1 envelope glycoprotein (gp120) [
15]. AEG-1 has been reported to be significantly overexpressed and function as a key oncogenic factor in various tumors such as breast cancer [
16], neuroblastoma [
17], hepatocellular carcinoma [
18], cervical cancer [
19] and gastric cancer [
20]. In CRC, ectopic expression of AEG-1 is observed in CRC tissues and high AEG-1 expression correlates with poor overall survival of patients [
21,
22]. Furthermore, inhibition of AEG-1 expression resulted in suppression of proliferation and invasiveness of CRC cells, with modulation of MMP2 or AMPK signaling [
23‐
25]. These findings suggest that AEG-1 promotes CRC. Therefore, in the current study, we investigated AEG-1 as the target for miR-217 to further explore the effects of miR-217/AEG-1 signaling on CRC.
Methods
Tissue samples and cell lines
Tissue samples were obtained from patients undergoing coloproctectomy according to the National Comprehensive Cancer Network (NCCN) guidelines for colon/rectal cancer (version 1. 2013). Samples were immediately snap-frozen and stored at −80 °C until RNA and protein extraction. All samples were identified as colorectal adenocarcinoma by two pathologists independently. All patients provided written informed consent before samples were collected and the study was approved by the local Research Ethics Committee of Peking University. The human CRC cell lines SW480, SW620, RKO, HT29, HCT116, and LoVo were purchased from the American Type Culture Collection (Manassas, VA, USA). NCM460 cells were purchased from INCELL Corporation (San Antonio, TX, USA). The genotypes of all cell lines were authenticated by DNA fingerprinting. All cells were cultured in RPMI1640 medium supplemented with 10 % fetal bovine serum (FBS) (all from Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C under 5 % CO2.
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Reverse transcription was performed with a reverse transcription kit (Takara, Japan). MiRNAs and potential target gene expression levels were measured by qRT-PCR with the SYBR Green PCR Kit (Takara) using the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Human U6 RNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was amplified as an internal control. The RNA expression levels were calculated according to 2
-ΔΔCt. MiR-217 expression was deemed to be high when the expression level was equal to or above the median of the cohort and low when it was below the median of the cohort [
26]. Primer sequences are shown in Additional file
1: Table S1. The universal reverse primers provided by Takara were used for amplification of U6 and miR-217.
Transfection
The miRNAs and siRNAs used in this study were designed and synthesized by RiboBio (Ribobio Co., Guangzhou, China). AEG-1 encoding plasmids were obtained from Invitrogen. Transfections with miRNA, siRNA or AEG-1 plasmids were performed using Lipofectamine 2000 (Invitrogen). CRC cells were seeded into 12-well plates before the transfection. The final concentration of miR-217 mimics or inhibitor or siRNA-AEG1 was 50 nM. The lentiviral miR-217 (LV-miR-217) and empty lentiviral (LV-miR-NC) vectors were generated by Genechem Company (Shanghai, China) and were used to transfect CRC cells according to the manufacturer’s instructions. All oligonucleotide sequences used in this experiment are listed in Additional file
1: Table S2.
Western blot assays
CRC cells were collected at 48 h after treatment with 50 nM miR-217 mimics or inhibitor or siRNA-AEG1 and corresponding controls. Protein extraction, SDS-PAGE gel electrophoresis and blotting were performed as previously described [
27]. Details of the primary detection antibodies are shown in Additional file
1: Table S3.
Cell proliferation and colony formation assays
The CCK8 colorimetric assays (Dojindo, Kyushu, Japan) were performed to estimate the cell proliferation rate according to the manufacturer’s protocol. The cells were incubated for 4 h after adding the CCK8 reagents. Proliferation at different time-points was assessed by measuring the absorbance at 450 nm using a microplate reader (Bio-Rad). The CCK8 assay was repeated three times with six replicates.
For colony formation assays, transfected cells were seeded into 6-well plate, incubated for 10 days and then stained with 0.1 % crystal violet. The colony assay was repeated three times using duplicate samples.
Evaluation of cell cycle distribution and apoptosis
These assays were performed by BD Biosciences flow cytometry as previously reported [
28]. For cell cycle assays, cells were collected and stained using BD cycletest
TM plus DNA reagent kit (BD Biosciences) according to the manufacturer’s instructions. For cell apoptosis analysis, cells were collected 72 h after transfection, and the assays were performed with the Alexa Fluor
R488 annexin V/Dead cell apoptosis kit (Invitrogen). Data were analyzed with FlowJo V7 software (Tree Star, Ashland, OR, USA).
Invasion assay
Transwell assays were performed to evaluate the invasive ability of CRC cells. Briefly, cells were seeded in the upper chamber (24-well plates, 8-μm pore size, Corning) with media containing 0.1 % bovine serum albumin and media containing 30 % FBS was placed in the lower chamber. After culture for 48 h, invasive cells at the bottom of the membrane were stained with 0.1 % crystal violet and were counted under a microscopic. Invasion assays were repeated three times using duplicate samples.
Dual-luciferase assay
MiR-217-binding region of AEG-1 was identified by TargetScan 6.2 (
http://www.targetscan.org/). SW480 cells were seeded in 96-well plates and cotransfected with total of 100 ng pMIR-REPORT Luciferase vector (Ribobio Co.) containing the AEG-1 3′UTR (0–1,500 bp) or mutated sequences plus 50 nM miR-217 mimics or negative control (NC) mimic according to the manufacturer’s instructions. After incubation for 48 h, luciferase activity was determined using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The relative luciferase activities were determined by normalizing to Renilla Luciferase activities.
CRC xenograft model
Four-week-old female BALB/c-nude mice (Vital River Laboratories, Beijing, China) were used to investigate SW480 cell tumorigenicity. A total of 200 μL cell suspensions (containing 1 × 107 SW480 cells) were subcutaneously injected into the right flank of the mice. Tumor volumes were measured every 4 days and calculated according to V = 0.5 × L (length) × W2 (width). At 32 days after the cell inoculation, mice were sacrificed and tumors were excised to measure the volume. All animal experiments were reviewed and approved by the Animal Research Committee of the Peking University People’s Hospital. Care and handling of the animals was performed in accordance with the guidelines of the Institutional and Animal Care and Use Committees.
Statistical analysis
Unless otherwise specified, all results were expressed as mean ± SD and analyzed using the SPSS 20.0 software (SPSS, Chicago, IL, USA). Differences between groups were assessed using Student’s t-test and Fisher’s exact test. The relationship between miR-217 expression and the clinicopathologic features of CRC was analyzed using the Pearson χ2 test. The differences between the two patient groups were analyzed by log-rank tests and the Kaplan–Meier method was used to calculate the overall survival. P < 0.05 was considered to indicate statistical significance.
Discussion
MiRNAs are known to be play a key role in tumorigenesis as a result of their involvement in many cellular processes including cell proliferation, differentiation, apoptosis and invasion [
29,
30]. In the present study, we focused on miR-217, which is abnormally expressed in a variety of cancer types [
8‐
10,
12]. To date, the evidence for aberrant expression of miR-217 in CRC has been obtained in microarray studies. In our study, qRT-PCR analysis demonstrated that miR-217 was significantly downregulated in CRC tissue samples and cancer cell lines. Furthermore, our study revealed, for the first time, the involvement of miR-217 in tumorigenesis through targeting AEG-1 targeting and that decreased miR-217 expression correlated with poor prognosis in patients with CRC. These findings implicate miR-217 as a novel prognostic marker in CRC.
Moreover, analysis of clinical data indicated that reduced expression of miR-217 in CRC patients correlated with poor tumor differentiation. In addition, Cox’s multivariate analysis indicated that miR-217 expression, TNM stage and distant metastasis act as an independent factor in the prediction of overall survival among patients with CRC.
In this study, we also showed, for the first time, that overexpression of miR-217 significantly repressed CRC cell proliferation, colony formation, and induced G0/G1 cell cycle arrest and apoptosis. Moreover, our in vivo studies confirmed that miR-217 overexpression remarkably suppressed CRC xenograft tumor growth in nude mice. These results imply that miR-217 acts as an inhibitor of colorectal tumorigenesis.
Metastasis, one of the most critical hallmarks of cancer, is the leading cause of cancer-related deaths worldwide, particular in CRC [
31,
32]. Accumulating evidence demonstrates the close correlation of invasive capacity and metastasis with miRNAs, such as miR-124 in nasopharyngeal carcinoma [
33], miR-153 in CRC [
34] and miR-335 in lung cancer [
35]. This evidence elucidating the role of miRNAs in CRC metastasis might represent the basis of a new therapeutic approach for CRC. The clinical outcomes in the patients in this study revealed that the expression level of miR-217 was closely correlated with CRC distant metastasis and also acted as an independent prognostic factor in patients with CRC. It is well-known that invasive tumors exist within a complex microenvironment composed of extracellular matrix (ECM) proteins, which play important roles in tumor invasion and metastasis [
36]. Thus, matrigel invasion assays were performed in our study to mimic this environment. The results showed that after overexpression of miR-217, the invasion capability of CRC cells was significantly reduced, indicating the involvement of miR-217 in CRC invasion and metastasis. Thus, it can be hypothesized that restoration of miR-217 in CRC might be a new therapeutic approach in CRC, especially in CRC with distant metastasis.
The effects of miRNAs are largely dependent on their regulation of the expression of many cancer-related genes through post-transcriptional repression [
37]. Using bioinformatics analysis, we found that miR-217 targeted multiple cancer-related genes that have been reported to have a close link with cancers, such as KRAS (pancreatic cancer) [
8], E2F3 (hepatocellular carcinoma) [
9], and DACH1 (breast cancer) [
38]. Interestingly, in this study, AEG-1 was predicted to be one of the target genes of miR-217. AEG-1 expression is frequently increased in multiple cancers including CRC [
21‐
23] and plays a critical role in oncogenic transformation and angiogenesis, which are essential to tumor cell development, growth, and metastatic progression [
39‐
41]. These studies provide important insights and a unique perspective on this multifunctional oncogene. In the current study, we evaluated the prognostic value of AEG-1 in CRC patients. Although the survival analysis showed no significant difference between the AEG-1-low and AEG-1-high groups, the median survival time was longer in the patients with low AEG-1 expression. Moreover, knockdown of AEG-1 repressed cell growth and invasion, induced G0/G1 arrest and apoptosis, which was similar to the effects of miR-217 overexpression. Thus, the results of our study indicate that AEG-1 acts as a tumor promoter in CRC.
We next used dual-luciferase assays to determine whether miR-217 binds directly to the 3′UTR of AEG-1 mRNA. Ectopic expression of miR-217 resulted in significant AEG-1 downregulation at both the mRNA and protein levels, whereas miR-217 silencing led to restoration of AEG-1 expression. Furthermore, the expression level of AEG-1 was inversely correlated with the miR-217 expression in both CRC and CRN tissues. Therefore, the results indicate that decreased AEG-1 expression represents a mechanism by which miR-217 plays a role in the progression of cancer. To further clarify this point, we performed a rescue experiment which demonstrated that AEG-1 overexpression significantly reversed miR-217-induced apoptosis, cell cycle arrest, proliferative inhibition and invasive suppression of SW620 cells. However, the subcutaneous xenograft model in our study cannot sufficiently represent clinical CRC, especially with regard to metastasis [
42]. The present study demonstrates that miR-217 remarkably represses the invasive ability of CRC cells
in vitro; therefore, further investigations in a metastasis model are required to clarify the effects of miR-217 on invasion and metastasis of CRC
in vivo.
Competing interests
The authors declare that we have no competing interests.
Authors' contributions
Conception and design: WB, SZL, WS
Development of methodology: WB, JKW, GZD
Acquisition of data: YY, ZJZ, SC
Analysis and interpretation of data: WB, ZG, WCY
Writing the manuscript: WB, SZL
Administrative, technical or material support: JKW, ZG, WCY, YYC
Study supervision: YYJ, WS
All authors read and approved the final manuscript.