MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene
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Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.
Colorectal cancer (CRC) is one of the most common malignances and the third leading cause of cancer-related death in the world, with an estimated incidence of 1 million new cases and a mortality of >600,000 deaths annually (1,2). Recent progress in diagnosis and therapy has helped to save the lives of many patients at early stages of this malignancy, but the prognosis for patients with advanced disease or metastasis is still very poor. Therefore, further investigation into the molecular pathogenesis of CRC and the consequential development of novel targeted therapeutics are needed. A series of studies have revealed that microRNAs (miRNAs) can regulate the expression of a variety of genes pivotal for tumor development and highlight a novel mechanism participating in CRC pathogenesis (3, 4, 5).
miRNAs are 21- to 23-nucleotide RNAs that negatively regulate gene expression by binding to the 3 untranslated regions (3 UTRs) of target transcripts, leading to mRNA degradation or inhibiting translation into protein. miRNAs are reported to play an important role in the pathogenesis of human cancers with disordered genome function (6,7). This result also applies to CRC; a subset of miRNAs was found to be aberrantly expressed in CRC, and most of the miRNAs are related to cell proliferation, apoptosis and tumor metastasis (4,8). By using miRNA arrays to compare the miRNA profiles in the CRC tissue samples and normal controls, we recently identified a promising candidate that is significantly downregulated in CRCs (miR-218). The identification and characterization of the role of novel miRNA in CRC may provide new insight into understanding the molecular mechanisms of disease development and is therefore crucial for the development of new therapy.
In the present study, we validated the downregulation of miR-218 by examining 48 paired CRC tumor and adjacent nontumor tissue samples. A series of in vitro and in vivo studies were then conducted to investigate the mechanisms and impact of miR-218 in colon cancer. We systematically discovered miR-218-mediated targets in silico by the algorithms TargetScan and MiRanda and experimentally validated that the repressions of BMI1 polycomb ring finger oncogene (BMI-1) was directly regulated by miR-218. We further demonstrated that ectopic expression of miR-218 in colon cancer cells led to reduced cell proliferation and induced apoptosis. Additionally, we demonstrate that miR-218 induces cell cycle arrest in the G2 phase of colon cancer cells by suppressing cyclin-dependent kinase 4 (CDK4) and up-regulating p53, two downstream targets of BMI-1.
Materials and Methods
Colon cancer cell lines HCT116, HT29, SW620 and LoVo were obtained from the American Type Culture Collection (Manassas, VA, USA). HCT116 and HT29 were cultured in McCoy’s 5A medium (Invitrogen; Life Technologies, Carlsbad, CA, USA), and SW620 and LoVo were cultured in RPMI 1640 containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). All the cells were cultured in a humidified 37°C incubator supplemented with 5% CO2.
Paired resected surgical specimens from primary tumor and adjacent nontumor sites were obtained from CRC patients who underwent surgery at the Prince of Wales Hospital, Hong Kong, according to a standard protocol, before any therapeutic intervention. The adjacent macroscopically nontumor mucosa at least 6 cm distant from the tumor was taken. The specimens were snap-frozen in liquid nitrogen and stored at −80°C for molecular analyses. The remaining tissue specimens were fixed in 10% formalin and embedded in paraffin for routine histological examination. All subjects provided informed consent before specimen collection. The study protocol was approved by the Ethics Committee of The Chinese University of Hong Kong.
Microdissection and RNA Extraction from Paraffin-Embedded Tissues
Five paraffin-embedded normal colon tissues were cut into 10-µm thickness sections by using a microtome. The muscularis mucosae in normal tissues were then excluded by laser-captured microdissection under a microscope. Total RNA was extracted from the five microdissected normal colon tissues by using a RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion, Austin, TX, USA).
Total RNA was extracted from tissue samples (10–30 mg) by using Trizol reagent (Invitrogen; Life Technologies). Total RNA was eluted in 50 µL nuclease-free water. RNA concentration was measured by Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA).
Reverse transcription for miRNA microarray was performed by using Megaplex Primer pools (Human Pools A v2.1and B v3.0; Applied Biosystems; Life Technologies). The cDNA products of miRNAs in the two pairs of tumor tissues and adjacent nontumor tissues of two CRC patients were used to perform miRNA array. The miRNA profile of 754 human miRNAs was obtained from each sample by using a TaqMan Human MicroRNA Array Set v2.0 (Applied Biosystems; Life Technologies). Briefly, 6 µL Megaplex RT product from each sample was added to 444 µL nuclease-free water and 450 µL TaqMan Universal PCR Master Mix with no AmpErase UNG (Applied Biosystems; Life Technologies). The reaction mix (100 µL) was loaded into each of the eight fill reservoirs of the array. Real-time quantitative polymerase chain reaction (PCR) was performed by using an Applied Biosystems 7900HT Real-Time PCR System (Life Technologies). Results were analyzed by the SDS RQ Manager 1.2 software (Applied Biosystems; Life Technologies).
Real-Time Quantitative PCR for miRNA and mRNA Expression Analyses
Quantitative real-time PCR (qRT-PCR) of individual miRNA was performed by using a TaqMan miRNA Reverse Transcription Kit (Applied Biosystems; Life Technologies) and TaqMan Human miRNA Assay kit (Applied Biosystems; Life Technologies). The comparative cycle threshold (Ct) method was used to calculate the relative abundance of miRNA compared with RNA U6 small nuclear 2 (RNU6B) expression (fold difference relative to RNU6B).
For BMI-1 mRNA quantification, RNA was reverse-transcribed with a random primer by using an ABI® Reverse Transcription Kit (Applied Biosystems; Life Technologies). mRNA levels were determined with qRT-PCR by using the SYBR Green master mixture on an HT7500 System (Applied Biosystems; Life Technologies) with the housekeeping gene GAPDH as an internal control. BMI-1 primers were as follows: forward 5′-GTGCT TTGTG GAGGG TACTT CAT-3′; reverse 5′-TTGGA CATCA CAAAT AGGAC AATAC TT-3′.
Total protein was extracted and protein concentration was measured by the Bradford DC protein assay (Bio-Rad, Hercules, CA, USA). Then, 20–40 µg protein from each sample were separated on 12% Bis-Tris polyacrylamide gel through electrophoresis and blotted onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). Blots were immunostained with primary antibody at 4°C overnight and secondary antibody at room temperature for 1 h. Proteins were visualized by using ECL Plus Western Blotting Detection Reagents (RPN2132; GE Healthcare).
Colony Formation Assay
Cells (1 × 105/well) were plated in a 24-well plate and transfected with premiR-218 or pre-miR control (control) at 30 nmol/L by using lipofectamine 2000 (Invitrogen; Life Technologies). After 48 h, the cells were collected and seeded (500–1,000/well) in a fresh 12-well plate for 10 d. The cells were stained with hematoxylin solution and the individual colony (>50 cells) was counted.
Cells (1 × 105/well) were collected 48 h after transfection with pre-miR-218 or control and were stained with Annexin V (allophycocyanin [APC] conjugated) and 7-aminoactinomycin D (7-AAD) according to the manufacturer’s instructions (BD Biosciences, Erembodegem, Belgium). Apoptosis was assessed by flow cytometry (BD FACSCalibur).
Vector Construction and Dual-Luciferase Reporter Assay
The potential miR-218 binding site in the BMI-1 3′UTR was predicted at position 1,470–1,476 from the transcriptional start site of BMI-1 (NM_005180) by TargetScan (www.targetscan.org) and miRanda (www.microRNA.org). Sequence of the 50-bp segment with the wild-type or mutant seed region was synthesized and cloned into the pMIR-REPORT luciferase vector (Applied Biosystems; Life Technologies). The mutant BMI-1 3′UTR sequence was prepared by mutating five nucleotides in the seed region. The synthesized oligos were shown as follows: wild-type: 5′-cgact ataag caaga caaag caata gaatT GTGCT Tcttt tgcag actgg ggaa-3′, 3′-tcgag ctgat attcg ttctg tttcg ttatc ttaAC ACGAA gaaaa cgtct gaccc cttga tc-5′; mutant type: 5′-cgact ataag caaga caaag caata gaatA GCTGT Acttt tgcag actgg ggaa-3′, 3′-tcgag ctgat attcg ttctg tttcg ttatc ttaTC GACAT gaaaa cgtct gaccc cttga t-5′.
Cells (1 × 105/well) transiently transfected with pre-miR-218 or control (at 30 nmol/L final concentration) were seeded in 24-well plates. pMIR-REPORT vector (195 ng/well) and pRL-cytomegalovirus (CMV) vector (5 ng/well) were cotransfected by using lipofectamine 2000 (Invitrogen; Life Technologies). Cells were harvested 48 h after transfection, and luciferase activities were analyzed by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
In Vivo Tumorigenicity
HCT116 cells (2 × 106 in 0.15 mL phosphate-buffered saline) were subcutaneously implanted into the left flank of the female nude mice (4 wks old) at d 0. After 5 d of implantation, control or miR-218 was injected into the tumor, and the injection was repeated every 3 d, respectively (n = 4 mice per group). miRNAs were prepared by preincubating 1.2 nmol miRNA with 10 µL Lipofectamine 2000 (Invitrogen; Life Technologies) for 15 min, and injections were made in a final volume of 100 µL in McCoy’s 5A medium (Sigma-Aldrich). Tumor sizes were measured every 3 d starting from the first injection with vernier calipers, and the tumor volume (V) was calculated as (L × w × w)/2, with L indicating length and w indicating width. The mice were euthanized when tumor size reached 12 mm in diameter and the tumors were excised. The animal studies and the experimental protocol were approved by the Department of Health of the Government of the Hong Kong Special Administrative Region and by the Committee on the Use of Live Animals in Teaching and Research of The Chinese University of Hong Kong.
Data were expressed as mean ± standard deviation (SD). Differences between two groups in luciferase reporter assay and comet assay colony formation assay were determined by a Student t test. Difference in cell growth curves and in vivo tumorigenicity were determined by repeated-measures analysis of variance. All analyses were performed by using SPSS for Windows 11.0.1 software. P < 0.05 was considered statistically significant.
All supplementary materials are available online at www.molmed.org.
miR-218 Was Downregulated from microRNA Array and Validated in the Primary CRC Tissues
Four patients with primary CRC were screened by using the miR array to identify miRNAs differentially expressed in CRC (data not shown). We identified 117 miRNAs downregulated in both samples. Among the candidate downregulated miRNAs (Supplementary Table S1), miR-218 showed a 2.5- to 10-fold decrease in expression level in the tumor samples compared with their adjacent nontumor samples.
miR-218 Inhibited the Expression of BMI-1 via Binding to Its 3′UTR
BMI-1 Is Upregulated in Primary CRC Tissues
BMI-1 was reported to be a potential oncogene in human cancers (9). To corroborate the potential importance of BMI-1 in primary CRCs, we compared the level of BMI-1 expression in 46 paired tumors versus the surrounding normal tissues. The expression of the BMI-1 mRNA accessed by real-time PCR was significantly increased in CRC tumors by about sixfold compared with the adjacent normal tissues (P < 0.0001; Figure 3D). BMI-1 is overexpressed in 84.8% (39/46) of tumors compared with their normal counterparts (see Figure 3D). Moreover, the enhanced level of BMI-1 mRNA was also detected in colon cancer cell lines compared with five normal colon tissues (Figure 3E).
BMI-1 Expression Correlates with miR-218 Expression in Primary CRC
To evaluate the correlation between BMI-1 and miR-218 expression in primary CRCs, we compared the expression of BMI-1 and miR-218 in 46 primary CRCs. Expression of BMI-1 and miR-218 exhibited a significant inverse correlation calculated by Pearson correlation (r = −0.823, P < 0.01), further supporting the miR-218 target status of BMI-1.
miR-218 Inhibits Cell Growth of Colon Cancer Cell Lines
miR-218 Induces Apoptosis in Colon Cancer Cell Lines
miR-218 Induces Cell Cycle Arrest
Concomitant with this inhibition of cell proliferation by miR-218, there was a significant increase in the number of cells accumulating in the G2 phase (P < 0.05) (Figure 5B). Thus, miR-218 reduced cell proliferative capacity with a G2 cell cycle arrest.
miR-218 Upregulates p53 via BMI-1 in HCT116
In the present study, we identified that miR-218 is a predominant downregulated miRNA by using an miRNA profiling assay. By analyzing more CRC patients, we confirmed that miR-218 expression was significantly decreased in CRC tissues when compared with the adjacent nontumor tissues (Figure 1). Reduced miR-218 expression was also demonstrated in colon cancer cell lines compared with the normal colon tissues. Consistent with our results, downregulation of miR-218 was reported in other human cancers, including gastric cancer (12), glioma (13), nasopharyngeal cancer (NPC) (14), lung cancer (15) and bladder cancer (16). Human miR-218 is encoded by two distinct genes (miR-218-1 and miR-218-2), which are processed into an identical mature sequence (www.mirbase.org). miR-218-1 and miR-218-2 are embedded in the intronic regions of the SLIT2 gene on chromosome 4p15.2 and SLIT3 on chromosome 5q35.1, respectively. Frequent deletion of the 4p15.1–15.3 region occurs in CRC (17). Loss of heterozygosity in this region therefore may be associated with the downregulation of miR-218 in CRC patients. On the other hand, the host genes of the SLIT family are often inactivated in CRC through their promoter hypermethylation (18). We found that miR-218 expression was restored in CRC cell lines upon administration of the DNA methylation inhibitor 5-aza-2′-deoxy-cytidine (data not shown). In keeping with our finding, miR-218 was reported to be downregulated by promoter hypermethylation in nasopharayngeal carcinoma and oral squamous cell carcinoma (19,20). These data indicated that the CpG island hypermethylation may also potentially contribute to the silence of miR-218 in human CRC. The downregulation of miR-218 in CRC indicated that it may play a role in CRC tumorigenesis as a potential tumor suppressor.
To identify the miR-218 target gene, we applied combined in silico seed site analysis and luciferase reporter assay. We demonstrated that BMI-1 is a direct target of miR-218, with the evidence that overexpression of miR-218 led to reduced luciferase activity of BMI-1 promoter and miR-218 downregulated BMI-1 expression (Figures 2, 3). BMI-1 was reported as an oncogene that regulates cell proliferation and transformation (21). BMI-1 is also crucial for self-renewal of stem cells and cancer initiation (22,23). We found that BMI-1 was overexpressed in CRC tumor tissues compared with adjacent normal tissues (Figure 3D). Enhanced BMI-1 expression was also observed in CRC cell lines. In keeping with our finding, other studies also reported that BMI-1 was overexpressed in CRC (24,25) and its overexpression was correlated with the malignant grades in pre-cancerous lesions (25) and a shorten survival of CRC patients (26). These results inferred that miR-218 targeted BMI-1 as a potential tumor suppresser in CRC.
We testified the putative tumor suppressor function of miR-218 in human CRC by both in vitro and in vivo assays. Overexpression of miR-218 in two colon cancer cell lines (HCT116 and HT29) showed significant growth-suppressing effect by inhibition of cell colony formation. In nude mice, colon cancer cells overexpressing miR-218 displayed a significantly lower growth rate than the control cancer cells. In addition, FACScan analysis revealed a significant increase of cell apoptosis in miR-218-reexpressed cells compared with the control cells. Moreover, overexpression of miR-218 can arrest the cell cycle in the G2 phase. Collectively, we indicate for the first time that miR-218 functions as a tumor suppressor in colon cancer. Other reports have shown that miR-218 acts as tumor-suppressive miRNA in several kinds of tumors, including gastric cancer (12), lung cancer (27), cervical cancer (28), head and neck cancer (29) and bladder cancer (16). Our findings have highlighted the importance of miRNA as a potential tumor suppressor in a solid cancer.
The growth-inhibitory activity was associated with rescuing BMI-1-mediated p53 suppression. The p53 protein was upregulated by overexpression of miR-218 in HCT116 (p53 wild-type) but not in HT29 (p53 mutant). This result was confirmed by enhanced p53 promoter luciferase activity in HCT116 cells after transfection with the miR-218 precursor. p53 is a well-known tumor suppressor gene; inactivation of p53 pathways is a common feature of many cancers (30,31). Other reports have found that BMI-1 inhibits c-Myc-induced apoptosis and c-Myc activates p53-dependent and -independent apoptosis pathways, depending on cancer cell type (32,33). In our investigation, miR-218 mediated BMI-1 down-regulation, which is accompanied by an induction of cell apoptosis. These results suggested that miR-218 suppresses BMI-1, which in turn induces apoptosis via a p53-dependent manner.
CDK4 belongs to the Ser/Thr protein kinase superfamily. The complexes formed by CDK4 and the D-type cyclins are involved in the control of cell proliferation and are required for G2 phase cell cycle progression, and the cyclin D3-CDK4 activity is necessary for cell cycle progression through the G2 phase into mitosis (34,35). We demonstrated that miR-218 inhibited BMI-1 downstream target CDK4 protein expression in colon cancer cell lines; thus, miR-218 mediated cell cycle arrest at the G2 checkpoint dependent, in part, on CDK4 inhibition. It was reported that BMI-1 deletion down-regulates CDK4 expression (10,36). In this regard, the suppression of BMI-1 may also contribute to the cell cycle arrest at the G2 checkpoint by miR-218. Recently, studies also demonstrated the tumor-suppressive role of miR-218 in other human cancers. miR-218 was reported to induce caspase-mediated apoptosis through interaction with the mechanistic target of rapamycin (serine/threonine kinase) (mTOR) component Rictor in oral cancer (20) and to activate tumor-suppressive genes Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) and claudin 6 (CLDN6) through directly inhibiting HoxB3 expression in breast cancer (37).
The potential clinical application of miR-218 in human cancer has been reported. Plasma miR-218 was significantly reduced in gastric cancer patients and might potentially serve as a noninvasive biomarker for gastric cancer detection (37). Patients with low miR-218 expression had shorter overall survival than patients with high miR-218 expression in lung cancer (38). However, there is no correlation between miR-218 expression and clinicopathological features in this study. The clinical implication of miR-218 in CRC remains to be investigated in the future with a large-scale sample size.
We have identified that miR-218 is downregulated in CRC. miR-218 suppressed colon cancer cell growth and increased cell cycle arrest and apoptosis, at least in part via inhibition of oncogene BMI-1. miR-218 is a candidate tumor-suppressive miRNA of CRC.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This project was supported by a National High-Tech R&D Program in China (863 Program, 2012AA02A506), National Natural Science Foundation of China (81072034) and an ITF fund in Hong Kong (project code ITS/276/11).
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