- Open Access
miR-133b, a muscle-specific microRNA, is a novel prognostic marker that participates in the progression of human colorectal cancer via regulation of CXCR4 expression
© Duan et al.; licensee BioMed Central Ltd. 2013
Received: 12 July 2013
Accepted: 5 December 2013
Published: 13 December 2013
MicroRNA-133b (miR-133b), which is a muscle-specific microRNA, has been reported to be downregulated in human colorectal carcinoma (CRC) when compared to adjacent non-tumor tissue. However, its diagnostic value and role in CRC have yet to be described. CXC chemokine receptor-4 (CXCR4), which participates in multiple cell processes such as cell invasion-related signaling pathways, was predicted to be a potential target of miR-133b. The aim of this study was to investigate the associations and functions of miR-133b and CXCR4 in CRC initiation and invasion.
Mature miR-133b and CXCR4 expression levels were detected in 31 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 6 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate CXCR4 as a putative target gene of miR-133b. Regulation of CXCR4 expression by miR-133b was assessed using qRT-PCR and Western blot analysis, and the effects of exogenous miR-133b and CXCR4 on cell invasion and migration were evaluated in vitro using the SW-480 and SW-620 CRC cell lines.
A significant downregulation of miR-133b was observed in 93.55% of CRC tissues, and the expression of miR-133b was much lower in metastatic tumors (stage C and D, stratified by the Modified Dukes Staging System) than in primary tumors (stage A and B). In contrast, CXCR4 protein expression significantly increased in 52.63% of CRC samples, and increased CXCR4 expression in CRC was associated with advanced tumor stage. CXCR4 was shown to be a direct target of miR-133b by luciferase reporter assays, and transfection of miR-133b mimics inhibited invasion and stimulated apoptosis of SW-480 and SW-620 CRC cells.
Our study demonstrated that downregulated miR-133b contributed to increased cell invasion and migration in CRC by negatively regulating CXCR4. These findings may be significant for the development of therapy target for CRC.
Colorectal cancer (CRC) is the third most common cancer in males and the second most common cancer in females worldwide and has high incidence and mortality rates . The number who are affected continues to rise, especially in most Asian countries . Despite gradually improved therapeutic schedules, post-operative recurrence and metastasis remain the two most challenging problems for prolonging patient survival time after surgery. Thus, it is necessary to understand the precise molecular mechanisms that modulate malignant transformation.
MicroRNAs (miRNAs), which are a class of endogenous, single-stranded RNA molecules of 20–25 nucleotides in length , have emerged as critical regulators of carcinogenesis and tumor progression over the last decade [4, 5] and are likely to be involved in widespread biological functions, such as cell proliferation, apoptosis, invasion, angiogenesis and metastasis [5, 6]. In addition, reports have increasingly shown the potential of using miRNAs as novel diagnostic markers and therapeutic targets [4, 7–9].
miR-133b, which is a miRNA commonly recognized as a muscle-specific molecule, participates in myoblast differentiation [10, 11] and myogenic-related diseases [12, 13]. Recent studies showed that miR-133b also plays a crucial role in the malignant progression of non-muscle-related diseases [14–16] such as cancer [14–19]. For example, Bandrés et al. revealed the deregulation of miR-133b alongside 12 deregulated miRNAs in 15 CRC cell lines and 6 paired human CRC specimens. Hu et al. uncovered receptor tyrosine kinase MET as one target of miR-133b in CRC and demonstrated its involvement in cell proliferation and apoptosis. Another study showed that the downregulation of miR-133b in CRC tissues, when compared to adjacent non-tumor tissues, was linked to poor survival . However, it remains undetermined how miR-133b functions in CRC pathogenesis and progression, especially in CRC invasion and metastasis.
The CXC chemokine receptor 4 (CXCR4) belongs to the G protein-coupled receptor (GPCRs) family [20, 21]. Through a specific interaction with its ligand CXCL12 (stromal cell-derived factor-1, SDF-1) , CXCR4 participates in the development of primary tumors and metastases . The dysregulated expression of CXCR4 was detected in several human cancers that included melanoma , breast , pancreatic  and CRC . In particular, as a versatile factor in human CRC, CXCR4 influences aspects such as proliferation , migration and invasion [27, 28]. Understanding the regulation network of CXCR4 would give us a deeper insight into the mechanisms underlying CRC metastasis and help in the development of new therapeutic regimens.
In this study, we found that CXCR4 was a direct target of miR-133b in colorectal cancer. We also demonstrated that miR-133b contributed to increased cell invasion by negatively regulating CXCR4 activity in CRC carcinogenesis and progression.
Decreased expression of miR-133b in human CRC showed significant diagnostic potential
We then examined the sensitivity and specificity of miR-133b. A receiver operating characteristic (ROC) curve analysis was performed using the relative expression of miR-133b, and the associated area under the curve (AUC) was used to confirm the diagnostic potency of the miRNA. As shown in Figure 1C, the AUC of miR-133b reached 0.8081 [95% confidence interval (CI): 0.6857-0.9306, P < 0.001], with a cut-off point of 77.42% sensitivity and 78.95% specificity. These results suggest that miR-133b can discriminate between CCA tissues and their paired adjacent normal tissues.
CXCR4 was a direct target of miR-133b
To gain insight into the biological role of miR-133b that underlies disease pathogenesis, we further investigated its downstream targets. Three bioinformatics algorithms, TargetScan , miRBase Target  and StarBase , were applied to search for the potential targets of miR-133b, and a number of potential targets were predicted. Among the predicted targets, CXCR4 was the most interesting. CXCR4 has been reported to be widely expressed and to exert large-scale effects in cancer cells by participating in multiple cellular processes, including cell invasion-related signaling . It has also been reported that upregulation of CXCR4 was found in CRC patients and increased the risk of recurrence and poor survival from CRC .
The inverse correlation between miR-133b and CXCR4 in CRC cell lines and clinical samples
To further validate the correlation between miR-133b and CXCR4, we then detected the expression levels of the CXCR4 protein in the six human CRC cell lines and in the clinical samples that were previously used for miR-133b detection. Intriguingly, the CXCR4 protein expression level in SW-620 was shown to be much higher than in SW-480 (Figure 3A) and was negatively coexpressed with the expression of miR-133b (Figure 3A). These results supported the hypothesis that CXCR4 is repressed by miR-133b. We then investigated the coexpression pattern between miR-133b and CXCR4 in the clinical samples. A panel of clinical samples that included CRC tissues and their corresponding adjacent non-neoplastic tissues was used.
Correlation analysis showed a significant relationship between these factors (Spearman r = -0.2840; p = 0.0456), and the results are shown in Figure 3B. The expression levels of miR-133b were significantly lower in CRC tumor tissues when compared to the NT group (Figure 1A). Conversely, the levels of CXCR4 protein were elevated in 52.63% (10/19) of the tumors when compared to their corresponding non-tumor tissues (Additional file 2: Figure S2). The remainder of the tested samples showed no significant differences between the two groups. Thus, a negative correlation exists between the level of miR-133b and the level of CXCR4 protein in CRC tumors.
Effects of miR-133b overexpression on cell proliferation and apoptosis by modulating CXCR4 levels
To investigate whether miR-133b functions as a tumor suppressor by promoting cell apoptosis and impairing proliferation, we performed overexpression and knockdown studies to characterize the effect of miR-133b on CRC proliferation using the miR-133b mimics, miR-133b inhibitor and siCXCR4 in SW-480 and SW-620 cells. The transfection efficiencies of siCXCR4 in both cell lines are shown in Additional file 1: Figure S1. The introduction of miR-133b or knocking down CXCR4 with siCXCR4 caused a remarkable inhibition of cell proliferation in SW-480 and SW-620 cells when compared to the controls (p < 0.05 at 96 h) (Additional file 3: Figure S3A and B). In contrast, when miR-133b activity was impeded by the miR-133b inhibitor, the cells presented strengthened proliferation ability (p < 0.05 at 96 h) (Additional file 3: Figure S3C and D).
Proliferation and apoptosis are two classic but crucial aspects of nearly all acquired diseases. Accordingly, fluorescence-activated cell sorting (FACS) analysis was used to assess whether miR-133b contributed to apoptosis in CRC cells. Apoptosis was measured after transfecting miR-133b and siCXCR4 into SW-480 and SW-620 cells for 48 hours, and this was followed by a 24-hour exposure to cisplatin at an appropriate concentration, as previously described  (The IC50 results are not shown). The results revealed a significant increase in apoptosis of SW-620 cells transfected with miR-133b mimics compared to the control transfected cells (the percentage of apoptotic cells increased from 21.31% to 88.37%; Figure 4B; p < 0.001).
The converse effect was observed in cells transfected with miR-133b inhibitor (Figure 4C). As observed in Figure 4C, the apoptotic rate in SW-480 cells transfected with the miR-133b inhibitor dropped from 18.77% to 10.67% (p < 0.01), and this apoptosis-promoting effect of siCXCR4 was corroborated in both cell lines. In SW-480 cells, apoptosis increased from 29.13% to 55.81% (p < 0.001), and in SW-620 cells, it increased from 20.69% to 50.09% (Figure 4D, p < 0.001). The apoptosis result was further confirmed using fluorescence microscopy, in which the pretreatment of cells was similar to that by flow cytometry analysis (Additional file 5: Figure S5). These results indicate that overexpression of miR-133b induced an aggravated apoptosis rate and an impaired proliferation of CRC cells.
Forced expression of exogenous miR-133b decreases CRC cell invasion and migration in vitro
CRC is one of the most common and lethal cancers and has a high relapse rate. Therefore, there is a strong need to develop novel, prognostic factors and therapeutic strategies. The outcome of CRC patients is determined primarily by the presence or absence of metastases. Thus, insight into the molecular mechanisms underlying the precise molecular mechanisms that modulate malignant transformation is required. Previous studies have shown that aberrant expression of miR-133b was found in CRC cancer tissues [14, 17] and that overexpression of miR-133b induced apoptosis and G1 cell-cycle arrest in CRC cells . Furthermore, miR-133b has reportedly been shown to be involved in the invasion of several other cancers. For instance, miR-133b was found to be downregulated in non-small cell lung cancer and modulate apoptosis and invasion , and overexpression of miR-133b has been shown to inhibit cell invasion activity in esophageal squamous cell carcinoma . However, the relationship between miR-133b expression and cell metastases in CRC has yet to be demonstrated.
In the present study, we investigated the expression patterns of miR-133b in CRC clinical samples and identified low miR-133b expression as a valid factor associated with advanced tumor stages. Further functional analysis revealed the involvement of miR-133b in the progression of human CRC, and transfection of miR-133b into two CRC cell lines, SW-480 and SW-620, significantly decreased tumor cell migration and invasion in vitro. These data provide the potential of miR-133b to serve as a molecular target for CRC therapy, especially for tumors with high degrees of metastasis. It is also worth noting that the outcome of CRC patients is highly relevant to the extent of local invasion; therefore, the metastases-related miR-133b might provide tumor progression and prognostic information in CRC patients who would need to be experimentally validated prospectively.
We revealed the involvement of miR-133b in the progression of human CRC via the regulation of CXCR4 expression. A significant correlation was also found between miR-133b and CXCR4 protein expression in tumor samples. The activation of CXCR4, a G protein-coupled receptor for CXCL12, induced tumor invasion and/or survival of cancer cells. CXCR4 has also been reported to be involved in a number of processes related to the immune system , the nervous system , angiogenesis , the hemopoietic system  and carcinogenesis [28, 43–46]. Therefore, it is a key receptor in the crosstalk between tumor cells and their microenvironment.
Our results demonstrated that the miR-133b/CXCR4 pair is involved in tumor growth and tumor cell apoptosis and controls cell migration and invasion. Intriguingly, CXCR4 has been regarded as an impressive anticancer target that suppresses the outgrowth of metastases in CRC . Moreover, previous reports have shown that the small non-peptide CXCR4 inhibitor ADM3100 effectively inhibited the invasion and metastasis activity of CRC , which strongly shows the potential of CXCR4 as a therapy target. Furthermore, we found that the miR-133b/CXCR4 interaction influenced CRC progression through modifying the VEGF and MMP-9 genes, both of which play significant roles in CRC, especially in migration and invasion [48, 49]. More importantly, we determined the downstream molecules of the miR-133b/CXCR4 interaction as was done in previous research on CXCR4 in CRC . This finding implies that miR-133b regulates CXCR4 to affect its classic underlying pathway, which highlights the potential of this miRNA to be used as a CXCR4 inhibitor in CRC treatment. Taken together, our research provides an alternative strategy for developing miRNA-based therapy via CXCR4 targeting in CRC, and this is considered more security for the natural and endogenous of miRNAs.
In conclusion, our current findings provide the first glimpse of the functional role of miR-133b in CRC carcinogenesis and progression through the negative regulation of CXCR4. We also identified the crucial role of this miRNA in tumor cell invasion. These results indicate that miR-133b may be a useful therapeutic target in CRC.
Materials and methods
Patients, tissues, cell lines and cultures
Detailed clinical information of CRC patients used in this study
Age at diagnosis
The HEK-293T and human CRC cell lines SW-480, SW-620, HCT-15, HCT-116, Caco-2 and RKO were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), maintained in a 37°C humidified incubator, and cultured in appropriate media as recommended by the supplier.
Wild-type and full-mutated miR-133b putative target segments comprising 59 bp of the 3′UTR (untranslated terminal region) of CXCR4 were synthesized by Invitrogen (Invitrogen, China) and cloned into the psiCHECK-2-CXCR4 vector (Promega, Madison, WI, USA) for miRNA functional analysis. These plasmids were designated psiCHECK-2-CXCR4 wt and psiCHECK-2-CXCR4 full mut, respectively. The psiCHECK-2-CXCR4 full-mutated vector introduced the full mutation into the miR-133b binding sites of the CXCR4 3′UTR. Additionally, we generated a luciferase vector containing the full length 3′UTR of CXCR4 by RT-PCR, and this was designated as psiCHECK-2-CXCR4 full length. Proper insertion was confirmed by sequencing, and all utilized primers are described in Additional file 6: Table S1.
The following oligonucleotides were purchased from GenePharma (GenePharma, Shanghai, China): miR-133b mimics; miRNA negative control (designated as miR-NC); miR-139 mimic as a positive control; miR-133b antisense with a sequence complementary to the mature miR-133b; and miRNA antisense negative control (designated as inhibitor-NC), which is a negative control for miR-133b antisense. The small interfering RNAs (siRNA) against the human CXCR4 (GenBank Access. NM_001008540.1 and NM_003467.2) transcripts (denoted as siCXCR4) and the negative control RNA duplex (denoted siRNA NC) were purchased from Guangzhou Ribo-Bio Co., Ltd (Guangzhou, China). The sequence of siCXCR4 is described in Additional file 6: Table S1. Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA) was used for reverse transfection of the small molecules as well as cotransfection of the miRNA mimics and reporter vectors at optimized concentrations (10–200 nM) according to the manufacturer’s recommendation. The plasmid pcDNA-6.2 containing GFP was used as a positive control for plasmid transfection, and Block it™ tagged with fluorescein was used as a positive control for oligonucleotide transfection. Twenty-four to 60 hours after transfection, the cells were harvested for the dual luciferase reporter assay, protein analysis or RNA extraction.
Luciferase target assays
Once 70-80% confluent in 48-well plates, HEK-293T cells were cotransfected with 50 ng/well of each luciferase reporter plasmid and 10 nM/well of either miR-133b mimic, miR-139 mimic or miR-NC, as described above. The lysates were collected 36 hours posttransfection to determine firefly and Renilla luciferase activity using the Dual-Luciferase Assay Kit (Promega, Madison, WI) following the manufacturer’s instructions. All experiments were performed in triplicate.
Total RNA extraction and quantitative reverse transcription-PCR (qRT-PCR) analysis
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Tissue was porphyrized in liquid nitrogen, and then the RNA was extracted with TRIzol.
The expression of mature miR-133b was determined using the Hairpin-it™ Assay kit (GenePharma, Shanghai, China) and normalized to U6-snRNA. A qRT-PCR for the CXCR4-mRNA was performed using the SYBR Premix ExTaq real-time PCR kit (Takara, Japan) according to the manufacturer’s instructions with GAPDH as the normalization controls, respectively. Each reaction was carried out in triplicate. To calculate the relative expression levels, we used the 2-∆∆CT-method. All primer sequences can be observed in Additional file 6: Table S1.
Protein extraction, western blotting and antibodies
Specimens were preprocessed as mentioned above, and total protein was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as recommended. Protein samples were lysed in buffer containing 1% DTT, 4% CHAPS, 7 M urea, 2 M thiourea and 2% ampholine. A volume of extract equivalent to 15 μg of total protein was separated in a 12% SDS-PAGE gel and then transferred to a methanol-activated PVDF membrane (Millipore, Beijing, China). The membranes were blocked with 5% BSA (bovine serum albumin, Sangon, Shanghai, China) and then incubated with primary antibody that selectively recognized CXCR4 (ab2074, Abcam, USA) at 4°C overnight. To determine the amounts of loaded proteins, membranes were also blotted with anti-GAPDH antibody (Proteintech Technology, Manchester, UK). Subsequently, we incubated the membranes with HRP-conjugated secondary anti mouse (Pierce) or rabbit (Sigma-Aldrich) antibody, and then protein bands were visualized by adding ECL Plus Western blotting detection reagents (Millipore) and exposure to Kodak film following the manufacturer’s instructions. Protein levels were normalized to GAPDH.
Cell proliferation and colony formation assays
Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Shanghai, China) as previously described. Cells were seeded as 5 replicates at a density of 6000/well in 100 μl of full medium in 96-well plates and transfected with miR-133b mimics (100 nM), miR-NC (100 nM), miR-133b inhibitor (200 nM), inhibitor-NC (200 nM), siCXCR4 (120 nM) or si-NC (120 nM) as described above. The cells were then incubated at 37°C, and the absorbance was measured at wavelengths of 480 nm and 630 nm on consecutive days for four days.
For colony formation assays, 1000 cells that had been transfected with oligonucleotides were suspended in 2 ml of full medium and then seeded in 6-well plates. The cells were washed with phosphate-buffered saline (PBS), fixed with methanol and stained with crystal violet (0.1% crystal violet in 20% methanol) after 12-day incubation. Colonies with more than 50 cells were counted, and five fields were counted for each plate. The assay was performed in triplicate for each cell line.
Apoptosis assay (fluorescence-activated cell sorting (FACS) analysis)
Cells were transfected with the small molecules for 48 hours followed by a 24-hour exposure to cisplatin at final concentrations of 2.5 μg/ml and 1 μg/ml, respectively. After trypsinization and washing with ice-cold PBS, the cell suspensions were stained using Annexin V/FITC and propidium iodide (PI) (Annexin V/PI Apoptosis Detection Kit, Lianke, China) and then analyzed by measuring the membrane redistribution of phosphatidylserine by flow cytometry (Beckman Coulter, USA). The experiments were performed in triplicate.
Cell migration and invasion assays
Migration and invasion assays were conducted using Transwell chambers (8 μm, Corning Costar Co., Cambridge, MA) according to the manufacturer’s instructions. Briefly, 24 hours after transfection, the cells were starved for 12 hours and then trypsinized and resuspended in serum-free medium to a final concentration of 2 × 105/ml (for the migration assays) or 4 × 105/ml (for the invasion assays). The cell suspension (200 μl) was then pipetted into the top chamber. Medium (600 μl) with 10% fetal bovine serum was added to the lower chamber as a chemoattractant. After 36-hour incubation, the cells on the upper side of the membrane were mechanically removed with cotton swabs, and cells that migrated to the lower surface were fixed with 100% methanol and stained with 0.1% crystal violet. The cells were counted in five fields for triplicate membranes at 10× magnification using a microscope (Zeiss).
Cell invasion assays were performed as described for the cell migration assay, but polycarbonate membranes coated with 45 μl of 300 μg/ml extracellular matrix (Matrigel; BD Biosciences, San Jose, CA) that was diluted with medium lacking FBS were used.
Scratch wound healing assay
Transfected SW-480 and SW-620 cells were cultured in 24-well plates for 24–48 hours in standard conditions until 70-80% confluency. Linear wound tracks were generated with sterile, 10-μl pipettes and maintained under standard conditions. The scratched cells were then rinsed twice with PBS to remove non-adherent cells, and fresh culture medium was added. Photographs of the centers of the gaps were taken using a phase-contrast microscope and the same magnification, 100×. The cell migration at 0, 24, and 48 h after scratching was evaluated by determining the wound distance at two random wound gap locations. Three independent scratch-wound experiments were used for calculations.
All statistical calculations were performed using GraphPad Prism (GraphPad Prism Software, Version 5.0, GraphPad, San Diego, CA) and SPSS (Statistical Package for the Social Scienes) PASW Statistics software (versions 17.0, USA).
Fisher’s exact test and the Mann–Whitney U-test were used to compare differences between two groups. The related clinical data after logarithmic transformation were used to analyze the diagnostic utility by receiver operating characteristic (ROC) curves. Discriminant analysis was conducted to find and build a model of predicted probability. The correlation between miR-133b and CXCR4 was determined by the Spearman rank correlation test. Youden’s index was used to predict the optimal cutoff point. The other data in each group were defined as the mean ± SD. All p values were two-tailed, and p < 0.05 was considered statistically significant.
This work was supported by the funds from National Science and Technology Department (973, 2011CB8113015 and 2011CBA0110) and National Science Foundation of China (No. 81270629).
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