CRC tissues with distant metastases (n = 32) and CRC tissues without distant metastases (n = 30) were obtained from 62 CRC patients who underwent initial surgery at Renmin Hospital of Wuhan University between June 2005 and January 2008. All the patients had a histological diagnosis of CRC. Following resection, the specimens were snap-frozen in liquid nitrogen and stored at -80 °C until RNA extraction. All the subjects involved in this study provided written informed consent. This project was approved by the ethics committee of Wuhan University.

The human HT-29, HCT116, SW480, SW620 and LoVo CRC cell lines, the 293T embryonic kidney cell line, and the NCM460 normal colonic epithelial cell line were purchased from the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a humidified 37 °C incubator supplemented with 5% CO₂.

Total RNA from the tissues and cells was extracted using Trizol reagent (Invitrogen, Carlsbad, California, United States). RNA quality and concentration were determined using the Nanodrop 2000 system (Thermo Fisher Scientific, Wilmington, United States). For analysis of the miR-132 level, a TaqMan MicroRNA Assay Kit (Applied Biosystems) was used, and U6 snRNA was used as the reference. For measurement of the ZEB2 mRNA level, a SYBR Premix Ex Taq^TM kit (Takara) was used, and β-actin expression was used as endogenous control. Real-time PCR was performed using the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, United States). Data were analyzed using the 2^-ΔΔct method.

Western blot analysis was performed as previously described[14]. Briefly, total cellular protein was isolated, and the protein concentration was determined using the Bradford DC protein assay (Bio-Rad, CA, United States). A total of 40 μg of protein was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were then incubated with the following primary antibodies: ZEB2 (1:1000; CST, United States), E-cadherin (1:1000; CST, United States), α-catenin (1:1000; CST, United States), vimentin (1:1000; CST, United States), fibronectin (1:1000; CST, United States) and GAPDH (1:2000; Santa Cruz Biotechnology, United States). Proteins were visualized using the ECL procedure (Amersham Biosciences, United States).

The hsa-miR-132 mimic, negative control (NC) oligonucleotides, has-miR-132 inhibitor and scramble oligonucleotides were purchased from Ribobio (Guangzhou, China). The cells were plated in a six-well plate the day before transfection. LoVo cells were transfected with the has-miR-132 mimic or NC (50 nmol/L), and HCT116 cells were infected with the has-miR-132 inhibitor or scramble oligonucleotides (100 nmol/L), using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, the cells were collected, and in vitro assays were performed.

The cell invasive potential was evaluated using specialized transwell chambers (8-μm pore; BD Biosciences). The cells (5 × 10⁴ cells suspended in 500 μL of serum-free medium) were added to the upper chamber of the inserts, which were coated with a Matrigel mix; fetal bovine serum (500 μL) was added to the bottom chamber as a chemoattractant. Twenty-four hours later, the non-invading cells on the upper surface were removed, and the cells that invaded to the bottom side of the membrane were fixed with methanol, stained with 0.1% crystal violet, air dried and subjected to digital image acquisition. The number of invasive cells was evaluated in five independent fields under a microscope. The mean of triplicate assays for each experimental condition was analyzed.

For the luciferase assays, the potential miR-132 binding site in the ZEB2 3′-UTR was predicted using TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org). The 3’-UTR of the ZEB2 mRNA and a mutant ZEB2 mRNA were synthesized and cloned into the Xba I site of a pGL3 basic vector (Promega, United States) downstream from the luciferase stop codon and were designated as pGL3-wt-ZEB2 and pGL3-mt-ZEB2, respectively. Then, 293T cells (1 × 10⁵ cells/well) were cultured in 24-well plates and co-transfected with the pGL3-Control (0.4 mg), pGL3-wt-ZEB2 (0.4 mg) or pGL3-mt-ZEB2 (0.4 mg) plasmid, the pRL-TK luciferase reporters (25 ng/well) and pcDNA-miR-132 (20 nmol/L) or pcDNA-miR-NC (20 nmol/L) using Lipofectamine 2000 (Invitrogen, United States). Forty-eight hours later, the cells were harvested, and luciferase activities were measured using a Dual-Luciferase Reporter Assay kit (Promega, United States).

The data were expressed as the mean ± SD. Statistical significance was analyzed using Student’s t-test (two-tailed). All statistical analyses were performed using SPSS 13.0 or the GraphPad Prism 5.0 software package. The Kaplan-Meier method and log-rank test were performed to analyze the prognostic significance. P < 0.05 was considered to be statistically significant in all tests.

First, we analyzed the expression of miR-132 in five CRC cell lines and the normal colonic cell line NCM460. MiR-132 was significantly down-regulated in CRC cell lines compared with the normal colonic cell line NCM460 (all P < 0.05) (Figure 1A). Among the six CRC cell lines, LoVo possessed the lowest miR-132 level, and HCT116 exhibited the highest miR-132 level (Figure 1A). To explore the expression of miR-132 in CRC tissues with different metastatic characteristics, the miR-132 level was measured in tissues with distant metastases (n = 32) and tissues without distant metastases (n = 30). Interestingly, miR-132 was markedly lower in CRC tissues with distant metastases than that in CRC tissues without distant metastases (10.52 ± 4.69 vs 23.11 ± 7.84) (P < 0.001; Figure 1B). To evaluate the clinical value of miR-132 in CRC patients, we divided the patients into two groups according to the median value (15.40) of the miR-132 level. The correlation between miR-132 and clinicopathological characteristics was then analyzed (Table 1). Low expression of miR-132 was significantly associated with a larger tumor size (P = 0.016), distant metastasis (P = 0.002) and TNM stage (P = 0.020). However, age, gender, CEA level, CA199 level, tumor site and differentiation showed no association with miR-132 expression (Table 1). Kaplan-Meier analysis showed patients with low miR-132 expression had significantly worse disease-free survival than patients with high miR-132 expression (P < 0.001, Figure 2).

Open in New Tab   Full Size Figure   Download Figure

Figure 1 miR-132 is significantly down-regulated in colorectal cancer cell lines and tissues with distant metastases. A: Relative expression level of miR-132 in colorectal cancer (CRC) cell lines and the normal colonic cell NCM460 (^aP < 0.05); B: Relative expression level of miR-132 in CRC tissues with distant metastases (n = 32) and CRC tissues without metastases (n = 30) (^bP < 0.01).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 miR-132 predicts disease-free survival in colorectal cancer patients. The Kaplan-Meier curve of disease-free survival in patients with high miR-132 expression (n = 31) and low miR-132 expression (n = 31) (P < 0.01).

Because miR-132 was significantly down-regulated in CRC tissues with distant metastases, we hypothesized that miR-132 could inhibit cell invasion. LoVo cells with low miR-132 expression were transfected with the miR-132 mimic to overexpress miR-132, whereas HCT116 cells with high miR-132 expression were transfected with the miR-132 inhibitor to knockdown endogenous miR-132. The efficiency of transfection was confirmed by real-time PCR (data not shown). As expected, ectopic expression of miR-132 reduced LoVo cell invasion by about 40% (P < 0.05; Figure 3A). By contrast, knockdown of miR-132 significantly increased the invasiveness of the HCT116 cells (P < 0.05; Figure 3B).

Open in New Tab   Full Size Figure   Download Figure

Figure 3 miR-132 inhibits colorectal cancer cell invasion. A: Ectopic expression of miR-132 inhibits cell invasion in LoVo cells (^aP < 0.05); B: Knockdown of miR-132 promotes cell invasion in HCT116 cells (^aP < 0.05).

EMT is known to be important for tumor metastasis. Thus, we investigated the role of miR-132 in EMT in CRC cells. As shown in Figure 4A, ectopic expression of miR-132 induced a morphological change from the spindle-shaped mesenchymal form to a cobblestone-shaped epithelial-like form in LoVo cells. By contrast, knockdown of miR-132 in HCT116 cells led to more spindle-shaped mesenchymal characteristics (Figure 4B). Moreover, the expression of a set of EMT-related protein markers was also altered along with the morphological changes. Overexpression of miR-132 increased the protein levels of epithelial markers (E-cadherin and α-catenin) but decreased the expression of mesenchymal markers (vimentin and fibronectin) in LoVo cells (Figure 4C). By contrast, knockdown of miR-132 resulted in a decreased level of epithelial markers (E-cadherin and α-catenin) but increased levels of mesenchymal markers (vimentin and fibronectin) in HCT116 cells (Figure 4D).

Open in New Tab   Full Size Figure   Download Figure

Figure 4 miR-132 regulates epithelial-mesenchymal transition in colorectal cancer cells. A: Ectopic expression of miR-132 leads to a cobblestone-shaped epithelial-like form in LoVo cells; B: Knockdown of miR-132 leads to spindle-shaped mesenchymal characteristics in HCT116 cells; C, D: Overexpression of miR-132 increased the protein levels of epithelial markers (E-cadherin and α-catenin) but decreased the expression of mesenchymal markers (vimentin and fibronectin) in LoVo cells, whereas knockdown of miR-132 resulted in decreased levels of epithelial markers (E-cadherin and α-catenin) and increased levels of mesenchymal markers (vimentin and fibronectin) in HCT116 cells.

To investigate the underlying mechanism of miR-132 in CRC, we first used TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org) to search for potential targets of miR-132. Because miR-132 could suppress CRC invasion, we focused on genes that could promote CRC metastasis. We found that the 3’-UTR of ZEB2 contains the complementary site for the seed region of miR-132. Additionally, ectopic expression of miR-132 could significantly reduce the ZEB2 mRNA and protein levels in LoVo and SW620 cells (Figure 5A and B). Furthermore, a luciferase activity assay demonstrated that miR-132 could significantly decrease the luciferase activity of the wild-type ZEB2 3’-UTR by 40%, a finding that was not observed for the mutant ZEB2 3’-UTR (Figure 5C).

Open in New Tab   Full Size Figure   Download Figure

Figure 5 ZEB2 is a direct target of miR-132 in colorectal cancer cells. A: Ectopic expression of miR-132 reduces the ZEB2 mRNA levels in LoVo and SW620 cells (^aP < 0.05); B: Ectopic expression of miR-132 decreases the ZEB2 protein levels in LoVo and SW620 cells; C: Co-transfection of pcDNA-miR-132 and pGL3-wt-EB2 reduced the luciferase activity in 293T cells (^aP < 0.05).

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in western countries. Although recent developments in therapeutic strategies have helped cure many patients with early stage disease, the prognosis of patients with advanced disease and metastasis remains poor. Further investigation into the underlying molecular mechanisms of CRC progression to identify biomarkers to distinguish CRC patients with or without a high risk of metastasis is of great importance.

MiRNAs have been found to be involved in the regulation of multiple pathological processes that contribute to tumorigenesis and metastasis, such as tumor cell proliferation, differentiation, apoptosis, and invasion. In CRC, studies have indicated that miRNAs play important roles in regulating tumor invasion and metastasis. Previously, Zhang et al reported that down-regulation of miR-132 by promoter methylation promotes pancreatic cancer development. Formosa et al found that miR-132 is silenced by promoter CpG island methylation, a process that contributes to prostate cancer progression and metastasis. However, the role of miR-132 in CRC progression and metastasis remains unclear and needs further exploration.

The authors found that miR-132 was significantly down-regulated in CRC tissues with metastasis compared with tissues without metastasis; the level of miR-132 was lower in CRC cell lines than in NCM460 cells (a normal colonic cell line). Ectopic expression of miR-132 markedly inhibited the invasiveness and epithelial-mesenchymal transition (EMT) in CRC cells. Further study indicated that ZEB2 (an EMT regulator) was a direct downstream target of miR-132. Collectively, these results demonstrated that miR-132 inhibited cell invasion and EMT in CRC cells by targeting ZEB2, thus providing a valuable target for cancer therapy.

The findings in this study indicated that miR-132 was significantly down-regulated in CRC with distant metastases. Moreover, miR-132 could predict disease-free survival and distant metastasis in CRC patients. Further investigation identified that the EMT regulator ZEB2 was a direct target of miR-132. Taken together, these data implicate that miR-132 might be used as a prognostic indicator and therapeutic target in CRC patients.

MiRNA: a small non-coding RNA molecule (approximately 22 nucleotides in length) found in plants, animals, and some viruses that functions in transcriptional and post-transcriptional regulation of gene expression; epithelial-mesenchymal transition: a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells.

The authors report on the biological and clinical significance of miR-132 in colorectal cancer, adding some information on a possible target of that microRNA. The results of the study are interesting and innovative. The sample size of the study is sufficiently large. The methods used are updated and well described, and the statistics are appropriate.