miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer
© Yu et al.; licensee BioMed Central Ltd. 2014
Received: 10 January 2014
Accepted: 15 May 2014
Published: 24 May 2014
Epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT.
The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC).
MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3′UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens.
Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer therapy.
MicroRNAs (miRNAs) are evolutionarily conserved, 20–22 nucleotide noncoding RNAs. They exert their function by binding to the 3′-untranslated region (3′UTR) of a subset of mRNAs resulting in reduced translation of proteins or degradation of the mRNAs . The importance of miRNAs in tumor development and progression has become increasingly evident . Some miRNAs have been shown to exert diverse functions in cancer cell proliferation, angiogenesis and epithelial-to-mesenchymal transition (EMT) [3, 4].
EMT is a complex trans-differentiation process in which epithelial cells lose junctional adhesion and adopt a mesenchymal phenotype and morphology. During the EMT process, epithelial cells can migrate through the extracellular matrix. In cancer, EMT allows neoplasic cells to become motile and invasive, and leave the primary epithelial tumor site, thereby contributing to the metastatic potential of carcinomas [5, 6].
Transforming growth factor-β (TGF-β) was first described as an inducer of EMT in normal mammary epithelial cells and has since been shown to mediate EMT in vitro in most epithelial cells [7, 8]. The process of EMT is also regulated by several transcription factors, including Twist (also known as Twist1), SNAIL, SLUG, ZEB1 and ZEB2, as reviewed in , which are transcriptional repressors of E-cadherin. Recent work has demonstrated some miRNAs play critical roles in EMT. Members of the miR-200 family are well-established EMT repressors through direct targeting of ZEB1 and ZEB2 [10, 11]. However, reports of the roles of other miRNAs in the regulation of EMT are limited.
Recently, using miRNA microarray analysis, we identified a group of differentially expressed miRNAs between mesenchymal-like cancer cells and epithelial-like cancer cells . Remarkably, miR-300 was down-regulated in cancer cells that have undergone EMT comparing with typical epithelial phenotype carcinoma cells, indicating miR-300 might be a regulator of EMT. Based on this finding, miR-300 was chosen for further investigation.
In this study, we verified that miR-300 was down-regulated during an EMT. The low expression of miR-300 plays an important role in EMT-mediated tumor metastasis. Furthermore, we show that Twist is a direct target of miR-300. Ectopic expression of miR-300 could repress invasion in vitro and experimental pulmonary metastases in vivo. In addition, the expression level of miR-300 is associated with metastatic pattern in oral squamous cell carcinoma.
Down-regulation of miR-300 in cells having undergone EMT
Overexpression of miR-300 blocks TGF-β induced EMT
Overexpression of miR-300 reverses the mesenchymal characteristics of HN-12 and MDA-MB-231 cells
Reducing miR-300 level induces EMT in HN-4 and MCF-7 cells
As a consequence of stable knockdown of miR-300, HN-4 and MCF-7 cells showed cellular changes consistent with EMT and increased invasiveness. HN-4 and MCF-7 cells with down-regulated level of miR-300 acquired a fibroblastoid appearance (Figure 3D). Western blot confirmed a strong reduction of E-cadherin expression and a concomitant induction of Vimentin in these cells (Figure 3E). In addition, downregulation of miR-300 led to increased cell invasion (Figure 3F). These results show that reducing endogenous miR-300 expression can efficiently induce EMT.
Twist is a direct target of miR-300
miR-300 inhibits the experimental lung metastasis in vivo
miR-300 expression is inversely correlated with Twist expression and metastasis in tumors from patients with oral cancer
Metastasis is the primary cause of mortality in most cancer patients . Thus, to understand the molecular mechanisms of metastasis is one of the most important issues in cancer research. Epithelial-mesenchymal transition (EMT), which enables epithelial cells to acquire invasive mesenchymal phenotype, is attracting increasing attention as an important mechanism for the initial step of metastasis [16, 17]. Recently, several miRNAs, such as the miR-200 family, have appeared as powerful master regulators of EMT. In this study, we observed that miR-300, a miRNA with so far unknown function in metastasis, played an important role as a suppressor of EMT in human epithelial cancer through direct targeting Twist, which, in turn, inhibited metastasis.
Previously, we identified a group of differentially expressed miRNAs between mesenchymal-like and epithelial-like cancer cells . In this study, these dysregulated miRNAs were verified by real-time PCR in two types of cancer cells, breast cancer cells and oral cancer cells. It was observed that miR-300 was significantly down-regulated both in natural mesenchymal phenotype cells (HN-12 cell and MDA-MB-231 cell) and EMT cell culture models by exposure to TGF-β compared with epithelial phenotype cells (HN-4 cell and MCF-7 cell). These results imply that miR-300 may function as an EMT regulator.
Furthermore, our data demonstrated that over-expression of miR-300 in cancer cells could prevent TGF-β-induced EMT. In addition, the ectopic expression of miR-300 is sufficient to reverse the mesenchymal characteristics and decrease the invasive abilities of mesenchymal phenotype cells. In contrast, by inhibition of miR-300, epithelial phenotype cells could be triggered to undergo EMT, which was accompanied by a more invasive property. Our findings showed that down-regulation of miR-300 is required for EMT initiation and maintenance. More importantly, the involvement of miR-300 in the regulation of EMT was observed in two different types of cancer, breast cancer and oral cancer, suggesting it is a common regulatory mechanism of tumor cell invasion rather than a tissue-specific one.Identifying targets is critical for understanding the biological effects of miRNA . In the present study, Twist was predicted as the target of miR-300 by TargetScan. This interaction between miR-300 and Twist mRNA has not been previously reported. Through dual-luciferase assays, we confirmed the Twist 3′UTR as a direct target of miR-300. The role of Twist in promoting EMT processes has been widely reported [19, 20]. Elevated Twist expression was also related with tumor invasion and metastasis in several solid cancers, such as oral cancer , esophageal squamous cell carcinomas , prostate cancer , and uterine cancer . Although Twist has been reported to promote EMT and tumor metastasis through repression of E-cadherin expression, few known miRNA regulators of Twist have been identified to date. In the present study, for the first time, we provided evidence that Twist is a direct target of miR-300. As Twist plays an essential role in EMT as well as tumor metastasis, our data establish a mechanistic link between miR-300, Twist, EMT and tumor metastasis.
Given the critical role of EMT in metastatic tumor formation, inhibition of EMT can be an important therapeutic strategy to inhibit tumor metastasis [24, 25]. As a matter of fact, we have shown that miR-300 could decrease in vitro invasive behavior of highly invasive cancer cells, suggesting it might be a rational approach of treatment against metastasis. Therefore, we subsequently turned our attention to the anti-metastatic efficacy of miR-300 in a mouse model of experimental lung metastasis. In accordance with our in vitro findings, ectopic expression of miR-300 in MDA-MB-231cells led to approximate 2.9-fold decrease in the number of lung metastasis tumor nodules compared with vector control cells, providing in vivo evidence that miR-300 has therapeutic potential for metastasis prevention. (HN-12 cells were also employed for animal study, however they failed to develop experimental metastasis model. Data not shown). In addition, levels of Twist protein in lung lysates from miR-300 treated mice were markedly reduced compared with control mice, further confirming that the Twist gene is a true target of miR-300. The miR-300 mediated inhibition of invasion and metastasis likely operates through its suppression effect on EMT.
To gain further insights into the role of miR-300 in oral cancer metastasis, the expression of miR-300 and Twist was detected in 75 oral cancer samples. A significant inverse correlation between miR-300 and Twist expression levels was observed, which confirms the functional interaction of miR-300 and its target Twist. Furthermore, significant lower levels of miR-300 and higher levels of Twist were found in patients with metastasis than that in patients without metastasis. Although low level expression of miR-300 was reported in some types of cancer [26–28], the exact role of miR-300 in cancerous transformation and progression is not clear. Our data provide evidence that down-regulation of miR-300 is involved in metastatic events, which is consistent with the function of miR-300 in modulating EMT. These results reveal a previously unknown function of miR-300 to prevent metastasis by suppressing EMT.
Two HNSCC cell lines (HN-12 and HN-4 cells) were obtained from the National Institutes of Health (NIH). Two human breast cancer cells (MDA-MB-231and MCF-7 cells) were purchased from American Type Culture Collection (ATCC). All cells were cultured in DMEM containing 10% (v/v) FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37°C, 5% CO2 in a humidified atmosphere. HN-4 cells are primary squamous cell carcinoma cells of the tongue with low invasive ability, while HN-12 cells were derived from a nodal metastasis in the patient from whom the HN-4 cells originated that have relatively high invasive ability . MDA-MB-231 and MCF-7 cells are commonly used breast cancer cell lines. MCF-7 cells have been found to exhibit low invasive abilities. By contrast, MDA-MB-231 cells have a high invasive potential . Induction of EMT in epithelial cancer cells was initiated by addition of 10 ng/ml TGF-β1 (Invitrogen) to the medium for up to 72 h.
RNA preparation and quantitative real-time PCR (qRT-PCR)
Primer sequences used in the present study
Twist 3′UTR F
Twist 3′UTR R
Twist qPCR F
Twist qPCR R
Immunofluorescence and confocal laser scanning microscopy
Confocal laser scanning microscopy was performed using a Leica TCS SP2 microscope (Leica Microsystems) as previously reported . Briefly, cells were grown on 10 mm glass coverslips in 24-well culture plates. After methanol fixation for 10 min, cells were incubated with an E-cadherin antibody (1:100, Invitrigen) or Vimentin antibody (1:100, Sigma), followed by incubation with goat-anti-mouse-Alexa 594 conjugated antibodies (1:500, Invitrogen). Cell nuclei were counterstained with DAPI (1:1000, Invitrogen). Confocal image series were recorded on a LEICA TCS SP2 laser scanning microscope.
Western blot analysis
Cells and murine lung tissue specimens were homogenized in extraction buffer (50 mM HEPES, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM PMSF,1 mM DTT supplemented with Protease inhibitor cocktail). Whole proteins were extracted by centrifugation (12,000 × g) for 10 min at 4°C. Protein concentration was determined using the BCA kit (Pierce). Equal amounts of protein (20 μg/lane) from the cell lysates were electrophoresed under nonreducing conditions on 10% acrylamide gels. After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 2 h in PBS plus 0.1% Tween-20 and 5% nonfat skim milk to block nonspecific binding. Subsequently, the membrane was incubated for 2 h with an antibody against E-cadherin (1:500, Invitrigen), Vimentin (1:500, Sigma) and Twist (1:400, Abcam), followed by incubation with fluorescent secondary antibodies (1:5000, IRDye 800 anti-mouse Molecular Probes, Rockland). Blots were stripped and reprobed by using anti-actin antibody (Santa Cruz Biotechnology). Images were acquired with the Odyssey infrared imaging system and analyzed by the software program as specified by Odyssey.
Transwell invasion assay
Cell invasion was studied using Matrigel-coated transwell inserts as described previously . Briefly, transwell inserts with 8 μm pores were coated with Matrigel (50 μg/well, BD Biosciences). 200 μl of cell suspension (1 × 106/ml) was added to the upper chambers, and 600 μl of DMEM medium containing 5% fetal bovine serum was placed in the lower wells. Thereafter, the cells were incubated for 24 h. Cells that had invaded to lower surface of the Matrigel-coated membrane were fixed with 70% ethanol, stained with hematoxylin, and counted in five random fields under a light microscope.
Transfection of miRNA mimics and inhibitors
A total of 2 × 105 cells were plated in triplicate overnight in antibiotic-free complete medium in 6-well plates. The cells were then transiently transfected with 200 μL containing 100 nM of a mature miRNA mimic or inhibitor using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 72 h after transfection, the total RNA and protein were collected for further analysis.
The sequences of miR-300 were as follows:
Sense: 5′- UAUACAAGGGCAGACUCUCUCU-3′.
Anti-sense: 5′- AGAGAGAGUCUGCCCUUGUAUA-3′.
The sequence of the miR-300 inhibitor was as follows:
Generation of stable cell lines with overexpression or knockdown of miR-300
The miR-300 precursor expression plasmid, inhibitor expression plasmid and non-targeting control plasmid were obtained from Genechem. Cells were plated in 6-well plates at a density of 5 × 105 cells per well, and incubated overnight before transfection. Plasmid transfections were performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. After transfection, cells were grown in the presence of Hygromycin (300 μg/ml) to generate stably transfected cell lines.
Bioinformatic prediction of miR-300 potential targets
Luciferase reporter assay
The Twist 3′ UTR segment, which contains the putative binding site for miR-300 was amplified by PCR from HN-12 cells genomic DNA and inserted into the SmaI and HindIII sites downstream of the firefly luciferase coding region of the pGL3 vector (Promega), resulting in pGL3-Twist 3′UTR. Primer sequences are in Table 1. Mutation from GTA to CAT was introduced in the potential miR-300 binding sites (named pGL3-Twist 3′UTR-mut) by using the QuickChange Stratagene method. The correct orientation of the insert was verified by sequencing. 4 × 104 293 T cells were cultured in 24-well plates and cotransfected with 0.15 μg of either pGL3-Twist 3′UTR or pGL3-Twist 3′UTR-mut together with 0.05 μg of the control vector containing renilla luciferase, pRL-TK (Promega) and 40 pmol of miR-300 mimics. Transfection was done using Lipofectamine 2000. Twenty-four hours after transfection, firefly and renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay (Promega) and firefly luciferase activity was normalized to renilla luciferase activity. Each transfection was repeated twice in triplicate. To assess the effect of the endogenous miR-300, luciferase activities were also measured in HN-4 and MCF-7 cells in the presence of miR-300 inhibitor.
To further validate direct targeting of Twist by miR-300, functional rescue experiment was performed by co-transfection with miR-300 mimic and plasmid constructs expressing Twist in HN-12 cells and MDA-MB-231 cells using Lipofectamine 2000 as described above. The expression plasmid pcDNA-Twist, encoding human Twist was purchased from Genechem. The presence of complete Twist coding regions was confirmed by DNA sequencing.
All animal studies were conducted in accordance with an Institutional Animal Care and Use Committee-approved protocol at the Shanghai Jiao Tong University. 5-week-old female nude BALB/c mice were used in the study. MDA-MB-231 cells stably transfected with miR-300 or with negative control vector (2 × 106 cells in 0.2 ml PBS) were injected into mice via the tail vein. Each treatment group consisted of 7 mice. After 6 weeks, the mice were necropsied, and the lungs were removed. Visible lung metastases were counted in Bouin’s fixed tissues with the aid of a dissecting microscope .
A total of seventy-five FFPE tissue specimens were obtained from patients who were diagnosed with oral squamous cell carcinoma (OSCC) and underwent surgical resection between 2006 and 2007 in the Department of Oral Pathology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, with (n = 35) or without metastasis (n = 40). Real-time PCR analysis for miR-300 and Twist mRNA was performed as described above. Primer sequences are in Table 1. Correlation analysis between miR-300 level and Twist mRNA expression was performed. The median value was used as cutoff point for separating miR-300 high and low expression cases.
All data are presented as the mean value ± SD. Differences between individual groups were analyzed by t-tests. P values less than 0.05 (two-sided) were considered statistically significant. In order to determine the correlation coefficient (R) and the significance (P) between the expression of miR-300 and Twist mRNA, the Pearson’s correlation algorithm was applied. Statistical analyses were performed using the SPSS 11.5 software (SPSS Inc).
This study was supported by the Chinese National Natural Science Foundation of China (Grant Number 81070845, 81272977), Shanghai Science and Technology Committee project (12140901100 and 12140901101).
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