MicroRNA-200c overexpression inhibits tumorigenicity and metastasis of CD117+CD44+ ovarian cancer stem cells by regulating epithelial-mesenchymal transition
- Dengyu Chen†1, 2,
- Yunxia Zhang†3,
- Jing Wang†3,
- Junsong Chen1,
- Cuiping Yang1,
- Kai Cai1,
- Xiaoying Wang1,
- Fangfang Shi4 and
- Jun Dou1Email author
© Chen et al.; licensee BioMed Central Ltd. 2013
Received: 16 May 2013
Accepted: 23 June 2013
Published: 10 July 2013
Cancer stem cells (CSCs) are believed to be ‘seed cell’ in cancer recurrence and metastasis. MicroRNAs (miRNAs) can play an important role in the progression of primary tumor towards metastasis by regulating the epithelial-mesenchymal transition (EMT). The goal of this study was to investigate the effect of miRNA-200c overexpression on the EMT, tumorigenicity and metastasis of epithelial ovarian cancer (EOC) CSCs.
The EOC CD117+CD44+CSCs were isolated from the human ovarian cancer cell line SKOV3 by using a magnetic-activated cell sorting system, and the lentivirus miR-200c transduced CSCs were then selected for the study. The assays of colony forming, wound healing, cellular migration in vitro and tumor progression in vivo were performed.
The miR-200c expression was reduced in the CD117+CD44+CSCs compared with the non-CD117+CD44+CSCs. However, the stable overexpression of the miR-200c in the CD117+CD44+CSCs resulted in a significant down-regulation of ZEB-1 and the Vimentin expression, an upregulation of the E-cadherin expression as well as a decrease of colony forming, migratory and invasion in vitro. Importantly, the miR-200c overexpression significantly inhibited the CD117+CD44+CSCs xenograft growth and lung metastasis in vivo in nude mice by inhibition of the EMT. In addition, the down-regulation of ZEB-1 showed the same efficacy as the miR-200c overexpression in the CD117+CD44+CSCs.
These findings from this study suggest that the miR-200c overexpression may be considered a critical approach for the EOC CD117+CD44+CSCs in clinical trials.
KeywordsEpithelial ovarian cancer Cancer stem cells MiRNAs-200c Epithelial- mesenchymal transition Metastasis
Ovarian cancer is the leading cause of mortality in gynecologic malignancy. The 5-year survival rate of stage III-IV ovarian cancer patients is about 20% [1, 2]. Ovarian cancer is currently treated with a combination of surgery and chemotherapy. Systemic chemotherapy is initially effective in 80% of patients, however, recurrent ovarian cancer’s responds to additional chemotherapy treatments becomes lower after each treatment cycle, as chemoresistance increases until the disease becomes incurable . It is therefore crucial to conduct an in-depth investigation of the biology of ovarian cancer. The major advance of tumor biology in recent years has been the discovery of the cancer stem cells (CSCs), which play pivotal roles in cancer progression and treatment resistance in various neoplastic diseases. CSCs may open up new possibilities of generating novel targets, diminishing resistance to chemoradiation and improving therapeutic efficacy .
Tumor metastasis has been considered the main cause of death in patients of various malignant tumors. Evidence from past studies has indicated that the CSC-like cells might be generated by processes that are related to aberrant activation of the epithelial- mesenchymal transition (EMT) that impacts cell differentiation and tumor metastatic potential. Therefore, an anti-EMT strategy would be a novel therapeutic option for treating aggressive cancers [5, 6]. There is increasing evidence that the microRNAs (miRNAs) have emerged as potential therapeutic candidates by virtue of their ability to down-regulate multiple targets involved in tumor progression and metastasis, and in tumor therapeutic resistance and relapse. In some studies, miR-200c was found to be down-regulated in ovarian cancer cell lines and in stage III ovarian tumors; the miR-200c down-regulation correlated with poor prognoses. However, restoration of the miR-200c served as a tumor suppressor by directly targeting the zinc-finger E-box binding homeobox 1 (ZEB1) to inhibit EMT and ovarian cancer metastasis [7–10].
The epithelial ovarian cancer (EOC) is genetically and epigenetically distinct from normal ovarian surface epithelial cells and is involved in the EMT during cancer initiation and progression including cancer metastasis and recurrence. ZEB1 is known to be associated with the EOC invasive and metastatic progression; ZEB1 is also known to be expressed in the EOC and be able to directly repress the epithelial marker E-cadherin to induce tumor cell invasive and metastatic progression [11–14]. However, much less information of the EMT is available about the miRNA in the EOC CSCs, and the exact molecular mechanism of modulating the EMT of the EOC CSCs is yet to be elucidated.
Our goal for this study was to assess the epigenetically regulation function of the miR-200c overexpression in the EMT, the tumorigenicity, and the metastasis of the EOC CD117+CD44+CSCs in vitro and in vivo. To accomplish this goal, we transduced the lentivirus miR-200c vector into the CD117+CD44+CSCs that were isolated from the human EOC SKOV3 cell line [15, 16]. We found in vitro a direct association between the miR-200c overexpression and the capability of the CD117+CD44+CSC in colony forming, migration and invasion. In particular, we noticed the evident relationship between the miR-200c and the ZEB1 expression. Our results suggested that the miR-200c overexpression, by modulating the EMT, specifically inhibited the ZEB1 expression in the CD117+CD44+CSCs and reduced cell tumorigenicity and lung metastasis in our nude mouse model.
Materials and methods
The human EOC SKOV-3 cell line was from an ovarian cancer patient of origin, which was a well-established ovarian cancer model system. The cell line was purchased from the Cellular Institute in Shanghai, China, and was maintained in complete media consisting of RPMI 1640, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum. The complete media were refreshed every 3 days to maintain the adherent cells.
Isolation of CD44+CD117+CSCs, transduction of lentivirus miR-200c and production of stable expression colonies
The CD44+CD117+CSCs were isolated from the SKOV3 cell line by using the magnetic- associated cell sorting (MACS) method as described previously [16, 17]. Briefly, the CD44+subsets were isolated by using the mouse antihuman CD44 antibody coupled to magnetic microbeads (Miltenyi Biotec., Germany); the resulting cells were then depleted of CD117−subsets by using the mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec., Germany). The resulting CD44+CD117+cells were labeled ‘EOC CD44+CD117+CSCs’ . These cells were further identified by using a flow cytometer (FCM, Beckman Coulter, USA) according to the manufacturer’s instructions. The anti-Human/ Mouse CD44 FITC and the anti-Human CD117 PE (eBioscience, USA) antibodies were used for the detection of the cells .
To generate the miR-200c expression lentivirus vector, we amplified an insert (full-length human miR-200c) by PCR from SKOV3 DNA. The lentivirus miR-200c was produced from the transient transfection of the HEK293T cells with pHAGE-CMV- miR-200c-IZsGreen, psPAX2, and pMD2.G plasmid DNAs plus Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. Forty-eight hours after the co-transfection, the lentivirus-bearing supernatants were collected and passed through a 0.45-mm filter. The CD44+CD117+CSCs were transduced with the pHAGE-CMV- miR-200c-IzsGreen lentivirus, and were selected by the IzsGreen expression [7, 19]. The stable expression colony was generated by limiting the dilution assay .
RNA isolation and quantitative RT-PCR
Total cellular RNA was isolated from a sample by using a Qiagen RNeasy Kit (Qiagen, CA). The sequences of the primers are as follows: the miR-200c-RT primer 5′-CTCAAC TGGTGTGGAGCGCATTCAGTTGAGTCCATCAT-3; the ZEB1 forward, 5′-GCACAAC CAAGGCAGAAGA-3′; reverse, 5′-CATTTGCAGTTGAGGCTGA-3′; the β-actin forward, 5′-GGACTTCGAGCAAGAGATGG-3′; reverse, 5′-AGCACTGTGTTGGCGTACAG-3′; U6: RT Primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGA CAAATATGGAAC-3′;forward, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′; URP Universal Reverse Primer, 5′-CCGGCAGGGTCCGAGGT-3′. The E-cadherin forward: CATTGCC ACACATACACTCTCTTCT, reverse: CGGTTACCGTGATCAAAATCTC; the Vimentin forward: GGAACAGCATGTCCAAATCG, reverse: GCACCTGTCTCCGGTACTCA. The qRT-PCR analysis was performed on an ABI step one plus real-time system (Applied Biosystems, USA) .
Short hairpin RNA sequence design and recombinant construction of shRNA1 targeting to ZEB1 gene
A short hairpin RNA sequence of the human ZEB1 was designed according to the ZEB1 DNA sequence (GenBank NO.NM_001128128.2)by the siDESIGN design software of Dharmacon Company. The shRNA sequences were as follows: The ZEB1-siRNA: forward, 5′- GATCCCCAG GAAGAGGAGGAGGATAATTCAAGAGATTATCCTCCTC CTCTTCCTTTTTTGGA AA-3′; reverse, 5′-AGCTTTTCCAAAAAAGGAAGAGGAGG AGGATAATCTCTTGAATTATCCTCCTCCTCTTCCTGGG-3′; the scramble-siRNA: forward, 5′-GATCCCCTTCT CCGAACGTGTCACGTTTCAAGAGAACG TGACACGT TC GGAGAATTTTTGGAAA −3′; reverse, 5′-AGCTTTCCAAAAATTCTCCGAACGTG TCACGTTCTCTTGAAACGTGACACGTTGGAGAAGGG-3′. All the primers were synthesized by Company of Gene and Technology of China in Shanghai. A pSUPER- EGFP1 (enhancement green fluorescent protein 1) vector was used to construct recombinants. The recombinant pSUPER-EGFP1- ZEB1-shRNA (shZEB1) was developed using the method described in a according to previous reports [1, 13]. A pSUPER- EGFP1-scrambled shRNA (scramble) was used for negative control. These recombinants were transfected by using the Lipofectamine™ 2000 reagent (Invitrogen, USA) per the manufacturer’s protocol .
Colony forming assay
The colony formation capabilities of the different CD44+CD117+CSCs were investigated. Colonies larger than 75 μm in diameter or containing more than 50 cells were counted as 1 positive colony according to our previous reports [20, 21]. About 500 cells per well were added into a six-well culture plate, with three wells per sample. After12-day incubation, the cells were washed twice with PBS and stained with the Giemsa solution. The plate clone formation efficiency was calculated as (number of colony /number of cells inoculated) × 100%.
The cells from the above-mentioned colonies were grown to confluence and were wounded by dragging a 1-mL pipette tip across their monolayer. The cells were washed to remove any cellular debris and were then allowed to migrate for 0, 24, and 48 hours, respectively, in a humidified 5% CO2 incubator at 37°C. Images were taken, using a DMI6000 inverted microscope (Leica Microsystems GmbH, Germany), at 0, 24, and 48 hours, respectively, after the wounding procedure [22, 23].
Matrigel invasion assay
The Matrigel invasion assay was done using the BD Biocoat Matrigel Invasion Chamber (pore size: 8 mm, 24-well; BD Biosciences, USA) following the manufacturer's protocol [17, 24]. From five randomly selected fields, the cells that had invaded through the membrane to the lower surface were counted under a light microscope.
Xenograft tumor model
Thirty-six five-to six-week-old female Balb/c athymic nu/nu mice were ordered from the Animal Center of Yang Zhou University of China and were raised at the Experimental Animal Center, Southeast University. All the animal experiments were performed in compliance with the guidelines of the Animal Research Ethics Board of Southeast University. The 36 mice were randomly divided into six groups of equal size (6) as follows: group 1 for CD44+CD117+CSCs with lentivirus miR-200c; group 2 for CD44+CD117+CSCs with lentivirus mock; group 3 for CD44+CD117+CSCs without lentivirus infection; group 4 for CD44+CD117+CSCs transfected with the shZEB1; group 5 for CD44+CD117+CSCs transfected with the scrambled control siRNA; group 6 for CD44+CD117+CSCs without transfection. Each mouse was subcutaneously (s.c.) injected in the back with 5 × 104 cells for the group it was in. Tumor formations in the mice were monitored every three days. Evaluation was also done of the tumor volume, tumor-free mice, and survival rates, respectively. A mouse was sacrificed when any of its tumors was over 1.4 cm in the largest diameter. The necropsy was performed on each animal for further analysis of the primary tumor along with possible metastases [18, 25].
Western blot analysis
Total cell lysates were prepared and analyzed by using the Western blot method as described before [26, 27]. Briefly, 1 × 106 CD44+CD117+CSCs were collected and lyzed in a protein extraction buffer according to the manufacturer’ s protocol. The PVDF membrane was blocked with 4% dry milk in the Tris-buffered saline with Tween-20 for 1 h at 20°C, and was incubated with the rabbit antibody specific to human ZEB1 (Santa cruz Biotechnology, CA, USA ) or with E-cadherin or with Vimentin (Bioworld Technology, USA) for overnight at 4°C. The membrane was then incubated with the anti-rabbit fluorescence secondary antibody for 1 h at 20°C. Immunoreactive bands were detected by the Odyssey scanning instrument (LICOR Odyssey, USA ).
4 μm-thin formalin fixed and paraffin-embedded slides were incubated with the rabbit antibody specific to human E-cadherin overnight at 4°C. The samples were then labeled with HRP-conjugated streptavidin (Invitrogen) and the chromogenic reaction that was developed using Liquid DAB Substrate Pack according to the manufacturer's instructions. E-cadherin-stained cells from random and non-overlapping fields were counted under a magnification of × 200 .
The lung tissues were removed from the mice and fixed in 10% formalin, and then embedded in paraffin. Tissue sections of 4 μm thin were cut and mounted on SuperFrost Plus glass slides, fixed in methanol, and stained in hematoxylin and eosin (HE) [29, 30].
Statistical comparisons of the results between groups were performed using the Student’s t-test method. P < 0.05 was considered significant statistically.
Phenotype identification, morphologic characteristics and miR-200c expression in lentivirus transduced CD44+CD117+CSCs
Effect of miR-200c overexpression on the capability of colony formation and cellular motility of CD44+ CD117+ CSCs
Overexpression of miR-200c reduced ovarian tumor formation and tumor burden
miR-200c inhibited ZEB1,Vimentin and enhanced E-Cadherin expression levels in tumor as well as decreased tumor lung metastasis in nude mouse model
Effects of down-regulation of ZEB1 on colony formation, migration, invasion of CD44+CD117+CSCs in vitro, tumorigenicity, and tumor metastasis in vivo
To characterize the function of the downregulation of ZEB1, we examined the expression of ZEB1, E-cadherin and Vimentin, respectively, in the tumor tissues of the EOC-bearing nude mice. The Western blot results indicated that the expression of ZEB1 and Vimentin was much lower, and the E-Cadherin expression was much higher in the mice injected with the shZEB1 CD44+CD117+CSCs than in the mice injected with the CD44+CD117+CSCs or with the scrambled CD44+CD117+CSCs (Figure 6D). Figures 6E to 5G show the quantities of the molecular expression from the gradation scanning analysis.
To assess the effect of the down-regulation of ZEB1 on tumor metastasis, we performed H&E staining of lung tissue sections. Compared with the mice of the two control groups, a few tumor metastasis were found in the lungs of the nude mice injected with the shZEB1 CD44+CD117+CSCs (Figure 6H and 6I). It is thus evident from the results that the EMT-phenotype tumor cell growth and metastasis were significantly inhibited in the mice injected with the shZEB1 CD44+CD117+CSCs compared with the mice injected with the CD44+CD117+CSCs or the scrambled CD44+CD117+CSCs.
EOC CSCs that undergo the EMT have demonstrated that the tumor cells are in general less differentiable, more invasive, more chemoresistant, and result in poor clinical outcomes . Numerous studies of EOC have focused on modulating the miR-200 family (including miR-200a, miR-200b, miR-200c, and miR-141) [31–34]. However, it is unknown whether the EOC CSCs, the “seed cells” in EOC, are closely associated with the miR-200 family expression.
The findings from our study demonstrated that the population of the rare CD44+CD117+CSCs (3.1%) existed in the human EOC SKOV3 cell line, and that the CD44+ CD117+CSCs showed lower expression of miR-200c than the non CD44+CD117+CSCs. With the stable miR-200c overexpression in the CD44+CD117+CSCs, the cells markedly decreased the colony forming capability. It is known that the tumor cell cloning efficiency is correlated positively with the cellular proliferation and self-renewal ability that may be associated with the cell tumorigenesis [23, 24]. The results from our colony forming assay indicated that the small subset of the CD44+CD117+CSCs had a strong colony forming capability, which signified that the CD44+CD117+CSCs might have powerful tumorigenesis in the mouse model. In our tumorigenesis analysis, we found that all 6 nude mice injected with the 5 × 104 CD44+CD117+CSCs developed tumors in 21 days after the injection. In comparison, for the group that was injected with the 5 × 104 CD44+ CD117+CSCs with the miR-200c overexpression, only 3 out of the 6 mice with equal injection of 5 × 104 cells developed tumors after 56 days into the observation; the tumor sizes of these 3 mice were also smaller than those of the control group mice. These results suggested that the miR-200c overexpression not only effectively decreased the colony forming capability but also obviously reduced the tumorigenicity and the tumor burden in our establishment mouse model.
In EOC, metastases account for the majority of deaths from gynecologic malignancies [35, 36], therefore, we next explored the relationship between the miR-200c overexpression and tumor metastases. The cell migration and invasion in vitro results indicated that the stable miR-200c overexpression in the CD44+CD117+CSCs reduced cell migration and invasion. It is well known that the cell migration and invasion in vitro are definitely associated with of cell metastases in vivo; this was confirmed by the lung metastasis in the mice in our study. The lung tumor metastasis in the mice injected with the CD44+CD117+CSCs with lentivirus miR-200c was markedly decreased. To study the efficacy of decreased tumor metastasis in the lungs of the mice in the study, we wanted to understand what molecular mechanism of reduced the tumor metastasis; we investigated this by detecting the characteristic biomarkers of E-cadherin (epithelial cells),Vimentin (mesenchymal cells), and ZEB1 (in association with EMT) in tumor tissues [9, 37]. We noticed that the enforced overexpression of miR200c in the CD44+CD117+CSCs significantly reduced the expressions of both ZEB1 and Vimentin, but increased the expression of E-cadherin in the RNA and the protein levels in tumor samples. Apparently, the miR-200c overexpression decreased the ZEB1 expression, which directly inhibited the EMT of the CD44+CD117+CSCs, and reduced the CSC metastasis potential. Our findings were in agreement with a recent report that the overexpression of miR-429, a member of the miR-200 family of microRNAs, in the mesenchymal-like ovarian cancer cells resulted in the mesenchymal–epithelial transition .
To assess the relationship between ZEB1 and miR-200c in the CD44+CD117+CSCs, we asked whether the down-regulation of ZEB1 would have similar effects as the miR-200c overexpression. We found that the down-regulation of the ZEB1 expression in the CD44+CD117+CSCs indeed had the similar effects as the miR-200c overexpression in the CD44+CD117+CSCs; this was reflected in the significant suppression of the tumorigenesis and tumor metastasis in the mice injected with the shZEB1 CD44+CD117+CSCs in comparison with the mice injected with the CD44+CD117+CSCs or with the CD44+CD117+CSCs with lentivirus mock. It is therefore reasonable to conclude that ZEB1 was essential for tumorigenesis and metastasis in xenografts transplantation experiments, and that the down-regulation of ZEB1 may not only be a useful biomarker of the EMT in the EOC CSCs, but also serve as a potential therapeutic target to inhibit EOC metastasis [33, 37].
In summary, the findings from our experiments demonstrate that the overexpression of miR-200c significantly reduced the CD117+CD44+CSCs xenograft growth and lung metastasis in vivo, partially through the reversal of the EMT phenotype. The down- regulation of the ZEB-1 expression in the CD117+CD44+CSCs induced the similar effects as the miR-200c overexpression. These findings may enable us to design a feasible strategy for the modulation of EMT in the CD44+CD117+CSCs for clinical EOC treatment.
This work was supported in part by the 973 National Nature Science Foundation of People’s Republic of China (2011CB933500) and part by the National Natural Science Foundation of China (No. 81071769, No. 81202372).
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