Figures
Abstract
MicroRNA-93, derived from a paralog (miR-106b-25) of the miR-17-92 cluster, is involved in the tumorigenesis and progression of many cancers such as breast, colorectal, hepatocellular, lung, ovarian, and pancreatic cancer. However, the role of miR-93 in endometrial carcinoma and the potential molecular mechanisms involved remain unknown. Our results showed that miR-93 was overexpressed in endometrial carcinoma tissues than normal endometrial tissues. The endometrial carcinoma cell lines HEC-1B and Ishikawa were transfected with miR-93-5P, after which cell migration and invasion ability and the expression of relevant molecules were detected. MiR-93 overexpression promoted cell migration and invasion, and downregulated E-cadherin expression while increasing N-cadherin expression. Dual-luciferase reporter assay showed that miR-93 may directly bind to the 3′ untranslated region of forkhead box A1 (FOXA1); furthermore, miR-93 overexpression downregulated FOXA1 expression while miR-93 inhibitor transfection upregulated FOXA1 expression at both mRNA and protein level. In addition, transfection with the most effective FOXA1 small interfering RNA promoted both endometrial cancer cell migration and invasion, and downregulated E-cadherin expression while upregulating N-cadherin expression. Therefore, we suggest that miR-93 may promote the process of epithelial–mesenchymal transition in endometrial carcinoma cells by targeting FOXA1.
Citation: Chen S, Chen X, Sun K-X, Xiu Y-L, Liu B-L, Feng M-X, et al. (2016) MicroRNA-93 Promotes Epithelial–Mesenchymal Transition of Endometrial Carcinoma Cells. PLoS ONE 11(11): e0165776. https://doi.org/10.1371/journal.pone.0165776
Editor: Zhiqian Zhang, Beijing Cancer Hospital, CHINA
Received: December 8, 2015; Accepted: October 18, 2016; Published: November 9, 2016
Copyright: © 2016 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work was supported by the National Natural Science Foundation of China (Yang Zhao, Nos. 81202049, 81472440; and Shuo Chen, No. 81602266).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Endometrial carcinoma is one of the most common tumors of the female reproductive system [1]. The incidence of endometrial carcinoma has dramatically increased over the past decades. Recurrence and metastasis are important causes of death in patients with endometrial cancer, and some studies have found that tumor invasion and metastasis are closely related to epithelial–mesenchymal transition (EMT) [2–6]. EMT is an intricate process where cancer cells demonstrate the loss of polarity and cell phenotype alteration from epithelial to mesenchymal morphology and it is considered a prerequisite for the typical tumor phenotypes of upregulated angiogenesis, enhanced cell motility, and extracellular matrix invasion [7–8]. In addition, it has been indicated that, in endometrial carcinoma, metastasis and recurrence are closely related with EMT [9–10].
MicroRNA (miRNA), a class of naturally occurring, small noncoding RNA, bind to the 3′ untranslated region (3′UTR) of target genes to downregulate their expression at post-transcription level by inducing the degradation of the targeted mRNA or inhibiting translation of the targeted mRNA and subsequently regulating diverse basic cellular functions such as proliferation, migration, invasion, and the EMT process [11–15]. In ovarian cancer, Ras homolog family member C (RhoC) overexpression promotes EMT and increased migration and invasion ability [16]. In our previous study, we found that miR-93 downregulated RhoC to inhibit cell migration and invasion in ovarian cancer [17]. However, the role of miR-93 in endometrial carcinoma and the potential molecular mechanisms involved remain unknown. This is the first study to investigate role of miR-93 in influencing the migration and invasion ability of endometrial carcinoma cells via EMT. We found that miR-93 promoted endometrial carcinoma cell EMT, migration, and invasion.
Materials and Methods
Cell culture and transfection
The endometrial carcinoma cell lines Ishikawa was purchased from Nanjing Keygen Biotech (Nanjing, China) and HEC-1B was purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). The Ishikawa cells were cultured in RPMI 1640 medium; the HEC-1B cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin / streptomycin. The two cell lines were propagated at 37°C in a humidified atmosphere of 5% CO2 in air. MiR-93 mimics, non-targeting control mimic, anti-miR-93 mimics, anti-miR-Control, three forkhead box A1 (FOXA1) small interfering RNA (siRNA) (siFOXA1, siFOXA1-1, siFOXA1-2), or scramble siRNA (Mock) (Sigma, Shanghai, China) were transiently transfected into the cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. For would healing and Transwell assay, the checkpoints was 24h after transfection, while 48h for western blot, RT-PCR and Immunofluorescence assays.
Wound healing assay
Cells were seeded at 1.0 × 106 cells/well in 6-well culture plates. After they had grown to confluence, the cell monolayer was scraped with a pipette tip (200 μL) to create a scratch, washed thrice with phosphate-buffered saline (PBS), and cultured in FBS-free medium. The cells were photographed at 0 and 24h, and the scratch area was measured using Image J software (National Institutes of Health, Bethesda, MD, USA). The migration rate = (area of original wound − area of wound at different times)/area of original wound × 100%.
Cell invasion assay
For the invasion assay, 5 × 104 cells were resuspended in serum-free medium and seeded into the top chambers of Matrigel-coated Transwell inserts (BD Bioscience, San Jose, CA, USA). The bottom compartment of the chamber contained complete medium as a chemoattractant. After 24-h incubation, cells on the top surface of the membrane were removed slightly by the dry cotton swabs, and cells on the bottom surface of the membrane were washed with PBS, fixed in formaldehyde, and stained with crystal violet and counted in five representative microscopic fields under Olympus fluorescence microscope (Tokyo, Japan) to quantify the extent of invasion, and three independent experiments were performed.
Immunofluorescence
Cells were cultured on glass coverslips, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 30 min at room temperature. After washing three times, 1% bovine serum albumin (BSA) was used for blocking. After 1 h, the cells were incubated with rabbit anti-human primary antibodies to E-cadherin and N-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:50 dilution at 4°C overnight. Thereafter, the cells were washed thrice with Tris-buffered saline with Tween 20 (TBST) and further incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibody (anti-rabbit, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. The nuclei were stained with 1 μg/mL diaminophenylindole (DAPI, 1:100; Sigma-Aldrich, St. Louis, MO, USA) for 5 min at 37°C. The coverslips were then mounted with SlowFade Gold Antifade Reagent (Invitrogen) and observed under a confocal laser microscope (Olympus, Tokyo, Japan).
Western blot
Western blotting was performed using a Bio-Rad protein assay kit (Hercules, CA, USA). Denatured proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels, and then transferred to Hybond membranes (Amersham, Freiburg, Germany). The membranes were blocked overnight in 5% fat-free milk in TBST. The membranes were incubated with primary antibodies against E-cadherin, N-cadherin, FOXA1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C at 1:500 or 1:2000 (GAPDH) dilutions. Thereafter, the membranes were washed thrice in TBST, and further incubated with secondary antibody (1:5000) at room temperature for 2 h. After applying electrochemiluminescence (ECL) Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the protein bands were visualized using X-ray film (Fujifilm, Tokyo, Japan).
Real-time reverse transcription–PCR (RT-PCR)
TRIzol reagent (Takara, Shiga, Japan) was used to isolate RNA from the tissues or cells. The RNA was reverse-transcribed to complementary DNA (cDNA) using an avian myeloblastosis virus transcriptase and random primers (Takara) according to the manufacturer’s protocol. The cDNA was amplified by RT-PCR with a SYBR Premix Ex Taq II kit (Takara). Denaturation was at 94°C for 30 sec, annealing was at 51°C for 60 sec, and extension was at 72°C for 60 sec. The data analysis was performed according to the sample threshold cycle (Ct) value from three independent experiments. 18sRNA was set as the inner control.
Dual-luciferase reporter assay
HEK293T cells were used to perform the dual-luciferase reporter assay. FOXA1 3′UTR clones or mutant clones were cotransfected with miR-93 using Lipofectamine 2000 24 h after plating. PGL-TK vector expressing Renilla luciferase was used as the transfection control. Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. For each transfection, the luciferase activity was averaged from three replicates.
Endometrial carcinoma specimens
57 endometrial carcinomas (ECs) and 12 normal endometrial specimens were collected from patients who had undergone surgical resection at the Department of Gynecology of the First Affiliated Hospital of China Medical University (Shenyang, Liaoning, China). None of the patients had preoperative chemotherapy or radiotherapy. Informed written consent was obtained from all participants and the research protocol was approved by the China Medical University Ethics Committee (No: 2016-32-2).
Statistical analysis
The results were analyzed using a double-sided Student’s t-test to compare the two independent groups and ANOVA (Analysis of Variance) with post-test to compare the three independent groups. Statistical evaluation was performed using SPSS v. 17.0 software (Chicago, IL, USA). A P-value < 0.05 was considered statistically significant.
Results
MiR-93 expression in endometrial carcinoma tissues and normal endometrial specimens
Expression levels of miR-93 in both endometrial carcinoma samples and normal samples were analyzed by RT-PCR (Fig 1A). As shown in Fig 1, miR-93 was highly expressed in EC tissue than in normal samples (P < 0.05; Fig 1B).
Expression levels of miR-93 in both endometrial carcinoma samples and normal samples were analyzed by RT-PCR (A). MiR-93 was highly expressed in EC tissue than in normal samples (B). *P < 0.05.
MiR-93 transfection promoted endometrial carcinoma cell migration and invasion
Endometrial carcinoma cells were transfected with miR-93 mimics to upregulate miR-93 expression. Quantitative RT-PCR (qRT-PCR) revealed significantly increased miR-93 levels (P < 0.05; Fig 2A).
Following miR-93 transfection, miR-93 levels were significantly increased (A), endometrial carcinoma cell lines exhibited faster migration (B), and stronger invasion (C) compared with the control and mock cells 24 hours after transfection. Three representative microscopic fields were photographed to quantify the extent of invasion and migration, and three independent experiments were performed. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, *P < 0.05.
The wound healing assay showed that cells transfected with miR-93 presented faster closing of the scratch wound as compared with the control cells (P < 0.05; Fig 2B). The Transwell assays showed that cells transfected with miR-93 had significantly increased invasive ability as compared with the control cells (P < 0.05; Fig 2C).
MiR-93 transfection downregulated E-cadherin expression and upregulated N-cadherin expression
Following miR-93 transfection, western blotting revealed significantly decreased E-cadherin expression and significantly increased N-cadherin expression (Fig 3A). We further investigated the distribution of E-cadherin and N-cadherin using cellular immunofluorescence (IF). Cellular IF demonstrated that E-cadherin was predominantly located at the membrane of normal and mock transfected endometrial cancer cells, while lack of periphery distribution was observed in miR-93 transfection cells (Fig 3B), while miR-93 transfection induced N-cadherin distribution at the membrane and cytoplasm (Fig 3C).
After miR-93 transfection, E-cadherin was downregulated and N-cadherin was upregulated in Western blot assay (A). Besides, Immunofluorescence assay was used to determine subcellular localization of E-cadherin and N- cadherin (B & C).
MiR-93 transfection downregulated FOXA1 expression
RhoC expression was not significantly different after the endometrial carcinoma cells had been transfected with miR-93 (Fig 4A). According to the microRNA.org prediction website, the FOXA1 3′UTR contains a complementary sequence of miR-93 (Fig 4B). The luciferase reporter assays demonstrated that miR-93 significantly decreased the relative luciferase activity of the wild-type FOXA1 3′UTR as compared with the mutant FOXA1 3′UTR (P < 0.05; Fig 4C). RT-PCR and western blotting showed that miR-93 transfection reduced FOXA1 expression (Fig 4D), while miR-93 inhibitor transfection induced FOXA1 expression (Fig 4E).
After miR-93 transfection the expression of RhoC had no significant differences (A) but FOXA1 was downregulated (D). According to a prediction website and Luciferase reporter assays demonstrated miR-93 could targets the 3’UTR of FOXA1 directly (B & C). MiR-93 inhibitor transfection induced FOXA1 expression (E).
SiFOXA1 transfection promoted endometrial carcinoma cell migration and invasion
Endometrial carcinoma cells were transfected with FOXA1 siRNAs (siFOXA1, siFOAX1-1, and siFOXA1-2) to downregulate FOXA1 expression. After transfection, we analyzed FOXA1 expression using RT-PCR and western blotting, and found significantly decreased FOXA1 expression levels (P < 0.05; Fig 5A), and siFOXA1 was the most effective siRNA in both two cell lines, thus it was used for the following experiments.
After siFOXA1 transfection, the expression levels of FOXA1 decreased significantly (A). Endometrial carcinoma cell lines exhibited faster migration (B), and stronger invasion (C) compared with the control and mock cells 24 hours after transfection. Three representative microscopic fields of each well (n = 3) were documented to quantify the extent of invasion and migration, and three independent experiments were performed. Data are expressed as the mean ± standard deviation, *P < 0.05.
The wound healing assay showed that cells transfected with siFOXA1 presented faster closing of the scratch wound as compared with the control (P < 0.05; Fig 5B). The Transwell assay showed that cells transfected with siFOXA1 demonstrated stronger invasive ability as compared with the control (P < 0.05; Fig 5C).
SiFOXA1 transfection downregulated E-cadherin expression and increased N-cadherin expression
Following siFOXA1 transfection, western blotting revealed decreased E-cadherin expression and increased N-cadherin expression (Fig 6A). We further investigated the distribution of E-cadherin and N-cadherin using cellular immunofluorescence (IF). Cellular IF demonstrated that E-cadherin was predominantly located at the membrane of normal and mock transfected endometrial cancer cells, while lack of periphery distribution was observed in siFOXA1 transfection cells (Fig 6B), while siFOXA1 transfection induced N-cadherin distribution at the membrane and cytoplasm (Fig 6C).
After siFOXA1 transfection, E-cadherin was downregulated and N-cadherin was upregulated in Western blot assay (A). Besides, Immunofluorescence assay was used to determine subcellular localization of E-cadherin and N- cadherin (B & C).
Discussion
It is well known that invasion and metastasis are important poor prognostic factors in malignancy. EMT is considered an important step in tumor invasion and metastasis, involving the transformation of epithelial cells to mesenchymal cells. Through EMT, the transformed epithelial cells gain mesenchymal traits that appear to contribute to metastasis. Individual cancer cells with a mesenchymal phenotype can cross the endothelial barriers and enter the blood and lymphatic circulation [18]. A critical molecular feature of this process is the downregulation of E-cadherin, an epithelial cell marker, and the upregulation of N-cadherin, an interstitial cell marker [19–21].
In other malignant tumors, many reports state that miRNAs can influence tumor invasion and metastasis by participating in the EMT process [22–26]. In ovarian cancer, RhoC overexpression promotes EMT, invasion, and metastasis, and RhoC knockout reverses the promoter role of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF) in EMT [16]. In a previous study, we found that miR-93 downregulates RhoC to inhibit migration and invasion in ovarian cancer [17]. However, the present study derived opposite results in that miR-93 was highly expressed in endometrial carcinoma tissues and, what’s more, miR-93 overexpression promoted endometrial carcinoma cell migration and invasion, and downregulated E-cadherin expression and increased N-cadherin expression, while RhoC expression in endometrial carcinoma cells was unchanged after miR-93 transfection. MiRNAs and their specific targets are dependent on specific cellular environments [27]. Therefore, we guess that the role of miR-93 in endometrial cancer may differ from its role in ovarian cancer and that it may plays a promoter role in EMT, migration, and invasion in endometrial carcinoma.
Next, we analyzed the potential molecular mechanism involved. According to the TargetScan and miRanda prediction websites, the 3′UTR of FOXA1 mRNA contains the complementary sequence of miR-93, which was confirmed by the luciferase reporter assays. MiR-93 transfection in the endometrial cancer cells downregulated FOXA1 expression while miR-93 inhibitor transfection upregulated FOXA1 expression significantly. FOXA1, which belongs to the FOX family of transcription factors, is a negative regulator of epithelial-mesenchymal transition [28–29]. FOXA1 suppresses EMT in pancreatic cancer [30], prostate cancer [31–32] and breast cancer [33–35]. Wang et al. reported that FOXA1 expression was lower in endometrial carcinoma tissue than in normal endometrial tissues and was positively related to the degree of differentiation of endometrial carcinoma, and FOXA1 overexpression inhibited endometrial cancer cell proliferation [36]. Immunohistochemistry has shown that FOXA1 expression is negatively related to lymph node metastasis, and downregulating FOXA1 promotes endometrial cancer cell proliferation and migration [37]. We transfected endometrial carcinoma cells with siFOXA1 to downregulate FOXA1 expression, and found upregulated N-cadherin expression, downregulated E-cadherin expression, and promoted migration and invasion ability, which demonstrates that FOXA1 acts as a suppressor in EMT. Above all, we suggest that miR-93 promote EMT, migration, and invasion in endometrial carcinoma cells through downregulating FOXA1.
Our findings report for the first time that miR-93 promotes endometrial carcinoma cell EMT, migration, and invasion via targeting downregulation of FOXA1, which differs from its anti-oncogene role in ovarian cancer. Targeted therapy with miRNA in malignant tumors is tissue-specific; therefore miRNA selection should be made with care. Further preclinical studies are necessary to determine the role of the same miRNA in different malignant tumors.
Author Contributions
- Conceptualization: YZ.
- Data curation: YZ XC K-XS SC M-XF Y-LX B-LL X-BS.
- Formal analysis: YZ.
- Funding acquisition: YZ SC.
- Methodology: YZ SC.
- Resources: XC K-XS SC.
- Software: XC K-XS.
- Validation: XC K-XS SC M-XF Y-LX B-LL X-BS.
- Writing – original draft: XC K-XS.
- Writing – review & editing: XC K-XS.
References
- 1. Yeramian A, Moreno-Bueno G, Dolcet X, Catasus L, Abal M, Colas E, et al. Endometrial carcinoma:molecular alterations involved in tumor development and progression. Oncogene. 2013; 32: 403–413. pmid:22430211
- 2. Christofori G. New signals from the invasive front. Nature. 2006; 441: 444–450. pmid:16724056
- 3. Meng X, Kong DH, Li N, Zong ZH, Liu BQ, Du ZX, et al. Knockdown of BAG3 induces epithelial-mesenchymal transition in thyroid cancer cells through ZEB1 activation. Cell Death and Disease. 2014; 5: e1092. pmid:24577090
- 4. Mitra A, Mishra L, Li S. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget. 2015; 6(13): 10697–711. pmid:25986923
- 5. Dong P, Kaneuchi M, Konno Y, Watari H, Sudo S, Sakuragi N. Emerging therapeutic biomarkers in endometrial cancer. Biomed Res Int. 2013; 2013: 130362. pmid:23819113
- 6. Chakrabarti R, Hwang J, Andres Blanco M, Wei Y, Lukačišin M, Romano RA, et al. Elf5 inhibits the epithelial-mesenchymal transition in mammary gland development and breast cancer metastasis by transcriptionally repressing Snail2. Nat Cell Biol. 2012; 14: 1212–1222. pmid:23086238
- 7. Azmi AS, Muqbil I, Wu J, Aboukameel A, Senapedis W, Baloglu E, et al. Philip and Ramzi M. Mohammad: Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition. Sci Rep. 2015; 5: 16077. pmid:26536918
- 8. Cheng CW, Chen PM, Hsieh YH, Weng CC, Chang CW, Yao CC, et al. Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in ERK-responsive lung cancer. Oncotarget. 2015 Dec 29; 6(42):44222–38. pmid:26528854
- 9. Zhao M, Fang W, Wang Y, Guo S, Shu L, Wang L, et al. Enolase-1 is a therapeutic target in endometrial carcinoma. Oncotarget. 2015; 6(17): 15610–27. pmid:25951350
- 10. Dong P, Konno Y, Watari H, Hosaka M, Noguchi M, Sakuragi N. The impact of microRNA-mediated PI3K/AKT signaling on epithelial-mesenchymal transition and cancer stemness in endometrial cancer. Journal of Translational Medicine J Transl Med. 2014; 12: 231. pmid:25141911
- 11. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat. Rev. Cancer. 2006; 6: 857–866. pmid:17060945
- 12. Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, Nair S. The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis. Nat. Cell Biol. 2008; 10(2): 202–10. pmid:18193036
- 13. Wang L, Song G, Tan W, Qi M, Zhang L, Chan J, et al. MiR-573 inhibits prostate cancer metastasis by regulating epithelial-mesenchymal transition. Oncolotarget. 2015 Nov 3; 6(34):35978–90.
- 14. Liu T, Nie F, Yang X, Wang X, Yuan Y, Lv Z, et al. MicroRNA-590 is an EMT-suppressive microRNA involved in the TGFβ signaling pathway. Mol Med Rep. 2015; 12(5): 7403–11. pmid:26459119
- 15. Ghahhari NM, Babashah S. Interplay between microRNAs and WNT/β-catenin signaling pathway regulates epithelial-mesenchymal transition in cancer. Eur J Cancer. 2015; 51(12): 1638–49. pmid:26025765
- 16. Gou WF, Zhao Y, Lu H, Yang XF, Xiu YL, Zhao S, et al. The role of RhoC in EMT of ovarian carcinoma cells. BMC Cancer. 2014; 14: 447.
- 17. Chen X, Chen S, Xiu YL, Sun KX, Zong ZH, Zhao Y. RhoC is a major target of microRNA-93-5P in epithelial ovarian carcinoma tumorigenesis and progression. Mol Cancer. 2015; 14(1): 31.
- 18. Wang SS, Jiang J, Liang XH, Tang YL. Links between cancer stem cells and epithelial–mesenchymal transition. Onco Targets Ther. 2015; 8: 2973–80. pmid:26527883
- 19. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED, et al. Epithelial—mesenchymal and mesenchymal—epithelial transitions in carcinoma progression. J Cell Physiol. 2007; 213: 374–383. pmid:17680632
- 20. Yang WN, Ai ZH, Wang J, Xu YL, Teng YC. Correlation between the overexpression of epidermal growth factor receptor and mesenchymal makers in endometrial carcinoma. Journal of Gynecol Oncol. 2014 Jan; 25(1):36–42.
- 21. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell. 2008; 14: 818–829. pmid:18539112
- 22. Ye Z, Zhao L, Li J, Chen W, Li X. miR-30d Blocked Transforming Growth Factor β1-Induced Epithelial-Mesenchymal Transition by Targeting Snail in Ovarian Cancer Cells. Int J Gynecol°Cancer. 2015; 9: 1574–81.
- 23. Long H, Wang Z, Chen J, Xiang T, Li Q, Diao X, et al. microRNA-214 promotes epithelial-mesenchymal transition and metastasis in lung adenocarcinoma by targeting the suppressor-of-fused protein (Sufu). Oncotarget. 2015 Nov 17; 6(36):38705–18. pmid:26462018
- 24. Chen SP, Liu BX, Xu J, Pei XF, Liao YJ, Yuan F, et al. MiR-449a suppresses the epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma by multiple targets. BMC°Cancer. 2015; 15 (1): 706.
- 25. De Mattos-Arruda L, Bottai G, Nuciforo PG, Di Tommaso L, Giovannetti E, Peg V, et al. MicroRNA-21 links EMT and inflammatory signals to confer resistance to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast cancer patients. Oncotarget. 2015 Nov 10; 6(35):37269–80. pmid:26452030
- 26. Qu J, Li M, An J, Zhao B, Zhong W, Gu Q, et al. MicroRNA-33b inhibits lung adenocarcinoma cell growth, invasion, and epithelial-mesenchymal transition by suppressing Wnt/β-catenin/ZEB1 signaling. Int J Oncol. 2015 Dec; 47(6):2141–52. pmid:26459797
- 27. Meltzer PS. Cancer genomics: Small RNAs with big impacts. Nature. 2005; 435:745–746. pmid:15944682
- 28. Hannenhalli S, Kaestner KH. The evolution of FOX genes and their role in development and disease. Nat Rev Genet. 2009; 10: 233–40. pmid:19274050
- 29. Huang JZ, Chen M, Zeng M, Xu SH, Zou FY, et al. Down-regulation of TRPS1 stimulates epithelial-mesenchymal transition and metastasis through repression of FOXA1. J Pathol. 2016 Mar 11. pmid:26969828
- 30. Song Y, Washington MK, Crawford HC. Loss of FOXA1/2 is essential for the EMT in pancreatic cancer. Cancer Res. 2010; 70(5): 2115–25. pmid:20160041
- 31. Liu YN, Abou-Kheir W, Yin JJ, Fang L, Hynes P, Casey O, et al. Critical and reciprocal regulation of KLF4 and SLUG in transforming growth factor β-initiated prostate cancer epithelial-mesenchymal transition. Mol Cell Biol. 2012; 32(5): 941–53. pmid:22203039
- 32. Jin HJ, Zhao JC, Ogden I, Bergan RC, Yu J. Androgen receptor-independent function of FOXA1 in prostate cancer metastasis. Cancer Res. 2013; 73(12): 3725–36. pmid:23539448
- 33. Lin YC, Lee YC, Li LH, Cheng CJ, Yang RB. Tumor suppressor SCUBE2 inhibits breast-cancer cell migration and invasion through the reversal of epithelial-mesenchymal transition. J°Cell°Sci. 2014; 127 (Pt 1): 85–100.
- 34. Zhang XL, Wang HS, Liu N, Ge LC. Bisphenol A stimulates the epithelial mesenchymal transition of estrogen negative breast cancer cells via FOXA1 signals. Arch Biochem Biophys. 2015; 585: 10–6. pmid:26363213
- 35. Wu S, Luo Z, Yu PJ, Xie H, He YW. Suberoylanilide hydroxamic acid (SAHA) promotes the epithelial mesenchymal transition of triple negative breast cancer cells via HDAC8/FOXA1 signals. Biol Chem. 2016 Jan; 397(1):75–83. pmid:26431101
- 36. Wang J, Bao W, Qiu M, Liao Y, Che Q, Yang T, et al. Forkhead-box A1 suppresses the progression of endometrial cancer via crosstalk with estrogen receptor α. Oncol Rep. 2014; 31(3): 1225–34. pmid:24452315
- 37. Qiu M, Bao W, Wang J, Yang T, He X, Liao Y, et al. FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer. BMC°Cancer. 2014; 14:78.