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MiR-498 regulated FOXO3 expression and inhibited the proliferation of human ovarian cancer cells
Biomedicine & Pharmacotherapy 72 (2015) 52–57
Available online at
ScienceDirect www.sciencedirect.com
Original article
MiR-498 regulated FOXO3 expression and inhibited the proliferation of human ovarian cancer cells Ruonan Liu a,*, Fenghua Liu a, Lei Li a, Miaomiao Sun b, Kuisheng Chen c a
Department of Gynecology, Cancer Hospital Affiliated to Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450008, People’s Republic of China Pathological Department, Cancer Hospital Affiliated to Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450008, People’s Republic of China c Department of Pathology and Pathological Physiology, Basic Medical College of Zhengzhou University, Zhengzhou 450052, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 March 2015 Accepted 3 April 2015
Ovarian cancer is one of the most common human malignancies and the fifth leading cause of cancer deaths in women. Thus, improved approaches for detection of ovarian cancer are urgently needed. Recently, microRNAs (miRNAs) have been suggested to be closely associated with ovarian cancer tumorigenesis. In the current study, our study showed that expression of miR-498 was markedly downregulated in ovarian cancer cells and ovarian cancer tissues compared with human ovary surface epithelial cells (HOSE) and the matched tumor adjacent normal tissues (ANT). Ectopic expression of miR498 suppressed cell proliferation of ovarian cancer cells, while i miR-498-in showed the opposite effect. Furthermore, upregulation of miR-498 in ovarian cancer cells resulted in blocking of their entry into the S transitional phase, which was caused by downregulation of the CDK regulator cyclin D1 and upregulation of cyclin-dependent kinase inhibitor p27. Additionally, we identified FOXO3 as a direct target of miR-498. Moreover, we demonstrated that miR-498 upregulated FOXO3 expression by directly targeting the FOXO3 30 -untranslated region. Thus, our findings suggested that miR-498 acted as a new tumor suppressor by targeting the FOXO3 gene and inhibiting cell proliferation of ovarian cancer. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: miR-498 Human ovarian cancer FOXO3 Cell proliferation
1. Introduction Ovarian cancer is generally accepted as the most lethal gynecological malignancy in women [1]. The majority of patients are diagnosed at advanced stage, which is the reason of high mortality rate of ovarian cancer [2]. Thus, there is urgent and of great interest to elucidate the potential mechanism that mediate the initiation and progression of ovarian cancer. The discovery of miRNAs and their function in tumor progression provide new sights for ovarian cancer research. MicroRNAs (miRNAs), small non-coding RNAs of 20–22 nucleotides, have been identified as a new type of gene expression regulators, which play essential roles in the biological and metabolic processes of cancer, including cell proliferation, apoptosis, invasion and differentiation [3–6]. Previous study revealed that miR-498 has been shown to be one of the important determinants of cell tumorigenicity in various kinds of tumors, including ovarian cancer [7–10]. However, the relationship * Corresponding author. Tel.: +86 371 65587320. E-mail address: [email protected] (R. Liu). http://dx.doi.org/10.1016/j.biopha.2015.04.005 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
between ovarian cancer and the expression of miR-498 has not yet been elucidated. In the present study, we investigated the biological effects and the potential mechanisms of miR-498 in ovarian cancer. Further investigations revealed that miR-498 directly targeted the 30 -UTR of FOXO3 to increase the expression of this gene, which in turn inhibited the proliferation of ovarian cancer. 2. Materials and methods 2.1. Clinical specimens Eight pairs of primary ovarian carcinomas tissues and the matched tumor adjacent normal tissues (ANT) were collected from patients in Department of Gynecology, Cancer Hospital Affiliated to Zhengzhou University, Henan cancer hospital (Zhengzhou, People’s Republic of China). The study was approved by the ethics committee of Cancer Hospital Affiliated to Zhengzhou University, Henan cancer hospital (Zhengzhou, People’s Republic of China). Written informed consent was obtained from all patients. Samples were snap frozen immediately and stored at 80 8C.
R. Liu et al. / Biomedicine & Pharmacotherapy 72 (2015) 52–57
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2.2. Cell culture
2.7. Cell cycle analysis
Human ovarian cancer cell lines A2780, CAOV-3, SKOV-3, HO8910 and ES-2 were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM, HyClone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone, USA), 100 units/ ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and HOSE (Pricells, Wuhan, China), a cell derived from human ovary surface epithelial cells, act as the normal control cell, was maintained in cell culture medium consisting of 1:1 Medium199 (Sigma–Aldrich) and MCDB105 medium (Sigma–Aldrich) with 10% heat-inactivated FBS and 10 ng/ml epidermal growth factor (Sigma–Aldrich). All cell lines were cultured in a humidified incubator in an atmosphere of 5% CO2 and 95% air at 37 8C.
Cells were harvested by trypsinization, washed in ice-cold phosphate-buffered saline (PBS) and fixed in 80% ice-cold ethanol in PBS. Before staining, cells were sedimented in a chilled centrifuge and resuspended in cold PBS. Bovine pancreatic RNase (Sigma–Aldrich) was added to a final concentration of 2 mg/ml, and cells were incubated at 37 8C for 30 min, followed by incubation with 20 mg/ml of propidium iodide (Sigma–Aldrich) for 20 min at room temperature. Cell cycle profiles of 5 104 cells were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).
2.3. Plasmids, small interfering RNA and transfection The miR-498 stem-loop (miR-498), inhibitors of miR-498 (miR498-in) and negative control were purchased from RiboBio (Guangzhou, China). FOXO3 ORFs with 30 -UTR was amplified using PCR and subcloned into pEGFP-N3 (Invitrogen, China). Plasmids were transfected into OC cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. For FOXO3 depletion, small interfering RNA (siRNA-FOXO3, HSH005759) was synthesized and purified by GeneCopoeia Co. (Guangzhou, China). Transfection of siRNAs was performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. 2.4. RNA extraction and real-time quantitative PCR Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription and quantitative PCR were performed using the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Dalian, China) by using the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers were synthesized by GeneCopoeia Co., FOXO3 (HQP000364), Cyclin D1 (HQP016204), p27 (HQP000342), GAPDH (HQP006940). Moreover, the cycle threshold (Ct) value was used for our analysis (DCt), and we determined the expression of small nuclear RNA U6 and GAPDH mRNA as internal controls to calculate the relative expression DD levels of miR-498, Cyclin D1, p27 via the 2 Ct method. 2.5. MTT assays and Colony formation For cell proliferation analysis, cell proliferation was examined by the MTT assay (Sigma–Aldrich) according to the standard protocol after transfection with miR-498 or miR-498-in or miR-NC for 24 h. For colony formation assay, HO8910 cells were plated into three 6-cm cell culture dishes (1 103 cells per well) and incubated in DMEM medium containing 10% FBS. After 2 weeks, the colonies were fixed with 10% formaldehyde for 15 min, stained with 1.0% crystal violet for 1 min. The number of colonies, defined as >50 cells/colony were counted. 2.6. Anchorage-independent growth assay Cells were trypsinized, and 1000 transfected cells were seeded in 2 ml complete medium plus 0.3% agar (Sigma). The agar-cell mixture was plated on top of a bottom layer consisting of 1% agar in complete medium. Cells were incubated at 37 8C. After 14 days, the colonies were stained with 0.1% Crystal Violet for staining and counted under microscope. Only cell colonies containing more than 50 cells were counted.
2.8. Luciferase assays Cells were co-transfected with 300 ng of miR-498 or miR-498in, 100 ng of the reporter vector containing the FOXO3 30 -UTR or the mutant 30 -UTR, and 25 ng of the Renilla luciferase vector as a transfection control. After 48 h of transfection, luciferase and Renilla activities were assayed, the cells were lysed and the fluorescence intensity was detected using the dual luciferase assay kit (Promega, Madison, WI, USA). 2.9. Western blotting The cells were lysed in RIPA lysis buffer (Sigma, St. Louis, MO, USA). Protein lysates were prepared, and protein concentration was measured using the BCA Protein Assay kit (Beyotime, China). Equal quantities of protein samples were loaded on 10% SDS-PAGE and transferred onto PVDF membranes, and Blocking was performed with 5% non-fat milk. The membrane was incubated overnight with anti-FOXO3, anti-Cyclin D1, anti-p27 (1:1000 dilution; Cell Signaling Technology) and anti-a-tubulin (Sigma– Aldrich). After washing and incubation with horseradish peroxidase-conjugated antibody (Sigma–Aldrich) for 1.5 h at room temperature, blotted proteins were detected using an enhanced chemiluminescence (ECL) system following the manufacturer’s protocol. 2.10. Statistical analysis All the statistics were expressed as mean standard deviation (SD) of three independent experiments and analysis by SPSS 18.0 software. Student’s t test was used to evaluate the significance of the differences between two groups of data in all the pertinent experiments. P < 0.05 was considered to be statistically significant. 3. Result 3.1. MiR-498 expression was downregulated in ovarian cancer cell lines and ovarian cancer tissues The expression pattern of miR-498 in human ovarian cancer has not been analyzed. We first evaluated the expression levels of miR498 in ovarian cancer tissues and cell lines by qRT-PCR. As shown in Fig. 1, compared with their non-cancerous counterparts, it was observed that miR-498 expression levels were lower in ovarian cancer tissues, it was also shown that miR-498 was downregulated in ovarian cancer cell lines compared with HOSE. Together, we conclude that miR-498 was abnormally downregulated in ovarian cancer tissues and ovarian cancer cell lines. 3.2. MiR-498 suppressed cell proliferation of ovarian cancer Next, to investigate the role of miR-498 in terms of cell proliferation, we transfected the HO8910 cells with miR-498
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Fig. 1. Expression of miR-498 in human ovarian cancer clinical specimens and cell lines. (A) Relative miR-498 expression levels in 8 paired primary ovarian cancer tissues and the matched tumor adjacent normal tissues (ANT) from the same patient were detected by PCR analysis. (B) Real-time PCR analysis of miR-498 expression in human ovary surface epithelial cells (HOSE) and OC cell lines, including A2780, CAOV-3, SKOV-3, ES-2 and HO8910. Experiments were repeated at least three times. Each bar represents the mean of three independent experiments. *P < 0.05.
mimics, miR-498-in or the respective controls. The transfection efficiency of miR-498 and miR-498-in was validated by qRT-PCR (Figs. 2A and 3A). To assess the potential role of miR-498 in ovarian cancer tumorigenesis, we determine whether miR-498 affects cell proliferation of ovarian cancers using MTT and colony formation assays. Our data indicated that the proliferation of HO8910 cells was decreased when cells were transfected with miR-498, while the miR-498-in showed the opposite effect (Figs. 2B, C and 3B, C). Using flow cytometry assays, we found that miR-498 overexpression decreased the percentage of cells in S phase and significantly increased the percentage of cells in G1/G0 (Fig. 2D), the opposite result was obtained when the cells were treated with miR-498-in (Fig. 3D). Collectively, our results suggest that miR-498 reduced ovarian cancer cell tumorigenicity in vitro.
3.3. MiR-498 directly restrained the expression of FOXO3 through direct targeting of FOXO3 mRNA 30 UTR and altered levels of proteins related to cell proliferation and cycle in ovarian cancer cells Potential target of miR-498 was predicted using bioinformatics methods. FOXO3 was selected as the target for further analysis. HO8910 cells were transiently transfected with miR-498 mimics, miR-498-in or the respective controls. Interestingly, we observed that miR-498 was capable of down-regulating FOXO3 protein expression, while miR-498-in clearly promoted its protein expression (Fig. 4B). To gain insight into the mechanism by which miR-498 inhibits FOXO3, we identified the miR-498 binding site in the FOXO3 mRNA 30 UTR (Fig. 4A) and constructed pGL3-FOXO3 and pGL3-FOXO3-mut
Fig. 2. MiR-498 upregulation inhibited cell proliferation of ovarian cancer. (A) Validation of miR-498 expression levels after transfection by PCR analysis. (B) MTT assays revealed that upregulation of miR-498 inhibited growth of HO8910 ovarian cancer cell line. (C) Representative micrographs (left) and quantification (right) of crystal violetstained cell colonies. (D) Flow cytometric analysis of the indicated HO8910 cells transfected with NC or miR-498. Each bar represents the mean of three independent experiments. *P < 0.05.
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Fig. 3. Inhibition of miR-498 promoted cell proliferation of ovarian cancer. (A) Validation of miR-498 expression levels after transfection by PCR analysis. (B) MTT assays revealed that inhibition of miR-498 promoted growth of HO8910 ovarian cancer cell line. (C) Representative micrographs (left) and quantification (right) of crystal violetstained cell colonies. (D) Flow cytometric analysis of the indicated HO8910 cells transfected with NC or miR-498-in. Each bar represents the mean of three independent experiments. *P < 0.05.
plasmids. Results of luciferase reporter assays showed that the cotransfection of miR-498 markedly increased the firefly luciferase activity of pGL3-FOXO3 but failed to influence the luciferase activity of pGL3-FOXO3-mut in HO8910 cells (Fig. 4C). Meanwhile, HO8910 cells transfected with miR-498-in resulted in suppressing firefly luciferase activity of the wild-type reporter but unaffected the mutant reporter (Fig. 4C). In summary, our data indicate that miR498 directly attenuated the expression of FOXO3 by targeting of its mRNA 30 UTR in ovarian cancer cell lines. Previous studies revealed that enhancing transcriptional activity of FOXO3 resulting in upregulation of cyclin-dependent kinase inhibitor p27 and downregulation of the CDK regulator cyclin D1 [11,12]. To investigate the mechanism underlying cell proliferation and cell cycle, we tested the effect of miR-498 on critical cell-proliferation and cell cycle related regulators cyclin D1 and p27. Results of real-time PCR and Western blotting analysis showed that cyclin D1 (mRNA and protein) levels were downregulated and p27 (mRNA and protein) levels were upregulated in miR-498-transfected HO8910 cells, compared to NC-transfected cells, while miR-498-in showed the opposite results (Fig. 4D and E). Altogether, our results indicated that miR-498 regulated expression of FOXO3, and then functionally modulated cell proliferation and cell cycle regulators, p27 and cyclin D1, thus relevant to cell proliferation and cell cycle. 3.4. FOXO3 is involved in miR-498-induced proliferation of ovarian cancer cells To further understand the role of FOXO3 in miR-498mediated pro-proliferation, miR-498-transfected HO8910 cells
were transfected with FOXO3-siRNA. As showed in Fig. 5A, results of Western blot analysis revealed that the FOXO3 expression in miR-498-transfected HO8910 cells was decreased after transfected with FOXO3-siRNA. Colony formation assays showed that the treatment with FOXO3-siRNA was able to reverse the miR-498-decreased proliferation (Fig. 5B), suggesting that miR-498 suppresses the proliferation of ovarian cancer cells by upregulating FOXO3. Therefore, our results demonstrate that miR-498 was able to inhibit the proliferation of ovarian cancer cells through direct targeting FOXO3. 4. Discussion The key finding of the current study is that miR-498 expression was markedly downregulated in ovarian cancer tissues and ovarian cancer cells as compared with that in the matched tumor adjacent normal tissues and human ovary surface epithelial cells. Our result revealed that ectopic expression of miR-498 could decrease the cell proliferation of ovarian cancer, while miR-498-in enhanced this effect. Result of dual-luciferase assay showed that FOXO3 was a direct of miR-498. Further experiment showed that upregulation of miR-498 in ovarian cancer cells led to downregulation of the cell-cycle regulator cyclin D1 and the downregulation of cyclin-dependent kinase (CDK) inhibitors p27 through downregulation of FOXO3 via directly targeting the 30 UTR of FOXO3. Taken together, these findings demonstrated that miR-498 may play an important role in carcinogenesis and progression of ovarian cancer. Numerous studies indicated that MicroRNAs (miRNAs), a class of small non-coding RNAs, potentially play critical roles in cell
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Fig. 4. MiR-498 increased FOXO3 expression by directly targeting the FOXO3 30 -UTR and altered levels of proteins related to cell proliferation and cell cycle in HO8910 cells. (A) Predicted miR-498 target sequence in the 30 -UTR of FOXO3 (FOXO3-30 -UTR) and positions of three mutated nucleotides (red) in the 30 -UTR of FOXO3 (FOXO3-30 -UTRmut). (B) Western blotting analysis of FOXO3 expression in cells transfected with miR-498 or the miR-498-in. a-Tubulin served as the loading control. (C) Luciferase reporter assay of the indicated cells transfected with the pGL3-FOXO3-30 -UTR reporter or pGL3-FOXO3-30 -UTR-mut reporter and miR-498 or miR-498-in with increasing amounts (10 and 50 nM) oligonucleotides. (D) Real-time PCR analysis of expression of cyclin D1 and p27 in indicated HO8910 cells. (E) Western blotting analysis of expression of cyclin D1 and p27 protein in HO8910 cells. a-Tubulin served as the loading control. *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
proliferation, cycle, differentiation, angiogenesis, invasion and migration of various types of human cancers [13–16]. MiRNAs regulate gene expression and are proposed as potential novel targets for anti-cancer therapies. However, it was uncertain whether dysregulation of miR-498 was associated with the progression of ovarian cancer. In this study, our results revealed that miR-498 was significantly down-regulated in ovarian cancer and inhibits cell proliferation of ovarian cancer in vitro and suggested miR-498 as a candidate tumor suppressor in the pathogenesis of ovarian cancer.
FOXO3 protein, a member of the forkhead transcriptional factor family, is critical in cell growth, proliferation, and development of cancer [17,18]. The levels of FOXO3 are highly expressed in most human cancers including ovarian cancer [19,20]. Through bioinformatics analysis, the tumor suppressor FOXO3 gene was indicated as a theoretical targeted gene of miR-498. Result of Western blotting analysis showed that overexpression of miR-498 resulted in the increase expression of FOXO3 protein. It has been demonstrated that the transcriptional factor FOXO3 can downregulate cyclin D1 and upregulate the expression of p27 at the
Fig. 5. FOXO3 upregulation is required for miR-498-induced proliferation of ovarian cancer cells. (A) Western blot analysis verified that silencing FOXO3 effectively decreased the expression of FOXO3 in miR-498-transfected HO8910 cells. (B) miR-498-transfected HO8910 cells after transfection with FOXO3-siRNAs promoted cell colonies formation. Each bar represents the mean of three independent experiments. *P < 0.05.
R. Liu et al. / Biomedicine & Pharmacotherapy 72 (2015) 52–57
transcriptional level [21,22]. Consistent with these reports, our result indicated that miR-498 exerts its functions by upregulating expression of FOXO3, which in turn may cause downregulating of expression of cyclin D1 and upregulating expression of p27, and then blocks cell cycle progression at G0/G1 phase, resulting in suppressing cell proliferation. Furthermore, FOXO3-silenced in HO8910-miR-498 cells had positive effect on cell proliferation, suggesting that direct up-regulation of FOXO3 is required for miR498-induced cell proliferation of ovarian cancer. In summary, the current study provides that an important link between miR-498-mediated proliferation of ovarian cancer cells and upregulation of FOXO3. Our findings revealed an essential role of miR-498 in the regulation of ovarian cancer cells proliferation. Therefore, all the results indicated that miR-498 was an antioncomiR and his implies miR-498 to be a potential mediator for novel miRNA replacement therapy for ovarian cancer. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by Department of Gynecology, Cancer Hospital Affiliated to Zhengzhou University, Henan Cancer Hospital. All authors designed the study together, performed the experiment together, analyzed the data and wrote the paper; all authors approved the final manuscript. References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9–29. [2] A. Kumar, J.N. Bakkum-Gamez, A.L. Weaver, M.E. McGree, W.A. Cliby, Impact of obesity on surgical and oncologic outcomes in ovarian cancer, Gynecol. Oncol. 135 (2014) 19–24. [3] G. Di Leva, M. Garofalo, C.M. Croce, MicroRNAs in cancer, Annu. Rev. Pathol. 9 (2014) 287–314. [4] E. Shoshan, A.K. Mobley, R.R. Braeuer, T. Kamiya, L. Huang, M.E. Vasquez, A. Salameh, H.J. Lee, S.J. Kim, C. Ivan, G. Velazquez-Torres, K.M. Nip, K. Zhu, D. Brooks, S.J. Jones, I. Birol, M. Mosqueda, Y.Y. Wen, A.K. Eterovic, A.K. Sood, P. Hwu, J.E. Gershenwald, A. Gordon Robertson, G.A. Calin, G. Markel, I.J. Fidler, M. Bar-Eli, Reduced adenosine-to-inosine mir-455-5p editing promotes melanoma growth and metastasis, Nat. Cell Biol. 17 (2015) 311–321.
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[5] X. Wan, X. Ding, S. Chen, H. Song, H. Jiang, Y. Fang, P. Li, J. Guo, The functional sites of miRNAs and lncRNAs in gastric carcinogenesis, Tumour Biol. 36 (2015) 521–532. [6] S.H. Chan, L.H. Wang, Regulation of cancer metastasis by microRNAs, J. Biomed. Sci. 22 (2015) 9. [7] V. Gopalan, R.A. Smith, A.K. Lam, Downregulation of microRNA-498 in colorectal cancers and its cellular effects, Exp. Cell Res. 330 (2015) 423–428. [8] R. Kasiappan, Z. Shen, A.K. Tse, U. Jinwal, J. Tang, P. Lungchukiet, Y. Sun, P. Kruk, S.V. Nicosia, X. Zhang, W. Bai, 1,25-Dihydroxyvitamin d3 suppresses telomerase expression and human cancer growth through microRNA-498, J. Biol. Chem. 287 (2012) 41297–41309. [9] S.K. Leivonen, K.K. Sahlberg, R. Makela, E.U. Due, O. Kallioniemi, A.L. BorresenDale, M. Perala, High-throughput screens identify microRNAs essential for HER2 positive breast cancer cell growth, Mol. Oncol. 8 (2014) 93–104. [10] L. Santarpia, G.A. Calin, L. Adam, L. Ye, A. Fusco, S. Giunti, C. Thaller, L. Paladini, X. Zhang, C. Jimenez, F. Trimarchi, A.K. El-Naggar, R.F. Gagel, A miRNA signature associated with human metastatic medullary thyroid carcinoma, EndocrineRelat. Cancer 20 (2013) 809–823. [11] C. Lin, Z. Wu, X. Lin, C. Yu, T. Shi, Y. Zeng, X. Wang, J. Li, L. Song, Knockdown of FLOT1 impairs cell proliferation and tumorigenicity in breast cancer through upregulation of FOXO3a, Clin. Cancer Res. 17 (2011) 3089–3099. [12] A. Cappellini, G. Tabellini, M. Zweyer, R. Bortul, P.L. Tazzari, A.M. Billi, F. Fala, L. Cocco, A.M. Martelli, The phosphoinositide 3-kinase/Akt pathway regulates cell cycle progression of HL60 human leukemia cells through cytoplasmic relocalization of the cyclin-dependent kinase inhibitor p27(Kip1) and control of cyclin d1 expression, Leukemia 17 (2003) 2157–2167. [13] Y. Kinose, K. Sawada, K. Nakamura, T. Kimura, The role of microRNAs in ovarian cancer, BioMed Res. Int. 2014 (2014) 249393. [14] S.D. Li, J.R. Zhang, Y.Q. Wang, X.P. Wan, The role of microRNAs in ovarian cancer initiation and progression, J. Cell. Mol. Med. 14 (2010) 2240–2249. [15] D. Mezzanzanica, M. Bagnoli, L. De Cecco, B. Valeri, S. Canevari, Role of microRNAs in ovarian cancer pathogenesis and potential clinical implications, Int. J. Biochem. Cell Biol. 42 (2010) 1262–1272. [16] G. Di Leva, C.M. Croce, The role of microRNAs in the tumorigenesis of ovarian cancer, Front. Oncol. 3 (2013) 153. [17] X. Zhao, Y. Liu, L. Du, L. He, B. Ni, J. Hu, D. Zhu, Q. Chen, Threonine 32 (Thr32) of FOXO3 is critical for TGF-beta-induced apoptosis via Bim in hepatocarcinoma cells, Protein Cell 6 (2015) 127–138. [18] J. Li, P. Li, T. Chen, G. Gao, X. Chen, Y. Du, R. Zhang, R. Yang, W. Zhao, S. Dun, F. Gao, G. Zhang, Expression of microRNA-96 and its potential functions by targeting FOXO3 in non-small cell lung cancer, Tumour Biol. 36 (2015) 685–692. [19] Z. Jin, L. Zheng, X. Xin, Y. Li, T. Hua, T. Wu, H. Wang, Upregulation of forkhead box o3 transcription is involved in c2-ceramide induced apoptosis and autophagy in ovarian cancer cells in vitro, Mol. Med. Rep. 10 (2014) 3099–3105. [20] M. Lu, Y. Zhao, F. Xu, Y. Wang, J. Xiang, D. Chen, The expression and prognosis of FOXO3a and skp2 in human ovarian cancer, Med. Oncol. (Northwood, London, Engl.) 29 (2012) 3409–3415. [21] C. Xie, L.B. Song, J.H. Wu, J. Li, J.P. Yun, J.M. Lai, D.Y. Xie, B.L. Lin, Y.F. Yuan, M. Li, Z.L. Gao, Upregulator of cell proliferation predicts poor prognosis in hepatocellular carcinoma and contributes to hepatocarcinogenesis by downregulating FOXO3a, PLoS ONE 7 (2012) e40607. [22] M. Schmidt, S. Fernandez de Mattos, A. van der Horst, R. Klompmaker, G.J. Kops, E.W. Lam, B.M. Burgering, R.H. Medema, Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D, Mol. Cell. Biol. 22 (2002) 7842–7852.
Available online at
ScienceDirect www.sciencedirect.com
Original article
MiR-498 regulated FOXO3 expression and inhibited the proliferation of human ovarian cancer cells Ruonan Liu a,*, Fenghua Liu a, Lei Li a, Miaomiao Sun b, Kuisheng Chen c a
Department of Gynecology, Cancer Hospital Affiliated to Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450008, People’s Republic of China Pathological Department, Cancer Hospital Affiliated to Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450008, People’s Republic of China c Department of Pathology and Pathological Physiology, Basic Medical College of Zhengzhou University, Zhengzhou 450052, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 March 2015 Accepted 3 April 2015
Ovarian cancer is one of the most common human malignancies and the fifth leading cause of cancer deaths in women. Thus, improved approaches for detection of ovarian cancer are urgently needed. Recently, microRNAs (miRNAs) have been suggested to be closely associated with ovarian cancer tumorigenesis. In the current study, our study showed that expression of miR-498 was markedly downregulated in ovarian cancer cells and ovarian cancer tissues compared with human ovary surface epithelial cells (HOSE) and the matched tumor adjacent normal tissues (ANT). Ectopic expression of miR498 suppressed cell proliferation of ovarian cancer cells, while i miR-498-in showed the opposite effect. Furthermore, upregulation of miR-498 in ovarian cancer cells resulted in blocking of their entry into the S transitional phase, which was caused by downregulation of the CDK regulator cyclin D1 and upregulation of cyclin-dependent kinase inhibitor p27. Additionally, we identified FOXO3 as a direct target of miR-498. Moreover, we demonstrated that miR-498 upregulated FOXO3 expression by directly targeting the FOXO3 30 -untranslated region. Thus, our findings suggested that miR-498 acted as a new tumor suppressor by targeting the FOXO3 gene and inhibiting cell proliferation of ovarian cancer. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: miR-498 Human ovarian cancer FOXO3 Cell proliferation
1. Introduction Ovarian cancer is generally accepted as the most lethal gynecological malignancy in women [1]. The majority of patients are diagnosed at advanced stage, which is the reason of high mortality rate of ovarian cancer [2]. Thus, there is urgent and of great interest to elucidate the potential mechanism that mediate the initiation and progression of ovarian cancer. The discovery of miRNAs and their function in tumor progression provide new sights for ovarian cancer research. MicroRNAs (miRNAs), small non-coding RNAs of 20–22 nucleotides, have been identified as a new type of gene expression regulators, which play essential roles in the biological and metabolic processes of cancer, including cell proliferation, apoptosis, invasion and differentiation [3–6]. Previous study revealed that miR-498 has been shown to be one of the important determinants of cell tumorigenicity in various kinds of tumors, including ovarian cancer [7–10]. However, the relationship * Corresponding author. Tel.: +86 371 65587320. E-mail address: [email protected] (R. Liu). http://dx.doi.org/10.1016/j.biopha.2015.04.005 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
between ovarian cancer and the expression of miR-498 has not yet been elucidated. In the present study, we investigated the biological effects and the potential mechanisms of miR-498 in ovarian cancer. Further investigations revealed that miR-498 directly targeted the 30 -UTR of FOXO3 to increase the expression of this gene, which in turn inhibited the proliferation of ovarian cancer. 2. Materials and methods 2.1. Clinical specimens Eight pairs of primary ovarian carcinomas tissues and the matched tumor adjacent normal tissues (ANT) were collected from patients in Department of Gynecology, Cancer Hospital Affiliated to Zhengzhou University, Henan cancer hospital (Zhengzhou, People’s Republic of China). The study was approved by the ethics committee of Cancer Hospital Affiliated to Zhengzhou University, Henan cancer hospital (Zhengzhou, People’s Republic of China). Written informed consent was obtained from all patients. Samples were snap frozen immediately and stored at 80 8C.
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2.2. Cell culture
2.7. Cell cycle analysis
Human ovarian cancer cell lines A2780, CAOV-3, SKOV-3, HO8910 and ES-2 were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM, HyClone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone, USA), 100 units/ ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and HOSE (Pricells, Wuhan, China), a cell derived from human ovary surface epithelial cells, act as the normal control cell, was maintained in cell culture medium consisting of 1:1 Medium199 (Sigma–Aldrich) and MCDB105 medium (Sigma–Aldrich) with 10% heat-inactivated FBS and 10 ng/ml epidermal growth factor (Sigma–Aldrich). All cell lines were cultured in a humidified incubator in an atmosphere of 5% CO2 and 95% air at 37 8C.
Cells were harvested by trypsinization, washed in ice-cold phosphate-buffered saline (PBS) and fixed in 80% ice-cold ethanol in PBS. Before staining, cells were sedimented in a chilled centrifuge and resuspended in cold PBS. Bovine pancreatic RNase (Sigma–Aldrich) was added to a final concentration of 2 mg/ml, and cells were incubated at 37 8C for 30 min, followed by incubation with 20 mg/ml of propidium iodide (Sigma–Aldrich) for 20 min at room temperature. Cell cycle profiles of 5 104 cells were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).
2.3. Plasmids, small interfering RNA and transfection The miR-498 stem-loop (miR-498), inhibitors of miR-498 (miR498-in) and negative control were purchased from RiboBio (Guangzhou, China). FOXO3 ORFs with 30 -UTR was amplified using PCR and subcloned into pEGFP-N3 (Invitrogen, China). Plasmids were transfected into OC cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. For FOXO3 depletion, small interfering RNA (siRNA-FOXO3, HSH005759) was synthesized and purified by GeneCopoeia Co. (Guangzhou, China). Transfection of siRNAs was performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. 2.4. RNA extraction and real-time quantitative PCR Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription and quantitative PCR were performed using the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Dalian, China) by using the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers were synthesized by GeneCopoeia Co., FOXO3 (HQP000364), Cyclin D1 (HQP016204), p27 (HQP000342), GAPDH (HQP006940). Moreover, the cycle threshold (Ct) value was used for our analysis (DCt), and we determined the expression of small nuclear RNA U6 and GAPDH mRNA as internal controls to calculate the relative expression DD levels of miR-498, Cyclin D1, p27 via the 2 Ct method. 2.5. MTT assays and Colony formation For cell proliferation analysis, cell proliferation was examined by the MTT assay (Sigma–Aldrich) according to the standard protocol after transfection with miR-498 or miR-498-in or miR-NC for 24 h. For colony formation assay, HO8910 cells were plated into three 6-cm cell culture dishes (1 103 cells per well) and incubated in DMEM medium containing 10% FBS. After 2 weeks, the colonies were fixed with 10% formaldehyde for 15 min, stained with 1.0% crystal violet for 1 min. The number of colonies, defined as >50 cells/colony were counted. 2.6. Anchorage-independent growth assay Cells were trypsinized, and 1000 transfected cells were seeded in 2 ml complete medium plus 0.3% agar (Sigma). The agar-cell mixture was plated on top of a bottom layer consisting of 1% agar in complete medium. Cells were incubated at 37 8C. After 14 days, the colonies were stained with 0.1% Crystal Violet for staining and counted under microscope. Only cell colonies containing more than 50 cells were counted.
2.8. Luciferase assays Cells were co-transfected with 300 ng of miR-498 or miR-498in, 100 ng of the reporter vector containing the FOXO3 30 -UTR or the mutant 30 -UTR, and 25 ng of the Renilla luciferase vector as a transfection control. After 48 h of transfection, luciferase and Renilla activities were assayed, the cells were lysed and the fluorescence intensity was detected using the dual luciferase assay kit (Promega, Madison, WI, USA). 2.9. Western blotting The cells were lysed in RIPA lysis buffer (Sigma, St. Louis, MO, USA). Protein lysates were prepared, and protein concentration was measured using the BCA Protein Assay kit (Beyotime, China). Equal quantities of protein samples were loaded on 10% SDS-PAGE and transferred onto PVDF membranes, and Blocking was performed with 5% non-fat milk. The membrane was incubated overnight with anti-FOXO3, anti-Cyclin D1, anti-p27 (1:1000 dilution; Cell Signaling Technology) and anti-a-tubulin (Sigma– Aldrich). After washing and incubation with horseradish peroxidase-conjugated antibody (Sigma–Aldrich) for 1.5 h at room temperature, blotted proteins were detected using an enhanced chemiluminescence (ECL) system following the manufacturer’s protocol. 2.10. Statistical analysis All the statistics were expressed as mean standard deviation (SD) of three independent experiments and analysis by SPSS 18.0 software. Student’s t test was used to evaluate the significance of the differences between two groups of data in all the pertinent experiments. P < 0.05 was considered to be statistically significant. 3. Result 3.1. MiR-498 expression was downregulated in ovarian cancer cell lines and ovarian cancer tissues The expression pattern of miR-498 in human ovarian cancer has not been analyzed. We first evaluated the expression levels of miR498 in ovarian cancer tissues and cell lines by qRT-PCR. As shown in Fig. 1, compared with their non-cancerous counterparts, it was observed that miR-498 expression levels were lower in ovarian cancer tissues, it was also shown that miR-498 was downregulated in ovarian cancer cell lines compared with HOSE. Together, we conclude that miR-498 was abnormally downregulated in ovarian cancer tissues and ovarian cancer cell lines. 3.2. MiR-498 suppressed cell proliferation of ovarian cancer Next, to investigate the role of miR-498 in terms of cell proliferation, we transfected the HO8910 cells with miR-498
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Fig. 1. Expression of miR-498 in human ovarian cancer clinical specimens and cell lines. (A) Relative miR-498 expression levels in 8 paired primary ovarian cancer tissues and the matched tumor adjacent normal tissues (ANT) from the same patient were detected by PCR analysis. (B) Real-time PCR analysis of miR-498 expression in human ovary surface epithelial cells (HOSE) and OC cell lines, including A2780, CAOV-3, SKOV-3, ES-2 and HO8910. Experiments were repeated at least three times. Each bar represents the mean of three independent experiments. *P < 0.05.
mimics, miR-498-in or the respective controls. The transfection efficiency of miR-498 and miR-498-in was validated by qRT-PCR (Figs. 2A and 3A). To assess the potential role of miR-498 in ovarian cancer tumorigenesis, we determine whether miR-498 affects cell proliferation of ovarian cancers using MTT and colony formation assays. Our data indicated that the proliferation of HO8910 cells was decreased when cells were transfected with miR-498, while the miR-498-in showed the opposite effect (Figs. 2B, C and 3B, C). Using flow cytometry assays, we found that miR-498 overexpression decreased the percentage of cells in S phase and significantly increased the percentage of cells in G1/G0 (Fig. 2D), the opposite result was obtained when the cells were treated with miR-498-in (Fig. 3D). Collectively, our results suggest that miR-498 reduced ovarian cancer cell tumorigenicity in vitro.
3.3. MiR-498 directly restrained the expression of FOXO3 through direct targeting of FOXO3 mRNA 30 UTR and altered levels of proteins related to cell proliferation and cycle in ovarian cancer cells Potential target of miR-498 was predicted using bioinformatics methods. FOXO3 was selected as the target for further analysis. HO8910 cells were transiently transfected with miR-498 mimics, miR-498-in or the respective controls. Interestingly, we observed that miR-498 was capable of down-regulating FOXO3 protein expression, while miR-498-in clearly promoted its protein expression (Fig. 4B). To gain insight into the mechanism by which miR-498 inhibits FOXO3, we identified the miR-498 binding site in the FOXO3 mRNA 30 UTR (Fig. 4A) and constructed pGL3-FOXO3 and pGL3-FOXO3-mut
Fig. 2. MiR-498 upregulation inhibited cell proliferation of ovarian cancer. (A) Validation of miR-498 expression levels after transfection by PCR analysis. (B) MTT assays revealed that upregulation of miR-498 inhibited growth of HO8910 ovarian cancer cell line. (C) Representative micrographs (left) and quantification (right) of crystal violetstained cell colonies. (D) Flow cytometric analysis of the indicated HO8910 cells transfected with NC or miR-498. Each bar represents the mean of three independent experiments. *P < 0.05.
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Fig. 3. Inhibition of miR-498 promoted cell proliferation of ovarian cancer. (A) Validation of miR-498 expression levels after transfection by PCR analysis. (B) MTT assays revealed that inhibition of miR-498 promoted growth of HO8910 ovarian cancer cell line. (C) Representative micrographs (left) and quantification (right) of crystal violetstained cell colonies. (D) Flow cytometric analysis of the indicated HO8910 cells transfected with NC or miR-498-in. Each bar represents the mean of three independent experiments. *P < 0.05.
plasmids. Results of luciferase reporter assays showed that the cotransfection of miR-498 markedly increased the firefly luciferase activity of pGL3-FOXO3 but failed to influence the luciferase activity of pGL3-FOXO3-mut in HO8910 cells (Fig. 4C). Meanwhile, HO8910 cells transfected with miR-498-in resulted in suppressing firefly luciferase activity of the wild-type reporter but unaffected the mutant reporter (Fig. 4C). In summary, our data indicate that miR498 directly attenuated the expression of FOXO3 by targeting of its mRNA 30 UTR in ovarian cancer cell lines. Previous studies revealed that enhancing transcriptional activity of FOXO3 resulting in upregulation of cyclin-dependent kinase inhibitor p27 and downregulation of the CDK regulator cyclin D1 [11,12]. To investigate the mechanism underlying cell proliferation and cell cycle, we tested the effect of miR-498 on critical cell-proliferation and cell cycle related regulators cyclin D1 and p27. Results of real-time PCR and Western blotting analysis showed that cyclin D1 (mRNA and protein) levels were downregulated and p27 (mRNA and protein) levels were upregulated in miR-498-transfected HO8910 cells, compared to NC-transfected cells, while miR-498-in showed the opposite results (Fig. 4D and E). Altogether, our results indicated that miR-498 regulated expression of FOXO3, and then functionally modulated cell proliferation and cell cycle regulators, p27 and cyclin D1, thus relevant to cell proliferation and cell cycle. 3.4. FOXO3 is involved in miR-498-induced proliferation of ovarian cancer cells To further understand the role of FOXO3 in miR-498mediated pro-proliferation, miR-498-transfected HO8910 cells
were transfected with FOXO3-siRNA. As showed in Fig. 5A, results of Western blot analysis revealed that the FOXO3 expression in miR-498-transfected HO8910 cells was decreased after transfected with FOXO3-siRNA. Colony formation assays showed that the treatment with FOXO3-siRNA was able to reverse the miR-498-decreased proliferation (Fig. 5B), suggesting that miR-498 suppresses the proliferation of ovarian cancer cells by upregulating FOXO3. Therefore, our results demonstrate that miR-498 was able to inhibit the proliferation of ovarian cancer cells through direct targeting FOXO3. 4. Discussion The key finding of the current study is that miR-498 expression was markedly downregulated in ovarian cancer tissues and ovarian cancer cells as compared with that in the matched tumor adjacent normal tissues and human ovary surface epithelial cells. Our result revealed that ectopic expression of miR-498 could decrease the cell proliferation of ovarian cancer, while miR-498-in enhanced this effect. Result of dual-luciferase assay showed that FOXO3 was a direct of miR-498. Further experiment showed that upregulation of miR-498 in ovarian cancer cells led to downregulation of the cell-cycle regulator cyclin D1 and the downregulation of cyclin-dependent kinase (CDK) inhibitors p27 through downregulation of FOXO3 via directly targeting the 30 UTR of FOXO3. Taken together, these findings demonstrated that miR-498 may play an important role in carcinogenesis and progression of ovarian cancer. Numerous studies indicated that MicroRNAs (miRNAs), a class of small non-coding RNAs, potentially play critical roles in cell
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Fig. 4. MiR-498 increased FOXO3 expression by directly targeting the FOXO3 30 -UTR and altered levels of proteins related to cell proliferation and cell cycle in HO8910 cells. (A) Predicted miR-498 target sequence in the 30 -UTR of FOXO3 (FOXO3-30 -UTR) and positions of three mutated nucleotides (red) in the 30 -UTR of FOXO3 (FOXO3-30 -UTRmut). (B) Western blotting analysis of FOXO3 expression in cells transfected with miR-498 or the miR-498-in. a-Tubulin served as the loading control. (C) Luciferase reporter assay of the indicated cells transfected with the pGL3-FOXO3-30 -UTR reporter or pGL3-FOXO3-30 -UTR-mut reporter and miR-498 or miR-498-in with increasing amounts (10 and 50 nM) oligonucleotides. (D) Real-time PCR analysis of expression of cyclin D1 and p27 in indicated HO8910 cells. (E) Western blotting analysis of expression of cyclin D1 and p27 protein in HO8910 cells. a-Tubulin served as the loading control. *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
proliferation, cycle, differentiation, angiogenesis, invasion and migration of various types of human cancers [13–16]. MiRNAs regulate gene expression and are proposed as potential novel targets for anti-cancer therapies. However, it was uncertain whether dysregulation of miR-498 was associated with the progression of ovarian cancer. In this study, our results revealed that miR-498 was significantly down-regulated in ovarian cancer and inhibits cell proliferation of ovarian cancer in vitro and suggested miR-498 as a candidate tumor suppressor in the pathogenesis of ovarian cancer.
FOXO3 protein, a member of the forkhead transcriptional factor family, is critical in cell growth, proliferation, and development of cancer [17,18]. The levels of FOXO3 are highly expressed in most human cancers including ovarian cancer [19,20]. Through bioinformatics analysis, the tumor suppressor FOXO3 gene was indicated as a theoretical targeted gene of miR-498. Result of Western blotting analysis showed that overexpression of miR-498 resulted in the increase expression of FOXO3 protein. It has been demonstrated that the transcriptional factor FOXO3 can downregulate cyclin D1 and upregulate the expression of p27 at the
Fig. 5. FOXO3 upregulation is required for miR-498-induced proliferation of ovarian cancer cells. (A) Western blot analysis verified that silencing FOXO3 effectively decreased the expression of FOXO3 in miR-498-transfected HO8910 cells. (B) miR-498-transfected HO8910 cells after transfection with FOXO3-siRNAs promoted cell colonies formation. Each bar represents the mean of three independent experiments. *P < 0.05.
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transcriptional level [21,22]. Consistent with these reports, our result indicated that miR-498 exerts its functions by upregulating expression of FOXO3, which in turn may cause downregulating of expression of cyclin D1 and upregulating expression of p27, and then blocks cell cycle progression at G0/G1 phase, resulting in suppressing cell proliferation. Furthermore, FOXO3-silenced in HO8910-miR-498 cells had positive effect on cell proliferation, suggesting that direct up-regulation of FOXO3 is required for miR498-induced cell proliferation of ovarian cancer. In summary, the current study provides that an important link between miR-498-mediated proliferation of ovarian cancer cells and upregulation of FOXO3. Our findings revealed an essential role of miR-498 in the regulation of ovarian cancer cells proliferation. Therefore, all the results indicated that miR-498 was an antioncomiR and his implies miR-498 to be a potential mediator for novel miRNA replacement therapy for ovarian cancer. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by Department of Gynecology, Cancer Hospital Affiliated to Zhengzhou University, Henan Cancer Hospital. All authors designed the study together, performed the experiment together, analyzed the data and wrote the paper; all authors approved the final manuscript. References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9–29. [2] A. Kumar, J.N. Bakkum-Gamez, A.L. Weaver, M.E. McGree, W.A. Cliby, Impact of obesity on surgical and oncologic outcomes in ovarian cancer, Gynecol. Oncol. 135 (2014) 19–24. [3] G. Di Leva, M. Garofalo, C.M. Croce, MicroRNAs in cancer, Annu. Rev. Pathol. 9 (2014) 287–314. [4] E. Shoshan, A.K. Mobley, R.R. Braeuer, T. Kamiya, L. Huang, M.E. Vasquez, A. Salameh, H.J. Lee, S.J. Kim, C. Ivan, G. Velazquez-Torres, K.M. Nip, K. Zhu, D. Brooks, S.J. Jones, I. Birol, M. Mosqueda, Y.Y. Wen, A.K. Eterovic, A.K. Sood, P. Hwu, J.E. Gershenwald, A. Gordon Robertson, G.A. Calin, G. Markel, I.J. Fidler, M. Bar-Eli, Reduced adenosine-to-inosine mir-455-5p editing promotes melanoma growth and metastasis, Nat. Cell Biol. 17 (2015) 311–321.
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