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MiR-23b targets cyclin G1 and suppresses ovarian cancer tumorigenesis and progression

MiR-23b targets cyclin G1 and suppresses ovarian cancer tumorigenesis and progression Background: It has been proposed that cyclin G1 (CCNG1) participates in p53-dependent G –S and G checkpoints 1 2 and might function as an oncogenic protein in the initiation and metastasis of ovarian carcinoma. MicroRNA 23b (miR-23b) is a critical regulatory factor in the progression of many cancer cell types that targets the relevant genes. Methods: MiR-23b expression in ovarian tissues was quantified by quantitative reverse transcription–PCR. The ovarian cancer cell lines OVCAR3, HO8910-PM, and SKOV3/DDP were transfected with miR-23b, after we assayed the cell phenotype and expression of the relevant molecules. Dual-luciferase reporter assay and a xenograft mouse model were used to examine the expression of miR-23b and its target gene CCNG1. Results: MIR23B mRNA expression was significantly lower in epithelial ovarian carcinoma and borderline tumors than in normal ovarian tissues and benign tumors, and miR-23b expression among ages and pathological subtypes was significantly different. CCNG1 mRNA expression was significantly lower in normal ovarian tissues than in benign tumors, borderline tumors, and ovarian carcinomas, and expression among pathological subtypes was significantly different. MiR-23b overexpression inhibited ovarian cancer cell proliferation, invasion, and migration, and induced apoptosis. Dual-luciferase reporter assay showed that miR-23b bound with the 3′ untranslated region of CCNG1. MiR-23b overexpression significantly downregulated CCNG1, urokinase, survivin, Bcl-xL, P70S6K, and matrix metallopeptidase-9 (MMP9) mRNA and protein expression. Furthermore, miR-23b inhibited tumor growth and suppressed CCNG1 expression in vitro. Conclusions: Our findings show that miR-23b may inhibit ovarian cancer tumorigenesis and progression by downregulating CCNG1 and the expression of the relevant genes. MiR-23b is a potentially novel application for regulating ovarian carcinoma progression. Keywords: miR-23b, CCNG1, Ovarian cancer, Tumorigenesis, Progression Background and acquired chemoresistance, and remarkable heterogen- The incidence of ovarian cancer is perpetually high: eity [5]; recurrence is frequently observed in up to 70 % of 200,000 new cases are diagnosed annually worldwide. cases [6]. In such patients, invasion, metastasis, and che- Each year, it constitutes 4 % of all cancers diagnosed in moresistance may play important roles. Consequently, in- women, and there are 6.6 new cases per 100,000 women creasingly sophisticated experiments have been performed per year [1, 2]. Globally, ovarian cancer caused 160,500 to study targeted treatments to improve the 5-year sur- deaths in 2010, an increase from the 113,600 in 1990 and vival rate of patients with ovarian cancer. the 140,200 in 2008 [3, 4]. It also remains the most lethal MicroRNAs (miRNAs) are a class of small non-coding gynecologic malignancy owing to late detection, intrinsic RNAs that negatively regulate gene expression at post- transcriptional level [7]. It has been demonstrated that a small number of miRNAs actively participate in regulat- * Correspondence: [email protected] ing tumor development; it has been shown that they play Department of Biochemistry and Molecular Biology, College of Basic different roles in different organs, particularly relating to Medicine, China Medical University, 100013 Shenyang, China Full list of author information is available at the end of the article © 2016 Yan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 2 of 10 cancer development [8–12]. It has been determined that at room temperature in the dark, 300 μL1×binding miR-23b mediates the various steps in the metastatic buffer together with 5 μL PI were added to each tube. process, including tumor growth, invasion, and even The samples were incubated for 30 min at room angiogenesis by repressing a cohort of prometastatic tar- temperature in the dark. Flow cytometry was per- gets [13]. MiR-23b is downregulated in many cancers formed within 1 h. and acts as a tumor suppressor [14–16]. From a clinical viewpoint, miR-23b has great potential as a diagnostic Wound healing assay and therapeutic agent in some tumors. Our previous Cells were seeded at 1 × 10 cells/well in 6-well cul- study showed that miR-23b was highly expressed in nor- ture plates. After they had grown to confluence, the mal ovarian tissues than ovarian carcinoma tissues, and monolayer was scratched with a pipette tip (200 μL). our predicted seed region in the 3′ untranslated regions The cells were washed with PBS three times and cul- (3′ UTR) of CCNG1 revealed that it’s a target of miR- tured in FBS-free medium, and then photographed at 23b, thus we investigated the involvement of CCNG1 0, 24, and 48 h. The scratched areas were measured and miR-23b in ovarian cancer for the first time. using ImageJ software, after which the wound healing rate was calculated. Methods Cell culture Cell invasion assay The ovarian cancer cell lines OVCAR3 and HO8910-PM 5×10 cells were resuspended in FBS-free medium and (highly invasive HO8910) were from ATCC. The seeded into the top chambers of Matrigel™-coated cisplatin-resistant SKOV3 (SKOV3/DDP) cell line was Transwell® inserts (BD Bioscience, San Jose, CA, USA). purchased from the Tumor Cell Bank of the Chinese The lower compartments of the chambers contained Academy of Medical Science (Peking, China). The cells 10 % v/v FBS as a chemoattractant. After 48-h incuba- were maintained in RPMI 1640 medium supplemented tion, the cells on the upper surface of the membrane with 10 % fetal bovine serum (FBS), 100 units/mL peni- were wiped away, and the cells on the lower surface of cillin and 100 μg/mL streptomycin in a humidified at- the membrane were washed, fixed, and stained with mosphere of 5 % CO at 37 °C. crystal violet to quantify the extent of invasion. Proliferation assay Ovarian carcinoma specimens We used 3-(4,5)-dimethylthiazol (−zyl)-3,5-diphenyltetra- Specimens from 50 ovarian epithelial carcinomas, 13 be- zolium bromide (MTT; China) to determine the number nign tumors, 5 borderline tumors, and 6 normal ovarian of viable cells. Briefly, approximately 2.5 × 10 cells/well tissues were collected from patients who underwent sur- were seeded in a 96-well plate and allowed to adhere. At gical resection at the Department of Gynecology of the different time points, 20 μL MTT solution was added to First Affiliated Hospital of China Medical University each well, and the plates were incubated for 4 h. Subse- (Shenyang, China). Two pathologists confirmed the tumor quently, the MTT solution was removed, and 150 μLdi- specimens independently. The China Medical University methyl sulfoxide was added and incubated for 10 min Ethics Committee approved the study. before the absorbance was measured at 490 nm. Real-time RT-PCR (real-time RT-PCR) Cell cycle analysis Real-time RT-PCR was performed from 2 μg total RNA After 48-h incubation, the cells were washed, collected, using AMV reverse transcriptase and random primers and fixed in 10 mL ice-cold ethanol (70 %) for 12 h. (Takara). The PCR primers were designed according to Then, the cells were washed, incubated with 5 μL RNase the GenBank sequences. The glyceraldehyde-3-phosphate (0.25 mg/mL) at 37 °C for 30 min, pelleted, resuspended dehydrogenase gene (GAPDH) was used as the internal in 50 μg/mL PI, and incubated for 15 min in the dark at control. The relative gene expression level (the amount of room temperature. Cell cycle analysis was performed by target gene normalized to the endogenous control gene) flow cytometric analysis. was calculated using the comparative threshold cycle -ΔΔCt method: 2 .Hairpin-it™ microRNA and U6 snRNA Apoptosis assay Normalization RT-PCR Quantitation (GenePharma) were Flow cytometry was performed following PI and FITC- used to examine mature miR-23b. labeled annexin V (KeyGen Biotech, Nanjing, China) staining according to the manufacturer’s protocol. Briefly, Western blotting after 48-h incubation, cells were washed and resuspended Proteins were separated by SDS-PAGE, transferred to in 200 μL 1× binding buffer at 1 × 10 cells/mL, and incu- polyvinylidene difluoride membranes and immunoblotted bated with 5 μL FITC–annexin V; after 15-min incubation overnight at 4 °C with the primary antibodies (1:500), Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 3 of 10 rinsed with TBST, and incubated with 1:5000 secondary Results antibodies conjugated to horseradish peroxidase (Dako). Correlation of MIR23B and cyclin G1 (CCNG1) mRNA After applying electrochemiluminescence (ECL) Plus expression with pathogenesis and aggressiveness of detection reagents (Santa Cruz Biotechnology), pro- ovarian carcinoma tein bands were visualized using X-ray film (Fujifilm, We quantified MIR23B and CCNG1 mRNA expression in Tokyo, Japan). GAPDH-specific monoclonal antibody normal ovary tissue, benign and borderline tumors, and (1:2000; Santa Cruz Biotechnology) was used as the primary ovarian carcinoma using real-time PCR. MIR23B internal control. mRNA expression was significantly lower in the ovarian carcinomas and borderline tumors than in the normal ovarian tissues and benign tumors (Fig. 1a, p <0.05), and In vivo nude mouse tumorigenicity assay there were significant differences in expression among The assay was performed to determine the effects of miR- ages (Fig. 1b, p < 0.05) and pathological subtypes (mucin- 23b overexpression on the tumorigenicity of OVCAR3 ous vs. other types, Fig. 1d, p < 0.05). However, there were cells in vivo. Mock or hsa-miR-23b–transfected OVCAR3 no significant differences among International Federation cells (1 × 10 ) were injected subcutaneously into the flanks of Gynecology and Obstetrics (FIGO) stages (I/II vs. III/ of 4-6-week-old male nude mice (n = 5). Once tumor IV, Fig. 1c, p > 0.05) and differentiation (well vs. poor and growth had been established in the mock-treated mice moderate, data not shown, p > 0.05) in ovarian carcinoma. (14 days post-injection), tumor sizes were measured every CCNG1 mRNA expression was significantly lower in the other day. Tumor volume (mean ± standard deviation) normal ovarian tissues and benign ovarian tumors than in was calculated as length × width /2. the ovarian carcinomas (Fig. 1e, p <0.05). Effects of miR-23b transfection on ovarian carcinoma cell Immunofluorescence (IF) staining phenotype in vitro 5-μm frozen section from each sample was fixed in acet- We transfected OVCAR3, HO8910-PM, SKOV3/DDP one at 4 °C overnight. After washing, sections were cells with miR-23b. The transfected cells exhibited blocked with 1 % bovine serum albumin for 30 min and significantly slower growth (Fig. 2a, p < 0.05) but incubated overnight at 4 °C with rabbit anti-human higher levels of mature miR-23b expression than the CCNG1 antibody (1:50). Then sections were incubated control and mock-transfected cells (Fig. 2b, p <0.05). with anti-rabbit IgG–FITC (1:100, Santa Cruz Biotech- MiR-23b overexpression significant induced G arrest nology) for 2 h at room temperature in the dark. Nuclei (Fig. 3a, p < 0.05), higher levels of apoptosis (Fig. 3b, were stained with diaminophenylindole (1 μg/mL; p < 0.05) and reduced cell migration (Fig. 4a, p < 0.05) Sigma-Aldrich) for 5 min at room temperature. Cover- and invasion (Fig. 4b, p < 0.05) compared to the con- slips were mounted and imaged using a laser confocal trol and mock-transfected cells. microscope (Olympus, Tokyo, Japan). Effects of miR-23b transfection on ovarian carcinoma cell genotype in vitro Dual-luciferase reporter assay The predicted seed region in the 3′ untranslated regions A293 cells were first seeded at 60–80 % confluence in (3′ UTR) of CCNG1 revealed that it is direct target 24-well plates for 12 h. For the dual-luciferase reporter of miR-23b (Fig. 5a); dual-luciferase reporter assay assays, cells were transiently transfected with Wt or mu- indicated that miR-23b significantly decreased the tated reporter plasmid and miR-23b or control-miR. relative luciferase activity of the wild-type CCNG1 3′ Firefly luciferase activity was measured using a Dual- UTR as compared with the mutant CCNG1 3′ UTR, Luciferase Assay (Promega) 48 h after transfection, and indicating that miR-23b may directly bind to the 3′ the results were normalized with Renilla luciferase. Each UTR of CCNG1 (Fig.5b).Reverse transcription reporter plasmid was transfected at least three times, (RT)-PCR (Fig. 5c, p < 0.05) and western blotting and each sample was assayed in triplicate. (Fig. 5d) showed that miR-23b overexpression reduced CCNG1, urokinase (uPA), P70S6K, Bcl-xL, survivin, and Statistical analysis matrix metallopeptidase 9 (MMP9) mRNA or protein Statistical evaluation was performed using Spearman’s expression. rank correlation coefficient to analyze ranked data; the Mann–Whitney U test was used to differentiate the MiR-23b inhibited tumor growth in vivo means of different groups. A p-value of < 0.05 was con- The tumor xenograft volume in nude mice treated with sidered statistically significant. SPSS v. 17.0 (SPSS) was miR-23b was smaller than that in the mock-treated mice used to analyze all data. (Fig. 6a, p < 0.05). From day 4 and week 2 onwards, the Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 4 of 10 Fig. 1 Correlation of MIR23B and CCNG1 mRNA expression with pathogenesis and aggressiveness of ovarian carcinoma. a MIR23B mRNA expression was significantly lower in the ovarian carcinomas and borderline ovarian tumors than in the normal ovarian tissues and benign tumors; b there were significant differences in expression among age and d pathological subtype (mucinous vs. other types) in the ovarian carcinoma tissues. c There were no significant differences among FIGO stage (I/II vs. III/IV) in ovarian carcinoma. e CCNG1 mRNA expression was significantly lower in the normal ovarian tissues and benign ovarian tumors than in the ovarian carcinomas. *p < 0.05 vs. normal ovarian tissues; p < 0.05 vs. benign ovarian tumors. No = Normal ovarian tissues, Be = benign ovarian tumors, Bo = borderline ovarian tumors, Om = omentum tumors, Mu = mucinous carcinoma, Ser = serous carcinoma tumor xenograft growth in the miR-23b–treated BALB/c Discussion mice was slower than that in the mock group (Fig. 6b, MiRNAs regulate their target genes by affecting base p < 0.05; p < 0.01, pairing to their 3′ UTRs, resulting in mRNA degrad- day 4 deviation of tumor xenograft volume [DV] and p < 0.05; p < 0.01), and the DV increased in ation or inhibition of translation [11]. An increasing week 2 DV the latter period. number of studies have revealed that miRNAs are prom- ising diagnostic and prognostic molecular biomarkers as well as therapeutic targets in cancer [17, 18]. Series of MiR-23b downregulated CCNG1 expression in tumor studies has reported that miR-23b acts as a tumor sup- xenografts in vivo pressor in different cancers [19]. Majid et al. showed that Immunofluorescence staining (IF) indicated decreased miR-23b has diagnostic/prognostic significance and dir- CCNG1 expression in the tumor xenografts of the miR- ectly targets the oncogenic ZEB1 in bladder cancer [14]. 23b–treated nude mice compared with that in the He et al. reported that miR-23b expression levels in pros- mock-treated nude mice (Fig. 7). tate carcinoma (PCa) tissues was significantly correlated Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 5 of 10 Fig. 2 Effects of miR-23b transfection on ovarian carcinoma cell proliferation in vitro. a Following miR-23b transfection, OVCAR3, HO8910-PM, SKOV3/DDP cell lines exhibited significantly slower growth and b higher levels of mature miR-23b expression than the control and mock- transfected cells. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, * p < 0.05 Fig. 3 Effects of miR-23b transfection on ovarian carcinoma cell cycle and apoptosis in vitro. MiR-23b transfection induced a G arrest and b early apoptosis in OVCAR3, HO8910-PM, and SKOV3/DDP cells as compared to the control and mock-transfected cells. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, * p < 0.05 Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 6 of 10 Fig. 4 Effects of miR-23b transfection on the invasive and metastatic ability of ovarian carcinoma cell lines. After transfection with the miR-23b mimic, ovarian carcinoma cells showed a lower migration and b slower invasion as compared with the control and mock-transfected cells. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, *p < 0.05 with that of peroxiredoxin 3 (PRDX3) and that miR-23b predicted seed region showed that miR-23b targets may be involved in the response of PCa cells to hypoxic CCNG1 3′ UTR, which was convinced by the dual- stress, therefore gene therapy using miRNA mimics may luciferase reporter assay. We also found that miR-23b be useful as PCa therapy [20]. Our results show that transfection decreased CCNG1 mRNA and protein ex- MIR23B mRNA expression was significantly lower in pression. CCNG1 was first identified as a p53-regulated ovarian carcinomas and borderline tumors than in normal transcript induced by DNA damage. It has been pro- ovarian tissues and benign tumors, and the expression posed that these events underpin CCNG1 participation among age and pathological subtypes (mucinous vs. other in the enforcement of the p53-dependent G –Sand G 1 2 types) was significantly different. These findings indicate checkpoints responsive to DNA damage [21]. Some have that miR-23b might affect ovarian epithelial carcinogen- suggested that CCNG1 might function as an oncogenic esis and the subsequent progression. Therefore, we ex- protein [22, 23] and play a pivotal role in the initiation plored the function and molecular mechanism of miR-23b and metastasis of hepatocellular carcinoma [24]. Russell in ovarian cancer cell lines. et al. reported that CCNG1 amplification is associated Ovarian cancer cells transfected with miR-23b had sig- with significantly shorter postsurgical survival in pa- nificantly slower growth than the negative control– and tients with ovarian cancer who have received adjuvant mock-transfected cells, and there was significantly in- chemotherapy with taxanes and platinum compounds duced G arrest and apoptosis and reduced cell invasion [21]. These results suggest that miR-23b may inhibit and migration, suggesting miR-23b may inhibit ovarian ovarian cancer tumorigenesis and progression by tar- carcinoma tumorigenesis and progression. Moreover, the geting CCNG1. Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 7 of 10 Fig. 5 Effects of miR-23b transfection on ovarian carcinoma cell genotype in vitro. a The predicted seed region in the 3′ UTR of CCNG1 revealed that CCNG1 was direct target of miR-23b, as predicted by microRNA.org; b dual-luciferase reporter assay indicated that miR-23b significantly decreased the relative luciferase activity of the wild-type CCNG1 3′ UTR as compared with the mutant CCNG1 3′ UTR, indicating that miR-23b may directly bind to the 3′ UTR of CCNG1. c & d MiR-23b overexpression reduced CCNG1, uPA, P70S6K, Bcl-xL, survivin and MMP9 mRNA or protein expression. * p < 0.05 In this study, we also found that miR-23b overexpres- and metastasis of cancer [31]. Wang et al. reported that sion downregulated uPA expression, which is in line uPA promoted carcinoma cell proliferation by stimulat- with the findings of Salvi et al. [15], who reported that ing P70S6K activation [32]. Survivin, a member of the miR-23b overexpression leads to uPA downregulation inhibitors of apoptosis protein family, is expressed dur- and decreased migration and proliferation ability in ing development and in various human cancers. Lee et hepatocellular carcinoma cells. Furthermore, miR-23b al. reported that downregulating survivin suppressed overexpression also downregulated the expression of uPA through the transcription factor JunB [33]. Ryan et P70S6K, survivin, Bcl-xL, and MMP9 mRNA and pro- al. reported that survivin expression in breast cancer tein. It has been established that uPA is integral to cell predicts clinical outcome and is associated with uPA differentiation, migration, tissue remodeling under [34]. Furthermore, Zhou et al. reported that Bcl-xL over- physiological and pathological conditions, and may be a expression strongly enhanced uPA in pancreatic ductal potential diagnostic biomarker and therapeutic target in adenocarcinoma cells [35]. Therefore, we suggest that cancer. Significant elevation of uPA protein levels in pri- miR-23b reduces cell proliferation by downregulating mary ovarian cancer tissue has been associated with CCNG1 and uPA/P70S6K expression, suppresses cell poor prognosis and disease progression [25–29]. The migration and invasion by downregulating uPA/MMP9 uPA system plays an important role in many patho- expression, and induces apoptosis by downregulating physiological processes, such as cell differentiation, mi- survivin and Bcl-xL/uPA expression. gration, tissue reconstruction, and matrix dissolution We found that CCNG1 mRNA expression was signifi- [30]. The aberrant expression of phosphorylated P70S6K cantly lower in the normal ovarian tissues and benign might contribute to the pathogenesis, growth, invasion, ovarian tumors than in the borderline ovarian tumors Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 8 of 10 Fig. 6 MiR-23b inhibited tumor growth in vivo. a Tumor xenograft volume in nude mice treated with hsa-miR-23b was smaller than that in mock- treated mice. b Tumor xenograft growth in miR-23b–treated nude mice was slower than that in the mock group from day 4 and week 2 onwards and ovarian carcinomas. Our subsequent in vivo potential suppressor of ovarian carcinoma by targeting tumor xenograft studies showed that miR-23b inhib- CCNG1. ited tumor growth and decreased CCNG1 expression. Our findings show that miR-23b may inhibit ovarian Recent studies have reported that CCNG1 gene ther- cancer tumorigenesis and progression by downregulating apy has been developed and has undergone phase I/II CCNG1 and the expression of the relevant genes, and clinical trials for treating colorectal and pancreatic that miR-23b is a potentially novel application for regu- cancer [36]. We suggest that miR-23b may also be a lating ovarian carcinoma progression. Fig. 7 MiR-23b downregulated CCNG1 expression in tumor xenografts in vivo. IF experiments indicated that CCNG1 expression in the tumor xenografts of miR-23b–treated nude mice were decreased compared with that in the mock-treated nude mice Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 9 of 10 Conclusion 8. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009;10:704–14. This is the first demonstration that miR-23b may inhibit 9. Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, Nair S, et al. The EOC tumorigenesis and progression by targeting CCNG1 microRNAs miR-373 and miR-520c promote tumor invasion and metastasis. and modulating the expression of the relevant genes. Nat Cell Biol. 2008;10:202–10. 10. Ma L, Teruya-Feldstein J, Weinberg RA. Tumor invasion and metastasis These findings indicate that miR-23b is a potential initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–8. suppressor of ovarian carcinoma tumorigenesis and pro- 11. Tao JQ, Zhi XF, Zhang XY, Fu M, Huang H, Fan Y, et al. miR-27b-3p gression. The involvement of miR-23b–mediated CCNG1 suppresses cell proliferation through targeting receptor tyrosine kinase like orphan receptor 1 in gastric cancer. J Exp Clin Cancer Res. 2015;34:139. downregulation in inhibiting EOC aggressiveness may 12. Xiang J, Bian CD, Wang H, Huang SS, Wu DL. MiR-203 down-regulates yield further insight into the molecular mechanisms Rap1A and suppresses cell proliferation, adhesion and invasion in prostate underlying cancer aggressiveness. cancer. J Exp Clin Cancer Res. 2015;34:8. 13. Zhang H, Hao Y, Yang J, Zhou Y, Li J, Yin S, et al. Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer Abbreviations metastasis. Nat Commun. 2011;2:554. CCNG1: cyclin G1; EOC: epithelial ovarian carcinoma; FIGO 14. Majid S, Dar AA, Saini S, Deng G, Chang I, Greene K, et al. MicroRNA-23b stages: International Federation of Gynecology and Obstetrics stages; functions as a tumor suppressor by regulating Zeb1 in bladder cancer. PLoS GAPDH: glyceraldehyde-3-phosphate dehydrogenase gene; One. 2013;8(7):e67686. IF: immunofluorescent staining; miR-23b: microRNA 23b; MMP9: matrix 15. Salvi A, Sabelli C, Moncini S, Venturin M, Arici B, Riva P, et al. MicroRNA-23b metallopeptidase-9; MTT: 3-(4,5)-dimethylthiazol (−zyl)-3,5- mediates urokinase and c-met down modulation and a decreased migration diphenyltetrazolium bromide; RT-PCR: real-time RT-PCR; SDS-PAGE: sodium of human hepatocellular carcinoma cells. FEBS J. 2009;276:2966–82. dodecyl sulfate–polyacrylamide gel electrophoresis; TBST: tris-buffered saline 16. Majid S, Dar AA, Saini S, Arora S, Shahryari V, Zaman MS, et al. miR-23b with tween 20®. represses proto-oncogene Src kinase and functions as methylation-silenced tumor suppressor with diagnostic and prognostic significance in prostate Competing interests cancer. Cancer Res. 2012;72:6435–46. The authors declare that they have no competing interests. 17. Guo J, Wang M, Liu XH. MicroRNA-195 suppresses tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma. J Exp Authors’ contributions Clin Cancer Res. 2015;34:91. ZH-Z and JY carried out the molecular genetic studies, participated in the in 18. Kawano M, Tanaka K, Itonaga I, Ikeda S, Iwasaki T, Tsumura H. microRNA-93 vivo tumor xenograft studies, and drafted the manuscript. JY J carried out promotes cell proliferation via targeting of PTEN in Osteosarcoma cells. J the immunoassays. XN-M and YL X participated in the design of the study Exp Clin Cancer Res. 2015;34:76. and performed the statistical analysis. ZH-Z conceived the study, participated 19. Donadelli M, Palmieri M. Roles for microRNA 23b in regulating autophagy in its design and coordination, and helped to draft the manuscript. All and development of pancreatic adenocarcinoma. Gastroenterology. 2013; authors read and approved the final manuscript. 145(5):936–8. 20. He HC, Zhu JG, Chen XB, Chen SM, Han ZD, Dai QS, et al. MicroRNA-23b Acknowledgements downregulates peroxiredoxin III in human prostate cancer. FEBS Lett. 2012; This work was supported by the Natural Scientific Foundation of China (Nos. 586(16):2451–8. 81472502), and Liaoning Science and Technology Grant (2013021077). 21. Russell P, Hennessy BT, Li J, Carey MS, Bast RC, Freeman T, et al. Cyclin G1 regulates the outcome of taxane-induced mitotic checkpoint arrest. Author details Oncogene. 2012;31:2450–60. 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MiR-23b targets cyclin G1 and suppresses ovarian cancer tumorigenesis and progression

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Springer Journals
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2016 Yan et al.
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1756-9966
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10.1186/s13046-016-0307-1
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Abstract

Background: It has been proposed that cyclin G1 (CCNG1) participates in p53-dependent G –S and G checkpoints 1 2 and might function as an oncogenic protein in the initiation and metastasis of ovarian carcinoma. MicroRNA 23b (miR-23b) is a critical regulatory factor in the progression of many cancer cell types that targets the relevant genes. Methods: MiR-23b expression in ovarian tissues was quantified by quantitative reverse transcription–PCR. The ovarian cancer cell lines OVCAR3, HO8910-PM, and SKOV3/DDP were transfected with miR-23b, after we assayed the cell phenotype and expression of the relevant molecules. Dual-luciferase reporter assay and a xenograft mouse model were used to examine the expression of miR-23b and its target gene CCNG1. Results: MIR23B mRNA expression was significantly lower in epithelial ovarian carcinoma and borderline tumors than in normal ovarian tissues and benign tumors, and miR-23b expression among ages and pathological subtypes was significantly different. CCNG1 mRNA expression was significantly lower in normal ovarian tissues than in benign tumors, borderline tumors, and ovarian carcinomas, and expression among pathological subtypes was significantly different. MiR-23b overexpression inhibited ovarian cancer cell proliferation, invasion, and migration, and induced apoptosis. Dual-luciferase reporter assay showed that miR-23b bound with the 3′ untranslated region of CCNG1. MiR-23b overexpression significantly downregulated CCNG1, urokinase, survivin, Bcl-xL, P70S6K, and matrix metallopeptidase-9 (MMP9) mRNA and protein expression. Furthermore, miR-23b inhibited tumor growth and suppressed CCNG1 expression in vitro. Conclusions: Our findings show that miR-23b may inhibit ovarian cancer tumorigenesis and progression by downregulating CCNG1 and the expression of the relevant genes. MiR-23b is a potentially novel application for regulating ovarian carcinoma progression. Keywords: miR-23b, CCNG1, Ovarian cancer, Tumorigenesis, Progression Background and acquired chemoresistance, and remarkable heterogen- The incidence of ovarian cancer is perpetually high: eity [5]; recurrence is frequently observed in up to 70 % of 200,000 new cases are diagnosed annually worldwide. cases [6]. In such patients, invasion, metastasis, and che- Each year, it constitutes 4 % of all cancers diagnosed in moresistance may play important roles. Consequently, in- women, and there are 6.6 new cases per 100,000 women creasingly sophisticated experiments have been performed per year [1, 2]. Globally, ovarian cancer caused 160,500 to study targeted treatments to improve the 5-year sur- deaths in 2010, an increase from the 113,600 in 1990 and vival rate of patients with ovarian cancer. the 140,200 in 2008 [3, 4]. It also remains the most lethal MicroRNAs (miRNAs) are a class of small non-coding gynecologic malignancy owing to late detection, intrinsic RNAs that negatively regulate gene expression at post- transcriptional level [7]. It has been demonstrated that a small number of miRNAs actively participate in regulat- * Correspondence: [email protected] ing tumor development; it has been shown that they play Department of Biochemistry and Molecular Biology, College of Basic different roles in different organs, particularly relating to Medicine, China Medical University, 100013 Shenyang, China Full list of author information is available at the end of the article © 2016 Yan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 2 of 10 cancer development [8–12]. It has been determined that at room temperature in the dark, 300 μL1×binding miR-23b mediates the various steps in the metastatic buffer together with 5 μL PI were added to each tube. process, including tumor growth, invasion, and even The samples were incubated for 30 min at room angiogenesis by repressing a cohort of prometastatic tar- temperature in the dark. Flow cytometry was per- gets [13]. MiR-23b is downregulated in many cancers formed within 1 h. and acts as a tumor suppressor [14–16]. From a clinical viewpoint, miR-23b has great potential as a diagnostic Wound healing assay and therapeutic agent in some tumors. Our previous Cells were seeded at 1 × 10 cells/well in 6-well cul- study showed that miR-23b was highly expressed in nor- ture plates. After they had grown to confluence, the mal ovarian tissues than ovarian carcinoma tissues, and monolayer was scratched with a pipette tip (200 μL). our predicted seed region in the 3′ untranslated regions The cells were washed with PBS three times and cul- (3′ UTR) of CCNG1 revealed that it’s a target of miR- tured in FBS-free medium, and then photographed at 23b, thus we investigated the involvement of CCNG1 0, 24, and 48 h. The scratched areas were measured and miR-23b in ovarian cancer for the first time. using ImageJ software, after which the wound healing rate was calculated. Methods Cell culture Cell invasion assay The ovarian cancer cell lines OVCAR3 and HO8910-PM 5×10 cells were resuspended in FBS-free medium and (highly invasive HO8910) were from ATCC. The seeded into the top chambers of Matrigel™-coated cisplatin-resistant SKOV3 (SKOV3/DDP) cell line was Transwell® inserts (BD Bioscience, San Jose, CA, USA). purchased from the Tumor Cell Bank of the Chinese The lower compartments of the chambers contained Academy of Medical Science (Peking, China). The cells 10 % v/v FBS as a chemoattractant. After 48-h incuba- were maintained in RPMI 1640 medium supplemented tion, the cells on the upper surface of the membrane with 10 % fetal bovine serum (FBS), 100 units/mL peni- were wiped away, and the cells on the lower surface of cillin and 100 μg/mL streptomycin in a humidified at- the membrane were washed, fixed, and stained with mosphere of 5 % CO at 37 °C. crystal violet to quantify the extent of invasion. Proliferation assay Ovarian carcinoma specimens We used 3-(4,5)-dimethylthiazol (−zyl)-3,5-diphenyltetra- Specimens from 50 ovarian epithelial carcinomas, 13 be- zolium bromide (MTT; China) to determine the number nign tumors, 5 borderline tumors, and 6 normal ovarian of viable cells. Briefly, approximately 2.5 × 10 cells/well tissues were collected from patients who underwent sur- were seeded in a 96-well plate and allowed to adhere. At gical resection at the Department of Gynecology of the different time points, 20 μL MTT solution was added to First Affiliated Hospital of China Medical University each well, and the plates were incubated for 4 h. Subse- (Shenyang, China). Two pathologists confirmed the tumor quently, the MTT solution was removed, and 150 μLdi- specimens independently. The China Medical University methyl sulfoxide was added and incubated for 10 min Ethics Committee approved the study. before the absorbance was measured at 490 nm. Real-time RT-PCR (real-time RT-PCR) Cell cycle analysis Real-time RT-PCR was performed from 2 μg total RNA After 48-h incubation, the cells were washed, collected, using AMV reverse transcriptase and random primers and fixed in 10 mL ice-cold ethanol (70 %) for 12 h. (Takara). The PCR primers were designed according to Then, the cells were washed, incubated with 5 μL RNase the GenBank sequences. The glyceraldehyde-3-phosphate (0.25 mg/mL) at 37 °C for 30 min, pelleted, resuspended dehydrogenase gene (GAPDH) was used as the internal in 50 μg/mL PI, and incubated for 15 min in the dark at control. The relative gene expression level (the amount of room temperature. Cell cycle analysis was performed by target gene normalized to the endogenous control gene) flow cytometric analysis. was calculated using the comparative threshold cycle -ΔΔCt method: 2 .Hairpin-it™ microRNA and U6 snRNA Apoptosis assay Normalization RT-PCR Quantitation (GenePharma) were Flow cytometry was performed following PI and FITC- used to examine mature miR-23b. labeled annexin V (KeyGen Biotech, Nanjing, China) staining according to the manufacturer’s protocol. Briefly, Western blotting after 48-h incubation, cells were washed and resuspended Proteins were separated by SDS-PAGE, transferred to in 200 μL 1× binding buffer at 1 × 10 cells/mL, and incu- polyvinylidene difluoride membranes and immunoblotted bated with 5 μL FITC–annexin V; after 15-min incubation overnight at 4 °C with the primary antibodies (1:500), Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 3 of 10 rinsed with TBST, and incubated with 1:5000 secondary Results antibodies conjugated to horseradish peroxidase (Dako). Correlation of MIR23B and cyclin G1 (CCNG1) mRNA After applying electrochemiluminescence (ECL) Plus expression with pathogenesis and aggressiveness of detection reagents (Santa Cruz Biotechnology), pro- ovarian carcinoma tein bands were visualized using X-ray film (Fujifilm, We quantified MIR23B and CCNG1 mRNA expression in Tokyo, Japan). GAPDH-specific monoclonal antibody normal ovary tissue, benign and borderline tumors, and (1:2000; Santa Cruz Biotechnology) was used as the primary ovarian carcinoma using real-time PCR. MIR23B internal control. mRNA expression was significantly lower in the ovarian carcinomas and borderline tumors than in the normal ovarian tissues and benign tumors (Fig. 1a, p <0.05), and In vivo nude mouse tumorigenicity assay there were significant differences in expression among The assay was performed to determine the effects of miR- ages (Fig. 1b, p < 0.05) and pathological subtypes (mucin- 23b overexpression on the tumorigenicity of OVCAR3 ous vs. other types, Fig. 1d, p < 0.05). However, there were cells in vivo. Mock or hsa-miR-23b–transfected OVCAR3 no significant differences among International Federation cells (1 × 10 ) were injected subcutaneously into the flanks of Gynecology and Obstetrics (FIGO) stages (I/II vs. III/ of 4-6-week-old male nude mice (n = 5). Once tumor IV, Fig. 1c, p > 0.05) and differentiation (well vs. poor and growth had been established in the mock-treated mice moderate, data not shown, p > 0.05) in ovarian carcinoma. (14 days post-injection), tumor sizes were measured every CCNG1 mRNA expression was significantly lower in the other day. Tumor volume (mean ± standard deviation) normal ovarian tissues and benign ovarian tumors than in was calculated as length × width /2. the ovarian carcinomas (Fig. 1e, p <0.05). Effects of miR-23b transfection on ovarian carcinoma cell Immunofluorescence (IF) staining phenotype in vitro 5-μm frozen section from each sample was fixed in acet- We transfected OVCAR3, HO8910-PM, SKOV3/DDP one at 4 °C overnight. After washing, sections were cells with miR-23b. The transfected cells exhibited blocked with 1 % bovine serum albumin for 30 min and significantly slower growth (Fig. 2a, p < 0.05) but incubated overnight at 4 °C with rabbit anti-human higher levels of mature miR-23b expression than the CCNG1 antibody (1:50). Then sections were incubated control and mock-transfected cells (Fig. 2b, p <0.05). with anti-rabbit IgG–FITC (1:100, Santa Cruz Biotech- MiR-23b overexpression significant induced G arrest nology) for 2 h at room temperature in the dark. Nuclei (Fig. 3a, p < 0.05), higher levels of apoptosis (Fig. 3b, were stained with diaminophenylindole (1 μg/mL; p < 0.05) and reduced cell migration (Fig. 4a, p < 0.05) Sigma-Aldrich) for 5 min at room temperature. Cover- and invasion (Fig. 4b, p < 0.05) compared to the con- slips were mounted and imaged using a laser confocal trol and mock-transfected cells. microscope (Olympus, Tokyo, Japan). Effects of miR-23b transfection on ovarian carcinoma cell genotype in vitro Dual-luciferase reporter assay The predicted seed region in the 3′ untranslated regions A293 cells were first seeded at 60–80 % confluence in (3′ UTR) of CCNG1 revealed that it is direct target 24-well plates for 12 h. For the dual-luciferase reporter of miR-23b (Fig. 5a); dual-luciferase reporter assay assays, cells were transiently transfected with Wt or mu- indicated that miR-23b significantly decreased the tated reporter plasmid and miR-23b or control-miR. relative luciferase activity of the wild-type CCNG1 3′ Firefly luciferase activity was measured using a Dual- UTR as compared with the mutant CCNG1 3′ UTR, Luciferase Assay (Promega) 48 h after transfection, and indicating that miR-23b may directly bind to the 3′ the results were normalized with Renilla luciferase. Each UTR of CCNG1 (Fig.5b).Reverse transcription reporter plasmid was transfected at least three times, (RT)-PCR (Fig. 5c, p < 0.05) and western blotting and each sample was assayed in triplicate. (Fig. 5d) showed that miR-23b overexpression reduced CCNG1, urokinase (uPA), P70S6K, Bcl-xL, survivin, and Statistical analysis matrix metallopeptidase 9 (MMP9) mRNA or protein Statistical evaluation was performed using Spearman’s expression. rank correlation coefficient to analyze ranked data; the Mann–Whitney U test was used to differentiate the MiR-23b inhibited tumor growth in vivo means of different groups. A p-value of < 0.05 was con- The tumor xenograft volume in nude mice treated with sidered statistically significant. SPSS v. 17.0 (SPSS) was miR-23b was smaller than that in the mock-treated mice used to analyze all data. (Fig. 6a, p < 0.05). From day 4 and week 2 onwards, the Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 4 of 10 Fig. 1 Correlation of MIR23B and CCNG1 mRNA expression with pathogenesis and aggressiveness of ovarian carcinoma. a MIR23B mRNA expression was significantly lower in the ovarian carcinomas and borderline ovarian tumors than in the normal ovarian tissues and benign tumors; b there were significant differences in expression among age and d pathological subtype (mucinous vs. other types) in the ovarian carcinoma tissues. c There were no significant differences among FIGO stage (I/II vs. III/IV) in ovarian carcinoma. e CCNG1 mRNA expression was significantly lower in the normal ovarian tissues and benign ovarian tumors than in the ovarian carcinomas. *p < 0.05 vs. normal ovarian tissues; p < 0.05 vs. benign ovarian tumors. No = Normal ovarian tissues, Be = benign ovarian tumors, Bo = borderline ovarian tumors, Om = omentum tumors, Mu = mucinous carcinoma, Ser = serous carcinoma tumor xenograft growth in the miR-23b–treated BALB/c Discussion mice was slower than that in the mock group (Fig. 6b, MiRNAs regulate their target genes by affecting base p < 0.05; p < 0.01, pairing to their 3′ UTRs, resulting in mRNA degrad- day 4 deviation of tumor xenograft volume [DV] and p < 0.05; p < 0.01), and the DV increased in ation or inhibition of translation [11]. An increasing week 2 DV the latter period. number of studies have revealed that miRNAs are prom- ising diagnostic and prognostic molecular biomarkers as well as therapeutic targets in cancer [17, 18]. Series of MiR-23b downregulated CCNG1 expression in tumor studies has reported that miR-23b acts as a tumor sup- xenografts in vivo pressor in different cancers [19]. Majid et al. showed that Immunofluorescence staining (IF) indicated decreased miR-23b has diagnostic/prognostic significance and dir- CCNG1 expression in the tumor xenografts of the miR- ectly targets the oncogenic ZEB1 in bladder cancer [14]. 23b–treated nude mice compared with that in the He et al. reported that miR-23b expression levels in pros- mock-treated nude mice (Fig. 7). tate carcinoma (PCa) tissues was significantly correlated Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 5 of 10 Fig. 2 Effects of miR-23b transfection on ovarian carcinoma cell proliferation in vitro. a Following miR-23b transfection, OVCAR3, HO8910-PM, SKOV3/DDP cell lines exhibited significantly slower growth and b higher levels of mature miR-23b expression than the control and mock- transfected cells. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, * p < 0.05 Fig. 3 Effects of miR-23b transfection on ovarian carcinoma cell cycle and apoptosis in vitro. MiR-23b transfection induced a G arrest and b early apoptosis in OVCAR3, HO8910-PM, and SKOV3/DDP cells as compared to the control and mock-transfected cells. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, * p < 0.05 Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 6 of 10 Fig. 4 Effects of miR-23b transfection on the invasive and metastatic ability of ovarian carcinoma cell lines. After transfection with the miR-23b mimic, ovarian carcinoma cells showed a lower migration and b slower invasion as compared with the control and mock-transfected cells. Results are representative of three separate experiments; data are expressed as the mean ± standard deviation, *p < 0.05 with that of peroxiredoxin 3 (PRDX3) and that miR-23b predicted seed region showed that miR-23b targets may be involved in the response of PCa cells to hypoxic CCNG1 3′ UTR, which was convinced by the dual- stress, therefore gene therapy using miRNA mimics may luciferase reporter assay. We also found that miR-23b be useful as PCa therapy [20]. Our results show that transfection decreased CCNG1 mRNA and protein ex- MIR23B mRNA expression was significantly lower in pression. CCNG1 was first identified as a p53-regulated ovarian carcinomas and borderline tumors than in normal transcript induced by DNA damage. It has been pro- ovarian tissues and benign tumors, and the expression posed that these events underpin CCNG1 participation among age and pathological subtypes (mucinous vs. other in the enforcement of the p53-dependent G –Sand G 1 2 types) was significantly different. These findings indicate checkpoints responsive to DNA damage [21]. Some have that miR-23b might affect ovarian epithelial carcinogen- suggested that CCNG1 might function as an oncogenic esis and the subsequent progression. Therefore, we ex- protein [22, 23] and play a pivotal role in the initiation plored the function and molecular mechanism of miR-23b and metastasis of hepatocellular carcinoma [24]. Russell in ovarian cancer cell lines. et al. reported that CCNG1 amplification is associated Ovarian cancer cells transfected with miR-23b had sig- with significantly shorter postsurgical survival in pa- nificantly slower growth than the negative control– and tients with ovarian cancer who have received adjuvant mock-transfected cells, and there was significantly in- chemotherapy with taxanes and platinum compounds duced G arrest and apoptosis and reduced cell invasion [21]. These results suggest that miR-23b may inhibit and migration, suggesting miR-23b may inhibit ovarian ovarian cancer tumorigenesis and progression by tar- carcinoma tumorigenesis and progression. Moreover, the geting CCNG1. Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 7 of 10 Fig. 5 Effects of miR-23b transfection on ovarian carcinoma cell genotype in vitro. a The predicted seed region in the 3′ UTR of CCNG1 revealed that CCNG1 was direct target of miR-23b, as predicted by microRNA.org; b dual-luciferase reporter assay indicated that miR-23b significantly decreased the relative luciferase activity of the wild-type CCNG1 3′ UTR as compared with the mutant CCNG1 3′ UTR, indicating that miR-23b may directly bind to the 3′ UTR of CCNG1. c & d MiR-23b overexpression reduced CCNG1, uPA, P70S6K, Bcl-xL, survivin and MMP9 mRNA or protein expression. * p < 0.05 In this study, we also found that miR-23b overexpres- and metastasis of cancer [31]. Wang et al. reported that sion downregulated uPA expression, which is in line uPA promoted carcinoma cell proliferation by stimulat- with the findings of Salvi et al. [15], who reported that ing P70S6K activation [32]. Survivin, a member of the miR-23b overexpression leads to uPA downregulation inhibitors of apoptosis protein family, is expressed dur- and decreased migration and proliferation ability in ing development and in various human cancers. Lee et hepatocellular carcinoma cells. Furthermore, miR-23b al. reported that downregulating survivin suppressed overexpression also downregulated the expression of uPA through the transcription factor JunB [33]. Ryan et P70S6K, survivin, Bcl-xL, and MMP9 mRNA and pro- al. reported that survivin expression in breast cancer tein. It has been established that uPA is integral to cell predicts clinical outcome and is associated with uPA differentiation, migration, tissue remodeling under [34]. Furthermore, Zhou et al. reported that Bcl-xL over- physiological and pathological conditions, and may be a expression strongly enhanced uPA in pancreatic ductal potential diagnostic biomarker and therapeutic target in adenocarcinoma cells [35]. Therefore, we suggest that cancer. Significant elevation of uPA protein levels in pri- miR-23b reduces cell proliferation by downregulating mary ovarian cancer tissue has been associated with CCNG1 and uPA/P70S6K expression, suppresses cell poor prognosis and disease progression [25–29]. The migration and invasion by downregulating uPA/MMP9 uPA system plays an important role in many patho- expression, and induces apoptosis by downregulating physiological processes, such as cell differentiation, mi- survivin and Bcl-xL/uPA expression. gration, tissue reconstruction, and matrix dissolution We found that CCNG1 mRNA expression was signifi- [30]. The aberrant expression of phosphorylated P70S6K cantly lower in the normal ovarian tissues and benign might contribute to the pathogenesis, growth, invasion, ovarian tumors than in the borderline ovarian tumors Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 8 of 10 Fig. 6 MiR-23b inhibited tumor growth in vivo. a Tumor xenograft volume in nude mice treated with hsa-miR-23b was smaller than that in mock- treated mice. b Tumor xenograft growth in miR-23b–treated nude mice was slower than that in the mock group from day 4 and week 2 onwards and ovarian carcinomas. Our subsequent in vivo potential suppressor of ovarian carcinoma by targeting tumor xenograft studies showed that miR-23b inhib- CCNG1. ited tumor growth and decreased CCNG1 expression. Our findings show that miR-23b may inhibit ovarian Recent studies have reported that CCNG1 gene ther- cancer tumorigenesis and progression by downregulating apy has been developed and has undergone phase I/II CCNG1 and the expression of the relevant genes, and clinical trials for treating colorectal and pancreatic that miR-23b is a potentially novel application for regu- cancer [36]. We suggest that miR-23b may also be a lating ovarian carcinoma progression. Fig. 7 MiR-23b downregulated CCNG1 expression in tumor xenografts in vivo. IF experiments indicated that CCNG1 expression in the tumor xenografts of miR-23b–treated nude mice were decreased compared with that in the mock-treated nude mice Yan et al. Journal of Experimental & Clinical Cancer Research (2016) 35:31 Page 9 of 10 Conclusion 8. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009;10:704–14. This is the first demonstration that miR-23b may inhibit 9. Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, Nair S, et al. The EOC tumorigenesis and progression by targeting CCNG1 microRNAs miR-373 and miR-520c promote tumor invasion and metastasis. and modulating the expression of the relevant genes. Nat Cell Biol. 2008;10:202–10. 10. Ma L, Teruya-Feldstein J, Weinberg RA. Tumor invasion and metastasis These findings indicate that miR-23b is a potential initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–8. suppressor of ovarian carcinoma tumorigenesis and pro- 11. Tao JQ, Zhi XF, Zhang XY, Fu M, Huang H, Fan Y, et al. miR-27b-3p gression. The involvement of miR-23b–mediated CCNG1 suppresses cell proliferation through targeting receptor tyrosine kinase like orphan receptor 1 in gastric cancer. J Exp Clin Cancer Res. 2015;34:139. downregulation in inhibiting EOC aggressiveness may 12. Xiang J, Bian CD, Wang H, Huang SS, Wu DL. MiR-203 down-regulates yield further insight into the molecular mechanisms Rap1A and suppresses cell proliferation, adhesion and invasion in prostate underlying cancer aggressiveness. cancer. J Exp Clin Cancer Res. 2015;34:8. 13. Zhang H, Hao Y, Yang J, Zhou Y, Li J, Yin S, et al. Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer Abbreviations metastasis. Nat Commun. 2011;2:554. CCNG1: cyclin G1; EOC: epithelial ovarian carcinoma; FIGO 14. Majid S, Dar AA, Saini S, Deng G, Chang I, Greene K, et al. MicroRNA-23b stages: International Federation of Gynecology and Obstetrics stages; functions as a tumor suppressor by regulating Zeb1 in bladder cancer. PLoS GAPDH: glyceraldehyde-3-phosphate dehydrogenase gene; One. 2013;8(7):e67686. IF: immunofluorescent staining; miR-23b: microRNA 23b; MMP9: matrix 15. Salvi A, Sabelli C, Moncini S, Venturin M, Arici B, Riva P, et al. MicroRNA-23b metallopeptidase-9; MTT: 3-(4,5)-dimethylthiazol (−zyl)-3,5- mediates urokinase and c-met down modulation and a decreased migration diphenyltetrazolium bromide; RT-PCR: real-time RT-PCR; SDS-PAGE: sodium of human hepatocellular carcinoma cells. FEBS J. 2009;276:2966–82. dodecyl sulfate–polyacrylamide gel electrophoresis; TBST: tris-buffered saline 16. Majid S, Dar AA, Saini S, Arora S, Shahryari V, Zaman MS, et al. miR-23b with tween 20®. represses proto-oncogene Src kinase and functions as methylation-silenced tumor suppressor with diagnostic and prognostic significance in prostate Competing interests cancer. Cancer Res. 2012;72:6435–46. The authors declare that they have no competing interests. 17. Guo J, Wang M, Liu XH. MicroRNA-195 suppresses tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma. J Exp Authors’ contributions Clin Cancer Res. 2015;34:91. ZH-Z and JY carried out the molecular genetic studies, participated in the in 18. Kawano M, Tanaka K, Itonaga I, Ikeda S, Iwasaki T, Tsumura H. microRNA-93 vivo tumor xenograft studies, and drafted the manuscript. JY J carried out promotes cell proliferation via targeting of PTEN in Osteosarcoma cells. J the immunoassays. XN-M and YL X participated in the design of the study Exp Clin Cancer Res. 2015;34:76. and performed the statistical analysis. ZH-Z conceived the study, participated 19. Donadelli M, Palmieri M. Roles for microRNA 23b in regulating autophagy in its design and coordination, and helped to draft the manuscript. All and development of pancreatic adenocarcinoma. Gastroenterology. 2013; authors read and approved the final manuscript. 145(5):936–8. 20. He HC, Zhu JG, Chen XB, Chen SM, Han ZD, Dai QS, et al. MicroRNA-23b Acknowledgements downregulates peroxiredoxin III in human prostate cancer. FEBS Lett. 2012; This work was supported by the Natural Scientific Foundation of China (Nos. 586(16):2451–8. 81472502), and Liaoning Science and Technology Grant (2013021077). 21. Russell P, Hennessy BT, Li J, Carey MS, Bast RC, Freeman T, et al. Cyclin G1 regulates the outcome of taxane-induced mitotic checkpoint arrest. Author details Oncogene. 2012;31:2450–60. 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Journal

Journal of Experimental & Clinical Cancer Research – Springer Journals

Published: Dec 1, 2016

Keywords: cancer research; immunology; apoptosis; oncology

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