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Diagnostic and prognostic potentials of microRNA-27a in osteosarcoma
Biomedicine & Pharmacotherapy 71 (2015) 222–226
Available online at
ScienceDirect www.sciencedirect.com
Original article
Diagnostic and prognostic potentials of microRNA-27a in osteosarcoma Jie Tang a, Hui Zhao b, Haikang Cai a, Haishan Wu b,* a b
Department of Orthopaedics, Xuhui Central Hospital, Shanghai, 200031, China Department of Joint Center, Changzheng Hospital, the Second Military Medical University, No. 440, North Chengdu Road, Shanghai, 200003, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2015 Accepted 23 January 2015
Aim: Growing evidence suggest that the microRNA (miR)-23a/24-2/27a cluster may play a crucial role in mammary tumorigenesis and act as a novel class of oncogenes. Among these members, miR-27a has been reported to promote proliferation, migration and invasion in human osteosarcoma cells. The aim of this study was to detect the serum levels of miR-27a in osteosarcoma patients and to investigate its associations with clinicopathological features and prognosis. Methods: miR-27a levels in sera from 166 osteosarcoma patients and 60 healthy controls were detected by real-time quantitative RT-PCR. Then, the associations of serum miR-27a level with clinicopathological factors or survival of osteosarcoma patients were further evaluated. Results: Compared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (P < 0.001). Importantly, miR-27a could efficiently screen osteosarcoma patients from healthy controls (Area under receiver operating characteristic curve, AUC = 0.867). Then, high miR27a expression was more frequently occurred in osteosarcoma patients with advanced clinical stage (P = 0.001), positive distant metastasis (P = 0.01) and poor response to chemotherapy (P = 0.008). In Kaplan–Meier survival analysis, high miR-27a expression was a significant indicator for poor overall survival (P = 0.006) as well as poor disease-free survival (P = 0.01). Furthermore, multivariate analysis demonstrated that miR-27a expression was an independent and significant prognostic factor to predict overall survival (P = 0.01) and disease-free survival (P = 0.03). Conclusion: miR-27a expression may be elevated in sera of osteosarcoma patients and in turn contributes to aggressive progression of this malignancy. Detection of serum miR-27a levels may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients. ß 2015 Published by Elsevier Masson SAS.
Keywords: Osteosarcoma microRNA-27a Serum Diagnosis Prognosis Real-time quantitative RT-PCR
1. Introduction Osteosarcoma, as the most common primary sarcoma of bone, is the leading cause of cancer-related death among children and adolescents [1]. It arises from osteoid tissues, and produces immature bone [2]. Normal bone tissues are often destroyed in this malignancy. The treatment for osteosarcoma is very challenging. Although the advancement of novel therapies, such as (neo) adjuvant chemotherapy, wide excision of tumors and amputation of the affected limbs, continue to emerge, osteosarcoma-related morbidity and mortality remain high [3]. More than 50% of patients who are chemoresistant have an extremely poor prognosis due to lung metastasis [4]. The 5-year overall and disease-free survival rates for osteosarcoma patients are around 50–60% [5]. Accumulating studies reported molecular alterations contributing to * Corresponding author. Tel.: +86 021 63520020; fax: +86 021 63520020. E-mail address: [email protected] (H. Wu). http://dx.doi.org/10.1016/j.biopha.2015.01.025 0753-3322/ß 2015 Published by Elsevier Masson SAS.
osteosarcoma development and progression. However, the etiology of osteosarcoma has not been fully elucidated due to the highly complex molecular mechanisms. Therefore, it is of great clinical significance to understand the fundamental molecular mechanisms in osteosarcoma pathogenesis that may help to identify diagnostic and prognostic biomarkers, and to optimize treatment strategies. In recent years, microRNAs (miRNAs), which play a critical role in cancers, have become a novel ‘‘hot topic’’ in this research field. MiRNAs, which are of 18–25 nucleotides in length, form an abundant class of endogenous noncoding small RNAs that are highly conserved in the genomes of most species, including plants, animals and DNA viruses [6]. These RNAs carry out their biological functions by binding to the 30 untranslated regions (UTRs) of their target mRNAs, thereby suppressing the translation of target mRNAs into proteins and/or directly inducing the degradation of target mRNAs [7]. Growing evidence showed that miRNAs play an important role in a number of biological processes, including
J. Tang et al. / Biomedicine & Pharmacotherapy 71 (2015) 222–226
embryogenesis, development, cell maintenance, cellular differentiation, proliferation, apoptosis and metabolism processes [8]. In recent years, accumulating studies have reported that miRNAs are not only involved in the initiation, progression and metastasis of human cancers but also influence tumor cells response to chemotherapeutic agents by acting as oncogenes and tumor suppressors [9,10]. miRNA expression profiles have been revealed to be capable of classify human cancers efficiently, implying that miRNAs have the potential to be useful diagnostic and prognostic tools [11]. More importantly, miRNAs have also been detected in human serum and plasma in remarkably stable forms, which makes the unique plasma/serum miRNA patterns possible to be noninvasive cancer biomarkers [12]. MiR-27a, located at chromosome 19p13.1, belongs to the miR-23a/24-2/27a cluster existing intergenically in the vertebrate genome [13]. Members of this cluster have been indicated to be involved in cell cycle control and differentiation of various cell types, and also have been demonstrated to play a crucial role in mammary tumorigenesis and act as a novel class of oncogenes [14,15]. The aberrant expression of miR27a has been observed in various human cancer types, which confirm it as an important regulator in carcinogenesis. However, its expression levels and biological functions in cancers are controversial. For example, its oncogenic role has been verified in breast cancer, hepatocellular carcinoma, gastric adenocarcinoma, colon cancer, renal cell carcinoma and cervical cancer; In contrast, miR27a also play tumor suppressive roles in acute leukemia, esophageal cancers, oral squamous cell carcinoma and non-small cell lung cancer [16–20]. The diverse effects of this miRNA suggest its context-dependent activities. Previous studies have reported that miR-27a expression could promote proliferation, migration and invasion of human osteosarcoma cells [21,22]. However, its expression pattern and clinical value in human osteosarcoma are still largely unknown. To address this problem, we here detected serum levels of miR-27a in 166 osteosarcoma patients and 60 healthy controls by real-time quantitative RT-PCR. Then, the associations of serum miR-27a level with clinicopathological factors or survival of osteosarcoma patients were further evaluated. We hypothesized that miR-27a expression in sera might be dysregulated at an early stage of osteosarcoma, and could effectively differentiate osteosarcoma patients from healthy controls, which may help to understand its roles in osteosarcoma.
223
distant metastasis, histological subtype of osteosarcoma, type of surgery, and histological response to pre-operative chemotherapy. The Huvos grading system was used to rate the level of tumor necrosis following preoperative chemotherapy [24]. The good responders were defined as patients whose tumors had 90% necrosis in response to preoperative chemotherapy as determined by histologic examination at the time of definitive surgery and poor responders had <90% necrosis. Clinical stage of these osteosarcoma patients were classified according to the Enneking Surgical Staging System. The clinicopathological information of the patients is shown in Table 1. At the same time, 60 healthy individuals (sex and age matched) were used as control group. The absence of disease in the control group was confirmed by physical examination, clinical history and routine laboratory tests. To detect the expression levels of miR-27a, serum samples (5 mL) were conventionally drawn from pre-therapeutic osteosarcoma patients and matched healthy individuals, respectively. Immediately after serum sample collection, the plasma and cellular elements were separated by centrifuging the blood at 1200 g for 15 min at 4 8C, and then stored in 1.5 mL RNase free tubes at 80 8C. All 166 osteosarcoma patients received follow-up. The median follow-up was 87 months (range: 10–152 months). During the follow-up period, 66 patients (66/166, 39.8%) died of disease. Distant metastases developed in 42 patients at a mean of 13.8 months (range 3–46 months) after the original diagnosis. Of these patients, 9 had bone metastases and 36 had lung metastases (3 patients had both bone and lung metastases). The median overall and disease-free survival of patients was 31 months (95% confidence interval [CI], 30.1–42.9 months) and 25 months (95%CI, 23.7–35.2 months), respectively. 2.2. RNA extraction and real-time quantitative RT-PCR assay The serum levels of miR-27a expression in osteosarcoma patients and healthy controls were detected by real-time Table 1 Association of serum miR-27a expression with clinicopathological features of osteosarcoma. Clinicopathological features
No. of cases
miR-27a expression High (n, %)
2. Materials and methods 2.1. Patients and tissue samples This study was approved by the Research Ethics Committee of Xuhui Central Hospital and Changzheng Hospital, China. Written informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards. A total of 166 patients with primary osteosarcoma, treated in the Department of Orthopaedics in Xuhui Central Hospital and Department of Joint Center in Changzheng Hospital, Shanghai, China, from 1998 to 2008, were enrolled retrospectively in this study. Patients were excluded if they had a previous or secondary malignancy. After establishing the diagnosis, all patients were treated with a preoperative chemotherapy lasting 4 months, using either the combination of an anthracycline (doxorubicin) and highdose methotrexate or the combination of etoposide, ifosfamide, and high-dose methotrexate. The postoperative treatment was determined by the histologic system established by Huvos et al. [23]. The following clinical parameters were analyzed: age, gender, site of tumor, tumor size, presence of pathological fracture and
Age 72 36 <55 55 94 50 Gender Male 96 51 Female 70 35 Tumor size (cm) >8 88 46 8 78 40 Anatomic location Tibia/femur 103 51 Elsewhere 63 35 Serum level of lactate dehydrogenase Elevated 90 46 Normal 76 40 Serum level of alkaline phosphatase Elevated 108 56 Normal 58 30 Clinical stage IIA 68 8 IIB/III 98 78 Distant metastasis Absent 124 50 Present 42 36 Response to chemotherapy Good 68 18 Poor 98 68
P
Low (n, %)
(50.00) (53.19)
36 (50.00) 44 (46.81)
NS
(53.13) (50.00)
45 (46.87) 35 (50.00)
NS
(52.27) (51.28)
42 (47.73) 38 (48.72)
NS
(49.51) (55.56)
52 (50.49) 28 (44.44)
NS
(51.11) (52.63)
44 (48.89) 36 (47.37)
NS
(51.85) (51.72)
52 (48.15) 28 (48.28)
NS
(11.76) (79.59)
60 (88.24) 20 (20.41)
0.001
(40.32) (85.71)
74 (59.68) 6 (14.29)
0.01
(26.47) (69.39)
50 (73.53) 30 (30.61)
0.008
224
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quantitative RT-PCR assay. Briefly, total RNA was extracted from 500 mL plasma of each sample using mirVana PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. RNA was dissolved in 100 mL of RNase free water. The purity and concentration of all RNA samples were evaluated by their absorbance ratio at 260/280 nm. A SYBR green qRT-PCR assay was used for miRNA quantification and Real-time PCR was performed on an ABI 7500 Real-Time PCR instrument (Applied Biosystems, Inc). For miR-27a analysis, the stem-loop RT primer was 50 -GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACG CGG AAC-30 and the amplifying primers were as follows: sense, 50 -TGC GGT TCA CAG TGG CTA AG-30 and antisense, 50 -CCA GTG CAG GGT CCG AGG T-30 . Primers for qRT-PCR of U6, an miRNA that was used as an internal control, were: sense, 50 -CTC GCT TCG GCA GCA CA-30 and antisense, 50 -AAC GCT TCA CGA ATT TGC GT-30 . Each sample was examined in triplicate and the relative expression DD level of miR-27a was normalized to that of U6 by the 2 Ct cycle threshold method [25]. 2.3. Statistical analysis The software of SPSS version 13.0 for Windows (SPSS Inc, IL, USA) and SAS 9.1 (SAS Institute, Cary, NC) was used for statistical analysis. Continuous variables were expressed as mean S.E. Mann–Whitney U test was used to compare the difference of serum miR-27a expression levels in osteosarcoma patients and healthy controls. The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnosis value of serum miR-27a level. The Chisquare test was used to show differences of categorical variables. Patient survival and their differences were determined by Kaplan– Meier method and log-rank test. Cox regression (Proportional hazard model) was adopted for multivariate analysis of prognostic factors. Differences were considered statistically significant when P was less than 0.05.
3. Results 3.1. Diagnostic value of serum miR-27a expression for osteosarcoma patients Compared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (osteosarcoma vs. controls: 3.87 1.01 vs. 2.09 0.46, P < 0.001, Figure 1A). As shown in Figure 1B, ROC curve analysis illustrated that the serum miR-27a level was a potential biomarker for screening
osteosarcoma patients from healthy controls with the area under the ROC curve (AUC) of 0.867, and the serum miR-27a level at 2.88 was the clear cutoff value to distinguish osteosarcoma patients from healthy controls. With this cutoff value, the sensitivity and specificity of the serum miR-27a level for osteosarcoma was 70.01% and 98.30%, respectively. 3.2. Elevated expression of miR-27a in sera associates with advanced clinicopathological features of osteosarcoma patients To investigate the associations of serum miR-27a expression with the clinicopathological features of osteosarcoma patients, we divided the patients into two groups using the median value of serum miR-27a expression levels in osteosarcoma patients as a cutoff point (3.70). Of 166 osteosarcoma specimens, 86 (51.81%) highly expressed miR-27a. As shown in Table 1, high miR-27a expression was more frequently occurred in osteosarcoma patients with advanced clinical stage (P = 0.001), positive distant metastasis (P = 0.01) and poor response to chemotherapy (P = 0.008). No significant differences were observed between serum miR-27a expression and patients’ age, gender, tumor size, anatomic location, serum levels of lactate dehydrogenase and alkaline phosphatase (all P > 0.05). 3.3. Elevated expression of miR-27a in sera associates with poor prognosis in osteosarcoma patients Using Kaplan–Meier method and log-rank test, the overall survival (Figure 2A, P < 0.001) and disease-free survival (Figure 2B, P < 0.001) of osteosarcoma patients with high miR-27a expression in sera were both significantly shorter than those with low miR27a expression. Besides, the survival benefits were also found in those with smaller tumor size (both P = 0.03), higher clinical stage (P = 0.01 and 0.006, respectively), without distant metastasis (P = 0.008 and 0.003, respectively), and better response to chemotherapy (both P = 0.02) for overall survival and disease-free survival. We further examined overall survival and disease-free survival using Cox regression hazard analyses to determine whether serum miR-27a expression could serve as a clinically useful prognostic assessment factor in osteosarcoma. Multivariate Cox regression analysis enrolling above-mentioned significant parameters revealed that serum miR-27a expression (RR 3.56, 95%CI, 1.87– 9.86, P = 0.01), clinical stage (RR 2.32, 95%CI, 1.10–7.01, P = 0.02), distant metastasis status (RR 3.72, 95%CI, 1.91–9.83, P = 0.01), and response to chemotherapy (RR 2.60, 95%CI, 1.52–8.21, P = 0.02)
Figure 1. Diagnostic value of serum miR-27a expression for osteosarcoma patients. (A) Serum miR-27a expression in 166 osteosarcoma patients and healthy controls were respectively detected by real-time quantitative RT-PCR assay. Compared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (osteosarcoma vs. controls: 3.87 1.01 vs. 2.09 0.46, P < 0.001). (B) ROC curve analysis illustrated that serum miR-27a expression was a potential biomarker for screening osteosarcoma patients from healthy controls (AUC = 0.867).
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Figure 2. Overall survival (A) and disease-free survival (B) curves for two groups defined by low and high expression of miR-27a in sera from osteosarcoma patients. The patients with high miR-27a expression had a significantly shorter 5-year overall and disease-free survival rate than those with low miR-27a expression (both P < 0.001).
were independent prognostic markers for overall survival of osteosarcoma patients (Table 2). Turning to disease-free survival, serum miR-27a expression (RR 3.19, 95%CI, 1.72–8.33, P = 0.01), clinical stage (RR 1.83, 95%CI, 0.71–8.12, P = 0.03) and metastasis status (RR 2.92, 95%CI, 1.32–10.62, P = 0.02) were also independent prognostic markers for disease-free survival of osteosarcoma patients (Table 2). 4. Discussion Aberrantly expressed circulating miRNAs have received a great deal of attention as potential sensitive and accurate biomarkers for cancer diagnosis and prognosis due to the following points: (1) miRNAs have high tissue- and tumor-specificity; (2) miRNAs have the inherent stability in patients’ sera and tissues; (3) Sampling is very easy by minimally invasive approaches; (4) Methods of isolation and detection are relatively cheap; (5) The aberrant expression of miRNAs has been reported to be associated with the development and progression of human malignancies [26]. In the current study, we detected the serum levels of miR-27a expression in osteosarcoma patients and healthy controls, and confirmed its elevated expression in sera of osteosarcoma patients. More importantly, the diagnostic value of serum miR-27a expression was also evaluated. Our data showed that miR-27a could efficiently screen osteosarcoma patients from healthy controls (AUC = 0.867). The AUC value is an indicator of the efficacy of the assessment system. An ideal test with perfect discrimination (100% sensitivity and 100% specificity) has an AUC of 1.0, whereas a noninformative prediction has the area 0.5, indicating that it may be achieved by mere guess. The closer to 1.0 the AUC of a test is, the higher the overall efficacy of the test will be [27]. Here, miR-27a could screen osteosarcoma patients from healthy controls with an
AUC value (0.867) approximating 1.0, suggesting that it had a relatively high ability to identify the true osteosarcoma patients. In addition, we focused on the potential relationship between the serum level of miR-27a and various clinicopathological characteristics of osteosarcoma patients, as well as disease-free survival and overall survival. It is worth noting that high levels of miR-27a appear to be significantly correlated with aggressive tumor progression and poor prognosis in patients with osteosarcoma. To the best of our knowledge, the current study represents the first demonstration that serum miR-27a expression may function as a diagnostic and prognostic marker for osteosarcoma patients. Similar with other members of the miR-23a/24-2/27a cluster, miR-27a has been reported to have both oncogenic and tumor suppressive functions in different cell lines and human cancer tissues. Tian et al. [28] indicated that miR-27a might act as an oncogene in laryngeal squamous cell carcinoma through suppressing the expression of PLK2 and serve as a useful biomarker in diagnosis and therapy in this malignancy; Li et al. [29] observed the upregulation of miR-27a in tumor tissues sampled from lung adenocarcinoma patients treated with cisplatin-based chemotherapy and proved that it might be correlated with low expression of RKIP, decreased sensitivity to cisplatin, and poor prognosis; Xu et al. [30] revealed that oncogenic miR-27a could play an important role in ovarian cancer cell growth and metastasis; Zhao et al. [17] confirmed that down-regulation of miR-27a could inhibit proliferation of gastric cancer cells in vitro and in vivo; Ma et al. [18] reported that miR-27a might play an oncogenic role by targeting Spry2 and modulating the malignant, biological behavior of pancreatic cancer cells; In breast cancer cells, miR-27a has been reported to directly suppress ZBTB10/RINZF expression and in turn upregulate VEGF and VEGF receptor, and overexpression of miR27a might be associated with poor outcomes of the patients
Table 2 Multivariate survival analysis of overall survival and disease-free survival in 166 osteosarcoma patients. Variables
Serum miR-27a expression Clinical stage Distant metastasis status Response to chemotherapy
Overall survival
Disease-free survival
RR
95%CI
P
RR
95%CI
P
3.56 2.32 3.72 2.60
1.87–9.86 1.10–7.01 1.91–9.83 1.52–8.21
0.01 0.02 0.01 0.02
3.19 1.83 2.92 0.93
1.72–8.33 0.71–8.12 1.32–10.62 0.30–1.71
0.01 0.03 0.02 NS
226
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[16]. Whereas, Bao et al. [19] identified the tumor suppressive role of miR-27a in colorectal carcinogenesis and progression by targeting SGPP1 and Smad2; In lung cancer, miR-27a was downregulated, directly targeted MET and EGFR, and suppressed their expression [20]. In the current study, we observed the elevated serum levels of miR-27a expression in osteosarcoma patients and confirmed its significant associations with aggressive clinicopathological features including advanced clinical stage, positive distant metastasis and poor response to chemotherapy, suggesting its oncogenic role in this malignancy. These findings were consistent with the previous study of Pan et al. [22], who revealed for the first time that miR-27a could function as an oncogene by targeting MAP2K4 in the osteosarcoma MG63 cell line. Although we here highlight the significant dysregulation and potentially novel diagnostic value for serum miR-27a expression in osteosarcoma patients, the detailed mechanisms underlying its oncogenic role in this disease remain to be further elucidated. Since the prediction of the clinical outcomes of osteosarcoma patients is an important prerequisite for better treatment stratification and personalized therapeutic regimens, our univariate analysis in the current study found that osteosarcoma patients with high miR-27a expression had both worse overall and diseasefree survival than those with low miR-27a expression significantly. Multivariate analysis further demonstrated that among all the factors analyzed, clinical stage, distant metastasis and serum miR27a expression were all independent prognostic factors for both overall and disease-free survival. These findings confirmed the prognostic value of serum miR-27a expression in osteosarcoma patients, implying that serum miR-27a expression could be used as a sufficient marker for further treatment stratification in the personalized therapeutic strategies in the future. In conclusion, our results imply that miR-27a expression may be elevated in sera of osteosarcoma patients and in turn contributes to aggressive progression of this malignancy. Detection of serum miR-27a levels may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients. Authors’ contributions WH conceived and designed the study, and approved the final version of the manuscript; TJ performed the experiments and wrote the manuscript; ZH and CH collected and analyzed the data. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer 2009;115:1531–43. [2] Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res 2009;152:3–13. [3] Bruland S, Bauer H, Alvegaard T, Smeland S. Treatment of osteosarcoma. The Scandinavian Sarcoma Group experience. Cancer Treat Res 2009;152:309–18.
[4] Federman N, Bernthal N, Eiber FC, Tap WD. The multidisciplinary management of osteosarcoma. Curr Treat Options Oncol 2009;10:82–93. [5] Bramer JA, van Linge JH, Grimer RJ, Scholten RJ. Prognostic factors in localized extremity osteosarcoma: a systematic review. Eur J Surg Oncol 2009;35: 1030–6. [6] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. [7] Dykxhoorn DM. MicroRNAs and metastasis: little RNAs go a long way. Cancer Res 2010;70:6401–6. [8] Wurdinger T, Costa FF. Molecular therapy in the microRNA era. Pharmacogenomics J 2007;7:297–304. [9] Meltzer PS. Cancer genomics: small RNAs with big impacts. Nature 2005;435:745–6. [10] Xue J, Niu J, Wu J, Wu ZH. MicroRNAs in cancer therapeutic response: friend and foe. World J Clin Oncol 2014;5:730–43. [11] Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006;6:857–66. [12] Cheng G. Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy. Adv Drug Deliv Rev 2015;81:75–93. [13] Xia J, Cheng L, Mei C, Ma J, Shi Y, Zeng F, et al. Genistein inhibits cell growth and invasion through regulation of miR-27a in pancreatic cancer cells. Curr Pharm Des 2014;20:5348–53. [14] Li X, Liu X, Xu W, Zhou P, Gao P, Jiang S, et al. c-MYC-regulated miR-23a/24-2/ 27a cluster promotes mammary carcinoma cell invasion and hepatic metastasis by targeting Sprouty2. J Biol Chem 2013;288:18121–33. [15] Chen Z, Ma T, Huang C, Zhang L, Lv X, Xu T, et al. MiR-27a modulates the MDR1/ P-glycoprotein expression by inhibiting FZD7/b-catenin pathway in hepatocellular carcinoma cells. Cell Signal 2013;25:2693–701. [16] Li X, Mertens-Talcott SU, Zhang S, Kim K, Ball J, Safe S. MicroRNA-27a indirectly regulates estrogen receptor {alpha} expression and hormone responsiveness in MCF-7 breast cancer cells. Endocrinology 2010;151:2462–73. [17] Zhao X, Yang L, Hu J. Down-regulation of miR-27a might inhibit proliferation and drug resistance of gastric cancer cells. J Exp Clin Cancer Res 2011;30:55. [18] Ma Y, Yu S, Zhao W, Lu Z, Chen J. miR-27a regulates the growth, colony formation and migration of pancreatic cancer cells by targeting Sprouty2. Cancer Lett 2010;298:150–8. [19] Bao Y, Chen Z, Guo Y, Feng Y, Li Z, Han W, et al. Tumor suppressor microRNA27a in colorectal carcinogenesis and progression by targeting SGPP1 and Smad2. PLoS One 2014;9:e105991. [20] Miao Y, Li J, Qiu X, Li Y, Wang Z, Luan Y. miR-27a regulates the self renewal of the H446 small cell lung cancer cell line in vitro. Oncol Rep 2013;29:161–8. [21] Jones KB, Salah Z, Del Mare S, Galasso M, Gaudio E, Nuovo GJ, et al. miRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res 2012;72:1865–77. [22] Pan W, Wang H, Jianwei R, Ye Z. MicroRNA-27a promotes proliferation, migration and invasion by targeting MAP2K4 in human osteosarcoma cells. Cell Physiol Biochem 2014;33:402–12. [23] Huvos AG. Osteosarcoma in adolescents and young adults: new developments and controversies. Commentary on pathology. Cancer Treat Res 1993;62: 375–7. [24] Rosen G, Caparros B, Huvos AG, Kosloff C, Nirenberg A, Cacavio A, et al. Preoperative chemotherapy for osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 1982;49:1221–30. [25] Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW. Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 2006;28:1601–13. [26] Croce C. Introduction to the role of microRNAs in cancer diagnosis, prognosis, and treatment. Cancer J 2012;18:213–4. [27] Baldi P, Brunak S, Chauvin Y, Andersen CA, Nielsen H. Assessing the accuracy of prediction algorithms for classification: an overview. Bioinformatics 2000;16:412–24. [28] Tian Y, Fu S, Qiu GB, Xu ZM, Liu N, Zhang XW, et al. MicroRNA-27a promotes proliferation and suppresses apoptosis by targeting PLK2 in laryngeal carcinoma. BMC Cancer 2014;14:678. [29] Li J, Wang Y, Song Y, Fu Z, Yu W. miR-27a regulates cisplatin resistance and metastasis by targeting RKIP in human lung adenocarcinoma cells. Mol Cancer 2014;13:193. [30] Xu L, Xiang J, Shen J, Zou X, Zhai S, Yin Y, et al. Oncogenic microRNA-27a is a target for genistein in ovarian cancer cells. Anticancer Agents Med Chem 2013;13:1126–32.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Diagnostic and prognostic potentials of microRNA-27a in osteosarcoma Jie Tang a, Hui Zhao b, Haikang Cai a, Haishan Wu b,* a b
Department of Orthopaedics, Xuhui Central Hospital, Shanghai, 200031, China Department of Joint Center, Changzheng Hospital, the Second Military Medical University, No. 440, North Chengdu Road, Shanghai, 200003, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2015 Accepted 23 January 2015
Aim: Growing evidence suggest that the microRNA (miR)-23a/24-2/27a cluster may play a crucial role in mammary tumorigenesis and act as a novel class of oncogenes. Among these members, miR-27a has been reported to promote proliferation, migration and invasion in human osteosarcoma cells. The aim of this study was to detect the serum levels of miR-27a in osteosarcoma patients and to investigate its associations with clinicopathological features and prognosis. Methods: miR-27a levels in sera from 166 osteosarcoma patients and 60 healthy controls were detected by real-time quantitative RT-PCR. Then, the associations of serum miR-27a level with clinicopathological factors or survival of osteosarcoma patients were further evaluated. Results: Compared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (P < 0.001). Importantly, miR-27a could efficiently screen osteosarcoma patients from healthy controls (Area under receiver operating characteristic curve, AUC = 0.867). Then, high miR27a expression was more frequently occurred in osteosarcoma patients with advanced clinical stage (P = 0.001), positive distant metastasis (P = 0.01) and poor response to chemotherapy (P = 0.008). In Kaplan–Meier survival analysis, high miR-27a expression was a significant indicator for poor overall survival (P = 0.006) as well as poor disease-free survival (P = 0.01). Furthermore, multivariate analysis demonstrated that miR-27a expression was an independent and significant prognostic factor to predict overall survival (P = 0.01) and disease-free survival (P = 0.03). Conclusion: miR-27a expression may be elevated in sera of osteosarcoma patients and in turn contributes to aggressive progression of this malignancy. Detection of serum miR-27a levels may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients. ß 2015 Published by Elsevier Masson SAS.
Keywords: Osteosarcoma microRNA-27a Serum Diagnosis Prognosis Real-time quantitative RT-PCR
1. Introduction Osteosarcoma, as the most common primary sarcoma of bone, is the leading cause of cancer-related death among children and adolescents [1]. It arises from osteoid tissues, and produces immature bone [2]. Normal bone tissues are often destroyed in this malignancy. The treatment for osteosarcoma is very challenging. Although the advancement of novel therapies, such as (neo) adjuvant chemotherapy, wide excision of tumors and amputation of the affected limbs, continue to emerge, osteosarcoma-related morbidity and mortality remain high [3]. More than 50% of patients who are chemoresistant have an extremely poor prognosis due to lung metastasis [4]. The 5-year overall and disease-free survival rates for osteosarcoma patients are around 50–60% [5]. Accumulating studies reported molecular alterations contributing to * Corresponding author. Tel.: +86 021 63520020; fax: +86 021 63520020. E-mail address: [email protected] (H. Wu). http://dx.doi.org/10.1016/j.biopha.2015.01.025 0753-3322/ß 2015 Published by Elsevier Masson SAS.
osteosarcoma development and progression. However, the etiology of osteosarcoma has not been fully elucidated due to the highly complex molecular mechanisms. Therefore, it is of great clinical significance to understand the fundamental molecular mechanisms in osteosarcoma pathogenesis that may help to identify diagnostic and prognostic biomarkers, and to optimize treatment strategies. In recent years, microRNAs (miRNAs), which play a critical role in cancers, have become a novel ‘‘hot topic’’ in this research field. MiRNAs, which are of 18–25 nucleotides in length, form an abundant class of endogenous noncoding small RNAs that are highly conserved in the genomes of most species, including plants, animals and DNA viruses [6]. These RNAs carry out their biological functions by binding to the 30 untranslated regions (UTRs) of their target mRNAs, thereby suppressing the translation of target mRNAs into proteins and/or directly inducing the degradation of target mRNAs [7]. Growing evidence showed that miRNAs play an important role in a number of biological processes, including
J. Tang et al. / Biomedicine & Pharmacotherapy 71 (2015) 222–226
embryogenesis, development, cell maintenance, cellular differentiation, proliferation, apoptosis and metabolism processes [8]. In recent years, accumulating studies have reported that miRNAs are not only involved in the initiation, progression and metastasis of human cancers but also influence tumor cells response to chemotherapeutic agents by acting as oncogenes and tumor suppressors [9,10]. miRNA expression profiles have been revealed to be capable of classify human cancers efficiently, implying that miRNAs have the potential to be useful diagnostic and prognostic tools [11]. More importantly, miRNAs have also been detected in human serum and plasma in remarkably stable forms, which makes the unique plasma/serum miRNA patterns possible to be noninvasive cancer biomarkers [12]. MiR-27a, located at chromosome 19p13.1, belongs to the miR-23a/24-2/27a cluster existing intergenically in the vertebrate genome [13]. Members of this cluster have been indicated to be involved in cell cycle control and differentiation of various cell types, and also have been demonstrated to play a crucial role in mammary tumorigenesis and act as a novel class of oncogenes [14,15]. The aberrant expression of miR27a has been observed in various human cancer types, which confirm it as an important regulator in carcinogenesis. However, its expression levels and biological functions in cancers are controversial. For example, its oncogenic role has been verified in breast cancer, hepatocellular carcinoma, gastric adenocarcinoma, colon cancer, renal cell carcinoma and cervical cancer; In contrast, miR27a also play tumor suppressive roles in acute leukemia, esophageal cancers, oral squamous cell carcinoma and non-small cell lung cancer [16–20]. The diverse effects of this miRNA suggest its context-dependent activities. Previous studies have reported that miR-27a expression could promote proliferation, migration and invasion of human osteosarcoma cells [21,22]. However, its expression pattern and clinical value in human osteosarcoma are still largely unknown. To address this problem, we here detected serum levels of miR-27a in 166 osteosarcoma patients and 60 healthy controls by real-time quantitative RT-PCR. Then, the associations of serum miR-27a level with clinicopathological factors or survival of osteosarcoma patients were further evaluated. We hypothesized that miR-27a expression in sera might be dysregulated at an early stage of osteosarcoma, and could effectively differentiate osteosarcoma patients from healthy controls, which may help to understand its roles in osteosarcoma.
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distant metastasis, histological subtype of osteosarcoma, type of surgery, and histological response to pre-operative chemotherapy. The Huvos grading system was used to rate the level of tumor necrosis following preoperative chemotherapy [24]. The good responders were defined as patients whose tumors had 90% necrosis in response to preoperative chemotherapy as determined by histologic examination at the time of definitive surgery and poor responders had <90% necrosis. Clinical stage of these osteosarcoma patients were classified according to the Enneking Surgical Staging System. The clinicopathological information of the patients is shown in Table 1. At the same time, 60 healthy individuals (sex and age matched) were used as control group. The absence of disease in the control group was confirmed by physical examination, clinical history and routine laboratory tests. To detect the expression levels of miR-27a, serum samples (5 mL) were conventionally drawn from pre-therapeutic osteosarcoma patients and matched healthy individuals, respectively. Immediately after serum sample collection, the plasma and cellular elements were separated by centrifuging the blood at 1200 g for 15 min at 4 8C, and then stored in 1.5 mL RNase free tubes at 80 8C. All 166 osteosarcoma patients received follow-up. The median follow-up was 87 months (range: 10–152 months). During the follow-up period, 66 patients (66/166, 39.8%) died of disease. Distant metastases developed in 42 patients at a mean of 13.8 months (range 3–46 months) after the original diagnosis. Of these patients, 9 had bone metastases and 36 had lung metastases (3 patients had both bone and lung metastases). The median overall and disease-free survival of patients was 31 months (95% confidence interval [CI], 30.1–42.9 months) and 25 months (95%CI, 23.7–35.2 months), respectively. 2.2. RNA extraction and real-time quantitative RT-PCR assay The serum levels of miR-27a expression in osteosarcoma patients and healthy controls were detected by real-time Table 1 Association of serum miR-27a expression with clinicopathological features of osteosarcoma. Clinicopathological features
No. of cases
miR-27a expression High (n, %)
2. Materials and methods 2.1. Patients and tissue samples This study was approved by the Research Ethics Committee of Xuhui Central Hospital and Changzheng Hospital, China. Written informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards. A total of 166 patients with primary osteosarcoma, treated in the Department of Orthopaedics in Xuhui Central Hospital and Department of Joint Center in Changzheng Hospital, Shanghai, China, from 1998 to 2008, were enrolled retrospectively in this study. Patients were excluded if they had a previous or secondary malignancy. After establishing the diagnosis, all patients were treated with a preoperative chemotherapy lasting 4 months, using either the combination of an anthracycline (doxorubicin) and highdose methotrexate or the combination of etoposide, ifosfamide, and high-dose methotrexate. The postoperative treatment was determined by the histologic system established by Huvos et al. [23]. The following clinical parameters were analyzed: age, gender, site of tumor, tumor size, presence of pathological fracture and
Age 72 36 <55 55 94 50 Gender Male 96 51 Female 70 35 Tumor size (cm) >8 88 46 8 78 40 Anatomic location Tibia/femur 103 51 Elsewhere 63 35 Serum level of lactate dehydrogenase Elevated 90 46 Normal 76 40 Serum level of alkaline phosphatase Elevated 108 56 Normal 58 30 Clinical stage IIA 68 8 IIB/III 98 78 Distant metastasis Absent 124 50 Present 42 36 Response to chemotherapy Good 68 18 Poor 98 68
P
Low (n, %)
(50.00) (53.19)
36 (50.00) 44 (46.81)
NS
(53.13) (50.00)
45 (46.87) 35 (50.00)
NS
(52.27) (51.28)
42 (47.73) 38 (48.72)
NS
(49.51) (55.56)
52 (50.49) 28 (44.44)
NS
(51.11) (52.63)
44 (48.89) 36 (47.37)
NS
(51.85) (51.72)
52 (48.15) 28 (48.28)
NS
(11.76) (79.59)
60 (88.24) 20 (20.41)
0.001
(40.32) (85.71)
74 (59.68) 6 (14.29)
0.01
(26.47) (69.39)
50 (73.53) 30 (30.61)
0.008
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quantitative RT-PCR assay. Briefly, total RNA was extracted from 500 mL plasma of each sample using mirVana PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. RNA was dissolved in 100 mL of RNase free water. The purity and concentration of all RNA samples were evaluated by their absorbance ratio at 260/280 nm. A SYBR green qRT-PCR assay was used for miRNA quantification and Real-time PCR was performed on an ABI 7500 Real-Time PCR instrument (Applied Biosystems, Inc). For miR-27a analysis, the stem-loop RT primer was 50 -GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACG CGG AAC-30 and the amplifying primers were as follows: sense, 50 -TGC GGT TCA CAG TGG CTA AG-30 and antisense, 50 -CCA GTG CAG GGT CCG AGG T-30 . Primers for qRT-PCR of U6, an miRNA that was used as an internal control, were: sense, 50 -CTC GCT TCG GCA GCA CA-30 and antisense, 50 -AAC GCT TCA CGA ATT TGC GT-30 . Each sample was examined in triplicate and the relative expression DD level of miR-27a was normalized to that of U6 by the 2 Ct cycle threshold method [25]. 2.3. Statistical analysis The software of SPSS version 13.0 for Windows (SPSS Inc, IL, USA) and SAS 9.1 (SAS Institute, Cary, NC) was used for statistical analysis. Continuous variables were expressed as mean S.E. Mann–Whitney U test was used to compare the difference of serum miR-27a expression levels in osteosarcoma patients and healthy controls. The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnosis value of serum miR-27a level. The Chisquare test was used to show differences of categorical variables. Patient survival and their differences were determined by Kaplan– Meier method and log-rank test. Cox regression (Proportional hazard model) was adopted for multivariate analysis of prognostic factors. Differences were considered statistically significant when P was less than 0.05.
3. Results 3.1. Diagnostic value of serum miR-27a expression for osteosarcoma patients Compared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (osteosarcoma vs. controls: 3.87 1.01 vs. 2.09 0.46, P < 0.001, Figure 1A). As shown in Figure 1B, ROC curve analysis illustrated that the serum miR-27a level was a potential biomarker for screening
osteosarcoma patients from healthy controls with the area under the ROC curve (AUC) of 0.867, and the serum miR-27a level at 2.88 was the clear cutoff value to distinguish osteosarcoma patients from healthy controls. With this cutoff value, the sensitivity and specificity of the serum miR-27a level for osteosarcoma was 70.01% and 98.30%, respectively. 3.2. Elevated expression of miR-27a in sera associates with advanced clinicopathological features of osteosarcoma patients To investigate the associations of serum miR-27a expression with the clinicopathological features of osteosarcoma patients, we divided the patients into two groups using the median value of serum miR-27a expression levels in osteosarcoma patients as a cutoff point (3.70). Of 166 osteosarcoma specimens, 86 (51.81%) highly expressed miR-27a. As shown in Table 1, high miR-27a expression was more frequently occurred in osteosarcoma patients with advanced clinical stage (P = 0.001), positive distant metastasis (P = 0.01) and poor response to chemotherapy (P = 0.008). No significant differences were observed between serum miR-27a expression and patients’ age, gender, tumor size, anatomic location, serum levels of lactate dehydrogenase and alkaline phosphatase (all P > 0.05). 3.3. Elevated expression of miR-27a in sera associates with poor prognosis in osteosarcoma patients Using Kaplan–Meier method and log-rank test, the overall survival (Figure 2A, P < 0.001) and disease-free survival (Figure 2B, P < 0.001) of osteosarcoma patients with high miR-27a expression in sera were both significantly shorter than those with low miR27a expression. Besides, the survival benefits were also found in those with smaller tumor size (both P = 0.03), higher clinical stage (P = 0.01 and 0.006, respectively), without distant metastasis (P = 0.008 and 0.003, respectively), and better response to chemotherapy (both P = 0.02) for overall survival and disease-free survival. We further examined overall survival and disease-free survival using Cox regression hazard analyses to determine whether serum miR-27a expression could serve as a clinically useful prognostic assessment factor in osteosarcoma. Multivariate Cox regression analysis enrolling above-mentioned significant parameters revealed that serum miR-27a expression (RR 3.56, 95%CI, 1.87– 9.86, P = 0.01), clinical stage (RR 2.32, 95%CI, 1.10–7.01, P = 0.02), distant metastasis status (RR 3.72, 95%CI, 1.91–9.83, P = 0.01), and response to chemotherapy (RR 2.60, 95%CI, 1.52–8.21, P = 0.02)
Figure 1. Diagnostic value of serum miR-27a expression for osteosarcoma patients. (A) Serum miR-27a expression in 166 osteosarcoma patients and healthy controls were respectively detected by real-time quantitative RT-PCR assay. Compared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (osteosarcoma vs. controls: 3.87 1.01 vs. 2.09 0.46, P < 0.001). (B) ROC curve analysis illustrated that serum miR-27a expression was a potential biomarker for screening osteosarcoma patients from healthy controls (AUC = 0.867).
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Figure 2. Overall survival (A) and disease-free survival (B) curves for two groups defined by low and high expression of miR-27a in sera from osteosarcoma patients. The patients with high miR-27a expression had a significantly shorter 5-year overall and disease-free survival rate than those with low miR-27a expression (both P < 0.001).
were independent prognostic markers for overall survival of osteosarcoma patients (Table 2). Turning to disease-free survival, serum miR-27a expression (RR 3.19, 95%CI, 1.72–8.33, P = 0.01), clinical stage (RR 1.83, 95%CI, 0.71–8.12, P = 0.03) and metastasis status (RR 2.92, 95%CI, 1.32–10.62, P = 0.02) were also independent prognostic markers for disease-free survival of osteosarcoma patients (Table 2). 4. Discussion Aberrantly expressed circulating miRNAs have received a great deal of attention as potential sensitive and accurate biomarkers for cancer diagnosis and prognosis due to the following points: (1) miRNAs have high tissue- and tumor-specificity; (2) miRNAs have the inherent stability in patients’ sera and tissues; (3) Sampling is very easy by minimally invasive approaches; (4) Methods of isolation and detection are relatively cheap; (5) The aberrant expression of miRNAs has been reported to be associated with the development and progression of human malignancies [26]. In the current study, we detected the serum levels of miR-27a expression in osteosarcoma patients and healthy controls, and confirmed its elevated expression in sera of osteosarcoma patients. More importantly, the diagnostic value of serum miR-27a expression was also evaluated. Our data showed that miR-27a could efficiently screen osteosarcoma patients from healthy controls (AUC = 0.867). The AUC value is an indicator of the efficacy of the assessment system. An ideal test with perfect discrimination (100% sensitivity and 100% specificity) has an AUC of 1.0, whereas a noninformative prediction has the area 0.5, indicating that it may be achieved by mere guess. The closer to 1.0 the AUC of a test is, the higher the overall efficacy of the test will be [27]. Here, miR-27a could screen osteosarcoma patients from healthy controls with an
AUC value (0.867) approximating 1.0, suggesting that it had a relatively high ability to identify the true osteosarcoma patients. In addition, we focused on the potential relationship between the serum level of miR-27a and various clinicopathological characteristics of osteosarcoma patients, as well as disease-free survival and overall survival. It is worth noting that high levels of miR-27a appear to be significantly correlated with aggressive tumor progression and poor prognosis in patients with osteosarcoma. To the best of our knowledge, the current study represents the first demonstration that serum miR-27a expression may function as a diagnostic and prognostic marker for osteosarcoma patients. Similar with other members of the miR-23a/24-2/27a cluster, miR-27a has been reported to have both oncogenic and tumor suppressive functions in different cell lines and human cancer tissues. Tian et al. [28] indicated that miR-27a might act as an oncogene in laryngeal squamous cell carcinoma through suppressing the expression of PLK2 and serve as a useful biomarker in diagnosis and therapy in this malignancy; Li et al. [29] observed the upregulation of miR-27a in tumor tissues sampled from lung adenocarcinoma patients treated with cisplatin-based chemotherapy and proved that it might be correlated with low expression of RKIP, decreased sensitivity to cisplatin, and poor prognosis; Xu et al. [30] revealed that oncogenic miR-27a could play an important role in ovarian cancer cell growth and metastasis; Zhao et al. [17] confirmed that down-regulation of miR-27a could inhibit proliferation of gastric cancer cells in vitro and in vivo; Ma et al. [18] reported that miR-27a might play an oncogenic role by targeting Spry2 and modulating the malignant, biological behavior of pancreatic cancer cells; In breast cancer cells, miR-27a has been reported to directly suppress ZBTB10/RINZF expression and in turn upregulate VEGF and VEGF receptor, and overexpression of miR27a might be associated with poor outcomes of the patients
Table 2 Multivariate survival analysis of overall survival and disease-free survival in 166 osteosarcoma patients. Variables
Serum miR-27a expression Clinical stage Distant metastasis status Response to chemotherapy
Overall survival
Disease-free survival
RR
95%CI
P
RR
95%CI
P
3.56 2.32 3.72 2.60
1.87–9.86 1.10–7.01 1.91–9.83 1.52–8.21
0.01 0.02 0.01 0.02
3.19 1.83 2.92 0.93
1.72–8.33 0.71–8.12 1.32–10.62 0.30–1.71
0.01 0.03 0.02 NS
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[16]. Whereas, Bao et al. [19] identified the tumor suppressive role of miR-27a in colorectal carcinogenesis and progression by targeting SGPP1 and Smad2; In lung cancer, miR-27a was downregulated, directly targeted MET and EGFR, and suppressed their expression [20]. In the current study, we observed the elevated serum levels of miR-27a expression in osteosarcoma patients and confirmed its significant associations with aggressive clinicopathological features including advanced clinical stage, positive distant metastasis and poor response to chemotherapy, suggesting its oncogenic role in this malignancy. These findings were consistent with the previous study of Pan et al. [22], who revealed for the first time that miR-27a could function as an oncogene by targeting MAP2K4 in the osteosarcoma MG63 cell line. Although we here highlight the significant dysregulation and potentially novel diagnostic value for serum miR-27a expression in osteosarcoma patients, the detailed mechanisms underlying its oncogenic role in this disease remain to be further elucidated. Since the prediction of the clinical outcomes of osteosarcoma patients is an important prerequisite for better treatment stratification and personalized therapeutic regimens, our univariate analysis in the current study found that osteosarcoma patients with high miR-27a expression had both worse overall and diseasefree survival than those with low miR-27a expression significantly. Multivariate analysis further demonstrated that among all the factors analyzed, clinical stage, distant metastasis and serum miR27a expression were all independent prognostic factors for both overall and disease-free survival. These findings confirmed the prognostic value of serum miR-27a expression in osteosarcoma patients, implying that serum miR-27a expression could be used as a sufficient marker for further treatment stratification in the personalized therapeutic strategies in the future. In conclusion, our results imply that miR-27a expression may be elevated in sera of osteosarcoma patients and in turn contributes to aggressive progression of this malignancy. Detection of serum miR-27a levels may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients. Authors’ contributions WH conceived and designed the study, and approved the final version of the manuscript; TJ performed the experiments and wrote the manuscript; ZH and CH collected and analyzed the data. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer 2009;115:1531–43. [2] Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res 2009;152:3–13. [3] Bruland S, Bauer H, Alvegaard T, Smeland S. Treatment of osteosarcoma. The Scandinavian Sarcoma Group experience. Cancer Treat Res 2009;152:309–18.
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