Down-regulation of miR-133a and miR-539 are associated with unfavorable prognosis in patients suffering from osteosarcoma

Mirghasemi et al. Cancer Cell Int (2015) 15:86 DOI 10.1186/s12935-015-0237-6 Open Access PRIMARY RESEARCH AR TI CL E Down‑regulation of miR‑133a and miR‑539 are associated with unfavorable prognosis in patients suffering from osteosarcoma Alireza Mirghasemi1, Afshin Taheriazam2, Seyyed Hasan Karbasy3, Ali Torkaman4, Mohammadreza Shakeri5, Emad Yahaghi6 and Aram Mokarizadeh7* RE TR AC TE D Abstract Background: MicroRNAs (miRNAs) play key roles in cancer development and progression. The purpose of the present study was to determine the expression levels of miR-133a and miR-539 in osteosarcoma patients and to further investigate the clinicopathological, and prognostic value of these miRNAs. Methods: The expression levels of miR-133a and miR-539 were determined by qRT-PCR. Associations between miRNAs expressions and various clinicopathological characteristics were analyzed. Survival rate was determined with Kaplan–Meier and statistically analyzed with the log-rank method between groups. Survival data were evaluated through multivariate Cox regression analysis Results: Our findings revealed that the miR-133a expression was significantly decreased in clinical osteosarcoma tissues compared to adjacent normal bone tissues. The expression level of miR-539 was decreased in clinical osteosarcoma tissues as compared to those adjacent normal tissues. Low expressions of miR-133a and miR-539 were significantly association with advanced TNM stage (P = 0.002; P = 0.001), and metastasis or recurrence (P = 0.001; P = 0.01). Furthermore, Kaplan–Meier survival analysis and log-rank test showed that the low expressions of miR-133a and miR-539 were correlated with the reduced overall survival of osteosarcoma patients. Multivariate Cox proportional hazards model showed that decreased expressions of miR-133a and miR-539 (P = 0.007; P = 0.02), TNM stage (P = 0.001; P = 0.002), and metastasis or recurrence (P = 0.005; P = 0.026) were independent prognostic markers of overall survival of patients. Conclusion: These results suggest that decreased miR-133a and miR-539 expressions may participate in the progression of osteosarcoma. Together, these results showed that miR-133a and miR-539 may have their role in both progression and prognosis of osteosarcoma. Keywords: MicRNA, Survival, Patient, Marker, Cancer Background One of the most common primary bone tumors in children and adolescents is osteosarcoma, which is most often localized in the metaphysis of the adolescent long *Correspondence: 7 Cellular & Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran Full list of author information is available at the end of the article bones [1]. More than 50 % of patients who are chemoresistant have an extremely poor prognosis due to lung metastasis [2]. The 5-year overall and disease-free survival rates for osteosarcoma patients are around 50–60 % [3]. Despite the advances in therapeutic strategies, there is dissatisfactory for most osteosarcoma patients with metastasis or recurrence. Therefore, it is required to identify biomarkers, and therapeutic targets for osteosarcoma. © 2015 Mirghasemi et al. 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. Mirghasemi et al. Cancer Cell Int (2015) 15:86 Page 2 of 5 Statistical analysis AR TI CL E Our data were evaluated using SPSS 19.0 software (SPSS Inc., USA). The differences between two groups were analyzed using the Student’s t-test. The statistical analysis of cases in groups were performed using the Chi square test. Moreover, log-rank test and Kaplan–Meier method were used to evaluate survival rate in patients with osteosarcoma. Multivariate analysis was performed to evaluate prognostic values using Cox proportional hazards model. P < 0.05 was used to indicate a statistically significant difference. Results As demonstrated by quantitative real-time PCR, the miR-133a expression was significantly decreased in clinical osteosarcoma tissues (median relative expression level ± SD: 3.74 ± 1.83) compared to adjacent normal bone tissues (median relative expression level ± SD: 15.23 ± 3.25, P < 0.001; Fig. 1). The expression level of miR-539 was decreased in clinical osteosarcoma tissues (median relative expression level ± SD: 2.23 ± 1.02) as compared to that adjacent normal tissues (mean ± SD: 5.68 ± 1.42, P < 0.0001; Fig. 2). Methods Patients TE D MicroRNAs (miRNAs) are endogenous 19e25 nt noncoding RNAs that can bind the 3′-untranslated region (3′-UTR) of specific genes to inhibit the translation of corresponding mRNA targets. Increasing evidence demonstrates that the deregulations of miRNAs are involved in various biological processes including proliferation, differentiation, and apoptosis [4]. Current evidences indicate that miRNAs have their role in tumorigenesis and provide new insights into the molecular mechanisms underlying carcinogenesis. Different miRNAs have been shown to participate in the initiation and progression of osteosarcoma [5–8]. Recently, it has been reported that miR-539 may inhibit cell proliferation through suppressing the MITF expression [9]. MiR-539 were confirmed to be down-regulated in osteosarcoma cell lines [10, 11]. MiR-133a is also shown to be an important regulator in osteogenesis, and its down-regulation has been reported in bone morphogenetic protein (BMP)-induced osteogenesis. Moreover, it can function as suppressor of RunX2 expression for inhibition of osteoblast differentiation [12]. Further investigations are required to identify miR-133a and miR-539 expression levels in clinical osteosarcoma patients and its potential roles in osteosarcoma carcinogenesis and progression. Therefore, we examined the clinical importance of miR-133a and miR-539 expressions in osteosarcoma tissue samples using real-time PCR. RE TR AC The samples of cancer tissue and corresponding noncancerous bone tissues were collected between 2010 and 2014 from 35 patients who were undergoing surgery in different hospitals of Tehran, Iran. None of the patients received radiotherapy and chemotherapy before the tissues were collected. Informed consents were obtained from all the patients. All specimens were obtained during surgery, frozen immediately in liquid nitrogen, and were stored at -80 °C. Furthermore, the diagnosis and the histological grading were proved by pathologists. The clinicopathological features are summarized in Tables 1 and 2. Real‑time quantitative PCR Total RNA was ex (...truncated)