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Down-regulation of miR-133a and miR-539 are associated with unfavorable prognosis in patients suffering from osteosarcoma
Article in Cancer Cell International · September 2015
DOI: 10.1186/s12935-015-0237-6
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PRIMARY RESEARCH
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*
Abstract
Background: MicroRNAs (miRNAs) play key roles in cancer development and progression. The purpose of the pre- sent 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 osteo- sarcoma 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 propor- tional 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 progres- sion of osteosarcoma. Together, these results showed that miR-133a and miR-539 may have their role in both progres- sion and prognosis of osteosarcoma.
Keywords: MicRNA, Survival, Patient, Marker, Cancer
© 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.
Background
One of the most common primary bone tumors in chil- dren and adolescents is osteosarcoma, which is most often localized in the metaphysis of the adolescent long
bones [1]. More than 50 % of patients who are chem- oresistant 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 strat- egies, there is dissatisfactory for most osteosarcoma patients with metastasis or recurrence. Therefore, it is required to identify biomarkers, and therapeutic targets for osteosarcoma.
Open Access
*Correspondence: [email protected]
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
Page 2 of 5 Mirghasemi et al. Cancer Cell Int (2015) 15:86
MicroRNAs (miRNAs) are endogenous 19e25 nt non- coding RNAs that can bind the 3′-untranslated region (3′-UTR) of specific genes to inhibit the translation of corresponding mRNA targets. Increasing evidence dem- onstrates that the deregulations of miRNAs are involved in various biological processes including proliferation, differentiation, and apoptosis [4]. Current evidences indi- cate 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 suppress- ing 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 osteogen- esis. 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 carcino- genesis and progression. Therefore, we examined the clin- ical importance of miR-133a and miR-539 expressions in osteosarcoma tissue samples using real-time PCR.
Methods Patients
The samples of cancer tissue and corresponding non- cancerous 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 tis- sues 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 his- tological grading were proved by pathologists. The clin- icopathological features are summarized in Tables 1 and 2.
Real‑time quantitative PCR
Total RNA was extracted using miRNeasy kit (Qia- gen) according to the manufacturer’s instructions. The expression levels of miRNAs quantitated using the TaqMan miRNA assey kit (Applied Biosystems). Real- timePCR was performed using Rotor Gene 6000 Real- Time PCR (Qiagen, Germany) with an invitrogen kit and a TaqMan universal PCR master mix. The relative expression levels of miRNAs were normalized to that of internal control U6 by using 2−Ct cycle threshold method [13].
Statistical analysis
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 osteo- sarcoma. 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 expres- sion level ± SD: 3.74 ± 1.83) compared to adjacent nor- mal 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 expres- sion level ± SD: 2.23 ± 1.02) as compared to that adja- cent normal tissues (mean ± SD: 5.68 ± 1.42, P < 0.0001;
Fig. 2).
Correlation of miRNAs expressions with the clinicopathological fetures
The patients with osteosarcoma were classified into two groups based on the median expression levels.
Low expression of miR-133a showed significant asso- ciation with advanced TNM stage (P = 0.002), and metastasis or recurrence (P = 0.001). But no significant association with other clinical factors (Table 1). On the other hand, low expression of miR-539 were significantly correlated with advanced TNM stage (P = 0.001), and metastasis or recurrence (P = 0.01). But no significant association with other clinical factors (Table 2). Further- more, 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 (log-rank test P < 0.001; Figs. 3, 4). 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 (Tables 3, 4).
Discussion
Various deregulated miRNAs, have been reported in osteosarcoma development [14]. Nevertheless, it is still an ongoing process to elucidate new important deregu- lated miRNAs and their roles in cancer carcinogenesis
and progression. In the present study, we found that the miR-133a was significantly decreased in clinical osteosar- coma tissues compared to adjacent normal bone. More- over, low expression of miR-133a showed significant Table 1 The association between miR-133a expression
and characteristics of patients suffering from osteosar- coma
Clinicopathological
features No. of
cases Expression
of miR‑133a P value of miR‑133a Low, 19 High, 16
Gender
Male 18 6 12 NS
Female 17 13 4
Age
≤40 20 9 11 NS
>40 15 10 5
Tumor diameter (cm)
≤5 21 10 11 NS
>5 14 9 5
Differentiation
Well and moderate 16 7 9 NS
Poor 19 12 7
TNM stage
I + II 15 5 10 0.002
III + IV 20 14 6
Metastasis or recurrence
Yes 19 16 3 0.001
No 16 3 13
Table 2 The association between miR-539 expression and characteristics of patients suffering from osteosar- coma
Clinicopathological
features No. of
cases, 35 Expression of
miR‑539 P value
Low, 21 High, 14 Gender
Male 15 9 6 NS
Female 20 12 8
Age
≤40 19 10 9 NS
>40 16 11 5
Tumor diameter (cm)
≤5 21 11 10 NS
>5 14 10 4
Differentiation
Well and moderate 18 9 9 NS
Poor 17 12 5
TNM stage
I + II 12 5 7 0.001
III + IV 23 16 7
Metastasis or recurrence
Yes 22 16 6 0.01
No 13 5 8
Fig. 1 The relative expression level of miR-133a between osteosar- coma bone tissue and noncancerous bone tissues
Fig. 2 The relative expression level of miR-539 between osteosar- coma bone tissue and noncancerous bone tissues
Fig. 3 Correlation between miR-133a expression and survival in patients
Page 4 of 5 Mirghasemi et al. Cancer Cell Int (2015) 15:86
association with advanced TNM stage, and metastasis or recurrence. Kaplan–Meier survival analysis and log-rank test showed that the low expression of miR-133a was cor- related with the reduced overall survival of osteosarcoma patients. Multivariate Cox proportional hazards model showed that decreased expressions of miR-133a, TNM stage, and metastasis were independent prognostic mark- ers of overall survival of patients. Recently, miR-133a is
also reported to be an important regulator in osteogen- esis, and miR-133a expression was proved to play an important role during osteoblast differentiation, by the finding that BMP2 treatment could decrease the expres- sion of miR-133a during osteoblast lineage commitment and osteogenesis [12]. Ji et al. [15] reported that miR- 133a expression was significantly suppressed in tumor tissues as compared to that in adjacent normal tissues.
They found that miR-133a expression in tumor tissues was significantly reverse-correlated with osteosarcoma clinical tumor and Kaplan–Meier survival analysis also revealed that low miR-133a expression in tumor tissues was significantly correlated with the reduced overall sur- vival of osteosarcoma patients.
They indicated that miR-133a might function as a tumor suppressor or an antionco-miRNA in cancer car- cinogenesis and progression. Previous reports revealed the roles of miR-133a in some other kinds of cancer, such as prostate cancer, bladder cancer, and esophagus cancer [16–18]. Among them, miR-133a expression is down- regulated in all these types of cancer, but the underlying mechanisms which mediate the down-regulation of miR- 133a in cancer required to further investigations.
Previous studies reported that the expression of Bcl-xL, is also associated with overall survival of osteosarcoma patients [19]. These results indicated that miR-133a may be effective to predict the prognosis of osteosarcoma patients. Human important anti-apoptotic molecules Bcl-xL and Mcl-1 are determined to be new direct tar- gets of miR-133a in osteosarcoma, indicating that miR- 133a may exert its pro-apoptotic function throughout inhibiting Bcl-xL and Mcl-1 expression. Human EGFR, TAGLN2, and FSCN1 reported to be molecular targets of miR-133a in other kinds of cancers [16–18]. The recent evidence indicated that cancer pathways may be regu- lated by miR-133a expression, and miR-133a may be a new therapeutic target to repress cancer progression.
In the present study, the expression level of miR-539 was decreased in clinical osteosarcoma tissues as com- pared to those adjacent normal tissues. Furthermore, low expression of miR-539 was significantly correlated with advanced TNM stage, and metastasis or recurrence.
Kaplan–Meier survival analysis and log-rank test showed that the low expression of miR-539 was correlated with the reduced overall survival of osteosarcoma patients.
Multivariate Cox proportional hazards model showed that decreased expressions of miR-539, TNM stage, and metastasis or recurrence were independent prognostic markers of overall survival of patients. Recently, it has been reported that miR-539 may inhibit cell proliferation through suppressing the MITF expression [9]. Moreover, miR-539 were confirmed to be down-regulated in osteo- sarcoma cell lines [10, 11].
Fig. 4 Correlation between miR-539 expression and survival in patients
Table 3 Multivariate analysis of the correlation of progno- sis miR-133a with clinicopathological factors
Clinicopathological characteristics HR 95 % CI P value
Gender 1.264 0.624–1.612 0.521
Age 1.365 0.725–1.937 0.4
TNM stage 3.618 1.319–10.261 0.001
Tumor diameter (cm) 0.89 0.615–1.523 0.2
Metastasis or recurrence 3.28 3.278–9.972 0.005
Differentiation 1.015 0.693–1.371 0.31
miR-133a expression 3.421 1.326–9.371 0.007
Table 4 Multivariate analysis of the correlation of progno- sis miR-539 with clinicopathological factors
Clinicopathological characteristics HR 95 % CI P value
Gender 1.03 0.532–1.762 0.541
Age 0.73 0.382–1.108 0.527
TNM stage 4.96 2.462–11.72 0.002
Tumor diameter (cm) 1.21 0.73–2.03 0.374
Metastasis or recurrence 3.77 1.538–8.725 0.026
Differentiation 1.316 1.035–2.441 0.3
miR-539 expression 3.68 2.153–10.227 0.02
There are few studies that have described the function of miR-539. One study indicated that miR-539 is among the factors sensing cellular biotin levels and targets holocarboxylase synthetase (HCS) [12]. Another study, showed that miR-539 suppress O-GlcNAcase expres- sion and play an important role in the failing heart [20].
Moreover, it has been reported that miR-539 participates in the regulation of mitochondrial activity and apoptosis through targeting PHB2 [21].
The expression of miR-539 was previously analyzed in human thyroid cancer cell lines and in normal thy- roids (NT). It has revealed that miR-539 expression was decreased compared to normal control [22]. Further- more, it has been found that miR-539 plays a suppres- sor role in thyroid cancer cell migration and invasion and miR-539 binding to the 3′-UTR region of CARMA1 inhibited the expression of CARMA1 in thyroid cancer cells. Mentioned study indicated that miR-539 is a novel regulator of migration and invasion in human thyroid cancer cells by targeting CARMA1 [22]. However, further studies are needed to verify the role miR-539 in develop- ing of different cancer types.
Conclusion
In conclusion, these results suggest that decreased miR- 133a and miR-539 expressions may participate in the pro- gression of osteosarcoma. Together, these results showed that miR-133a and miR-539 may have their role in both pro- gression and prognosis of osteosarcoma. However, further studies are required to identify the role of these miRNAs in progression and prognosis of osteosarcoma by their targets.
Authors’ contributions
AM, AT, SHK, AT, MSH, and EY participated in sample collection and processing, and participated in design of the study and coordination and helped to draft the manuscript and AM participated in writing. All authors read and approved the final manuscript.
Author details
1 Department of Orthopedics, Qom University of Medical Sciences, Qom, Iran. 2 Department of Orthopedics Surgery, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran. 3 Department of Anesthesiology, Birjand University of Medical Sciences, Birjand, Iran. 4 Department of Orthopedics, Firoozgar Hospital, Iran University of Medical Sciences, Tehran, Iran. 5 Depart- ment of Orthopaedic and Trauma Surgery, Birjand University of Medical Sci- ences, Birjand, Iran. 6 Department of Molecular Biology, Baqiyatallah University of Medical Sciences, Tehran, Iran. 7 Cellular & Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran.
Acknowledgements This work had no fund.
Compliance with ethical guidelines Competing interests
The authors declare that they have no competing interests.
Received: 23 July 2015 Accepted: 1 September 2015
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