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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 405-409

Role of taurine upregulated gene 1 as a predictor of poor outcome in osteosarcoma


Department of Orthopedics, No. 187 Hospital of the People's Liberation Army, Haikou, Hainan 571159 Province, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Qi Chen
Department of Orthopedics, No. 187 Hospital of the People's Liberation Army, Haikou, Hainan 571159 Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.172585

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 > Abstract 

Aims: Previous studies have found that long noncoding RNA taurine upregulated gene 1 (TUG1) can regulate osteosarcoma cells apoptosis and proliferation; the aim of this study was to investigate the clinical significance of TUG1 in osteosarcoma.
Subjects and Methods: The expression of TUG1 was detected by real-time and quantitative polymerase chain reaction assay in 94 pairs of tumor tissues and corresponding noncancerous bone tissues of osteosarcoma patients. Its correlations with clinicopathologic features were analyzed, and the significance of TUG1 as a prognostic factor was determined.
Results: This study shows that the expression of TUG1 in osteosarcoma tissues was significantly higher than that in adjacent normal bone tissues. Upregulation of TUG1 was significantly correlated with the larger tumor size and advanced tumor-node-metastases stage of osteosarcoma patients. Kaplan–Meier curve showed a decreased overall survival time of osteosarcoma patients with high TUG1 expression. Moreover, univariate and multivariate analyses suggested that low-expression level of TUG1 was an independent poor prognostic indicator for osteosarcoma patients.
Conclusions: In conclusion, our data support TUG1 as a potential prognostic predictor and gene therapy target with its high expression in tumor tissues and its association with carcinogenesis and progression in osteosarcoma.

Keywords: Long non-coding RNA, osteosarcoma, real-time and quantitative polymerase chain reaction, survival, taurine upregulated gene 1


How to cite this article:
Wang Q, Chen Q. Role of taurine upregulated gene 1 as a predictor of poor outcome in osteosarcoma. J Can Res Ther 2018;14, Suppl S2:405-9

How to cite this URL:
Wang Q, Chen Q. Role of taurine upregulated gene 1 as a predictor of poor outcome in osteosarcoma. J Can Res Ther [serial online] 2018 [cited 2019 Jul 20];14:405-9. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/405/172585


 > Introduction Top


Osteosarcoma is the most primary malignant tumor of bone mainly arising from the metaphysis of the long bones of adolescents.[1] Due to the advances in therapeutic strategies including radical surgery excision and adjuvant polychemotherapy, the 5-year survival rate of osteosarcomas patients has been remarkably improved.[2],[3] However, most of the patients still have a risk of local relapse or distant metastasis and the outcome remains dissatisfactory.[3] Therefore, understanding the molecular mechanisms involved in osteosarcoma carcinogenesis as well as identification of effective prognostic and therapeutic markers are of great clinical significance.

Long non-coding RNAs (lncRNAs) are a family of endogenous noncoding RNA molecules great than 200 nucleotides in length.[4] LncRNAs are widely expressed in eukaryotic cells and play essential roles in the regulation of target gene at epigenetic, transcriptional, and posttranscriptional level.[5] It has been pointed out that aberrant lncRNAs expression are associated with the initiation and development of human tumor entities, and several lncRNAs that can function either as oncogene or tumor suppressor have been identified.[6] Recently, some studies have demonstrated that abnormal expression of various lncRNAs, such as H19,[7] loc285194,[8] and MALAT1,[9] play important roles in osteosarcoma carcinogenesis and progression.

LncRNA taurine upregulated gene 1 (TUG1) is located on chromosome 22q12.2, a region frequently altered in lung cancer,[10] chondrosarcoma,[11] astrocytomas,[12] and hepatocellular carcinoma.[13] Previous studies identified TUG1 as a functional lncRNA – depletion of TUG1 suppresses mouse retinal development.[14] Recently, TUG1 was found downregulated in human malignancies including osteosarcoma, and down-regulation of TUG1 inhibits osteosarcoma cell proliferation and promotes apoptosis.[15] However, little is known about the association between TUG1 expression levels and clinicopathological parameters and prognosis in osteosarcoma. Therefore, the aim of this study was to investigate the clinical significance of TUG1 in patients with osteosarcoma.


 > Subjects and Methods Top


Patients and tissue samples

Ninety-four pairs of osteosarcoma tumor tissues and paired normal adjacent bone tissues from patients underwent resection of primary osteosarcoma at our hospital were collected between 2008 and 2010. The approval for the study was obtained from the Ethics Committee of our hospital. Written informed consent was obtained from all participants. All tissue samples were immediately snap frozen in liquid nitrogen after surgery and stored at −80°C. All patients' slides were reviewed to confirm the diagnosis and to classify the tumor according to the sixth edition of the tumor node metastases (TNM) classification of the International Union against Cancer. The clinicopathologic features of the patients with osteosarcoma are shown in [Table 1]. Patients with evidence of other diseases were excluded from this study. All patients completed the standard therapeutic regimen including neoadjuvant chemotherapy and surgical resection with wide or radical margin followed by adjuvant chemotherapy.
Table 1: The relationship between TUG1 expression and clinicopathologic parameters

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Cell culture and transfection

Human osteosarcoma cell lines U2OS, MG-63, and SAOS-2, and human normal bone cell line hFOB were purchased from cell bank of Chinese Academy of Sciences. Cells were cultured in Dulbecco's modified Eagle medium/high glucose supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA) maintain at 37°C in a humidified atmosphere with 5% CO2.

Real-time and quantitative polymerase chain reaction for taurine upregulated gene 1

TUG1 expression in tissues and cells was performed by quantitative real-time polymerase chain reaction (RT-qPCR). Briefly, total RNAs were isolated from the tissues and cells by TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. RT-qPCR) kits were used to evaluate the TUG1 expression from tissue samples. QPCR was performed using the SYBR PrimeScript RT-PCR kit (Takara, Ohtsu, Japan) in an Applied Biosystems 7900 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA). Primers for TUG1: Forward 5'-CTGAAGAAAGGCAA CATC-3'; reverse 5'-GTAGGCTACTACAGGATTTG-3'; and β-actin: Forward 5'-CCACTGGCATCGTGA TGGA-3'; reverse 5'-CGCTCGGTGAGGATCTTCAT-3'. Each sample was examined in triplicate, and the mean values were calculated. Relative gene expression was calculated using the comparative cycle threshold (2ΔΔCt) method, with β-actin served as an endogenous control for normalization.

Statistical analysis

All statistical analyses were performed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). The significance of between the group differences was estimated using Student's t-test, one-way ANOVA, and χ2 test as appropriate. Overall survival (OS) was calculated as the time interval from the date of surgery to either the day of the last follow-up or cancer-related death. OS rates were calculated using the Kaplan–Meier method, with the log-rank test, applied for comparison. Variables with a value of P < 0.05 in univariate analysis were used in a subsequent multivariate analysis, based on the Cox proportional hazards model. Two-sided P values were calculated, and P < 0.05 indicated statistical significance.


 > Results Top


Taurine upregulated gene 1 show high expression in osteosarcoma

Using RNA isolated from tissues, we performed qRT-PCR to detect the expression levels of TUG1 in osteosarcoma. After normalizing our results to β-actin expression, TUG1 expression was found significantly higher in tumor tissues compared with adjacent normal bone tissues [P = 0.013, [Figure 1]a. The mean level of TUG1 expression in osteosarcoma tissues was 3.091 ± 2.611, significantly higher than that in normal adjacent bone tissues (2.449 ± 1.360). In addition, TUG1 expression was significantly increased in three osteosarcoma cell lines (MG-63, U2OS, and SAOS-2), as compared with hFOB cell line [Figure 1]b, all P < 0.05].
Figure 1: Relative NEAT1 expression in osteosarcoma. (a) Quantitative polymerase chain reaction (qPCR) analysis was performed to detect the relative taurine upregulated gene 1 expression in 94 pairs of osteosarcoma and corresponding noncancerous bone tissues. (b) Comparison of taurine upregulated gene 1 expression levels between three osteosarcoma cell lines and normal hFOB cells by qPCR analysis (*P < 0.05). β-actin was used as an internal control

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Correlations between taurine upregulated gene 1 expression and clinicopathologic factors of osteosarcoma

To assess the correlation of TUG1 expression with clinicopathologic data, all cases were categorized as low or high in relation to the median value. Chi-square tests results showed that high TUG1 expression in osteosarcoma was significantly correlated with larger tumor size (P = 0.037) and advanced TNM stage (P = 0.025). However, no correlation was observed between TUG1 expression and other parameters such as age (P = 0.345), gender (P = 0.360), location (P = 0.259), pathological type (P = 0.636), or metastasis (P = 0.289) in osteosarcoma [Table 1].

Correlation between taurine upregulated gene 1 expression and osteosarcoma patient prognosis

We then investigate the prognostic value of TUG1 expression in osteosarcoma patients. OS curves were plotted according to TUG1 expression levels using the Kaplan–Meier method and log-rank test. During follow-up, 58 patients died of OS. As presented in [Figure 2], osteosarcoma patients with high TUG1 expression are correlated with shorter OS (P = 0.016). Univariate analysis of OS revealed that the relative level of TUG1 expression (P = 0.016), tumor size (P = 0.046), metastasis (P = 0.027), and clinical stage (P = 0.000) were prognostic indicators [Table 2]. Furthermore, multivariate analysis revealed that in addition to the tumor stage (P = 0.030), TUG1 expression (P = 0.035) was an independent prognostic indicator for OS in osteosarcoma patients [Table 2].
Figure 2: Kaplan–Meier survival curve of overall survival according to taurine upregulated gene 1 level. Kaplan–Meier survival curve and log-rank test showing osteosarcoma patients with high taurine upregulated gene 1 expression had a significantly worse prognosis than those with low taurine upregulated gene 1 expression (P = 0.016)

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Table 2: Univariate and multivariate analysis of different prognostic factors for overall survival in 94 patients with osteosarcoma

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 > Discussion Top


Osteosarcoma is the most common type of primary malignancy deriving from primitive bone-forming mesenchyme, which mainly arising from the metaphysis of the long bones of adolescents and young adults.[1] As the eighth leading cancer, osteosarcoma shows an incidence of 4.4/million, with the 5-year survival rate about 60–70% after surgery.[2],[3] Although great improvements have been brought in adjuvant treatments including radiotherapy and chemotherapy, local relapse, and distant metastases are still common in a significant proportion of osteosarcoma patients, the survival time for osteosarcoma remains dismal.[3] Hence, more efforts are needed to identify novel biomarkers for risk prognostication and therapy.

In the recent years, cumulative studies reveal that besides of small noncoding RNA such as miRNAs and piRNA, lncRNAs can also function as the kinds of crucial regulators in tumor paradigm, which have dramatically altered our understanding of the physiology and biology of human tumor entities.[4],[5],[6] A large number of studies have shown that dysregulation of lncRNAs participates in the carcinogenesis of osteosarcoma.[7],[8],[9] Here, we focused on lncRNA-TUG1, which was previously reported to upregulate in varies of malignant tumors, such as bladder cancer,[16] nonsmall cell lung cancer,[17] hepatocellular carcinoma,[18] and esophageal squamous cell carcinoma.[19] In the present study, by assessing the expression level of TUG1 in 94 osteosarcoma patients using qRT-PCR, we identified that TUG1 was significantly increased in osteosarcoma tissues compared with adjacent normal bone tissues, which was consistent with Zhang's finding.[15] Meanwhile, its remarkable higher expression was also presented in three osteosarcoma cell lines when compared with that in normal bone cell line.

Then, we analyzed the relationship between TUG1 expression and clinicopathologic features of osteosarcoma patients. Results showed that the expression of TUG1 was closely associated with tumor size and TNM stage of osteosarcoma. As is known, dysregulated genes may be an indicator of the intrinsic characteristics of cancer progression, and we found that patients with high TUG1 expression had a shorter OS rate than those in the low TUG1 group. By further univariate and multivariate Cox regression analyses, the expression of TUG1 was demonstrated to be an independent prognosis predictor for osteosarcoma patients. Taken together, these results suggested that TUG1 may potentially a useful biomarker for prognostication, which would be not only helpful for risk stratification but also offer patients more optimized therapeutic schedule.

It has been shown that downregulation of TUG1 inhibits osteosarcoma cell proliferation and promotes apoptosis.[15] Previous studies also have shown that several lncRNAs, such as HOTAIR, exhibit modulating function via binding to polycomb repressive complex 2 (PRC2),[20] or miRNAs.[21],[22] TUG1, which is induced by p53, can also bind to PRC2 and miRNAs and then play pivotal suppressor role in cancer cell growth.[23],[24],[25] It was also reported that TUG1 promotes cell growth and apoptosis by epigenetically silencing of KLF2;[18] by form a double-negative feedback loop with miR-145, TUG1 promotes epithelial to mesenchymal transition and radioresistance in human bladder cancer cells.[26] However, the molecular mechanisms underlying how upregulation of TUG1 influenced the development and progression of osteosarcoma have not been fully stated, further studies are required to solve this problem.

Owing to its retrospective nature, this study has some limitations. A wide range of prospective, randomized, and controlled studies are needed to identify the predictors of TUG1 in osteosarcoma patients. A certain follow-up duration is required to validate important evaluation indexes. Thus, our results should be validated in a large-scale and long-term study.


 > Conclusions Top


We demonstrated that TUG1 expression was significantly upregulated in osteosarcoma tissues and cell lines compared to their normal counterparts. In addition, TUG1 expression levels decrease with larger tumor size and advanced TNM stage. Kaplan–Meier analysis indicated that osteosarcoma patients with higher TUG1 expression levels had worse OS rates. Moreover, high expression of TUG1 is an independent predictor of OS in osteosarcoma patients. Together, these results suggested that TUG1 might represent a novel prognostic indicator and a target for gene therapy in osteosarcoma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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