Serum miR-9 as a prognostic biomarker in patients with osteosarcoma

Abstract

Objectives

To quantify microRNA-9 (miR-9) concentrations in the serum of patients with osteosarcoma; to explore its relationship with clinicopathological characteristics and prognosis of osteosarcoma.

Method

Serum miR-9 was quantified via real-time reverse transcription–polymerase chain reaction in patients with osteosarcoma and healthy control subjects. Overall survival was evaluated using the Kaplan–Meier method.

Results

Serum miR-9 was significantly upregulated in patients with osteosarcoma (n = 118) compared with healthy control subjects (n = 60); its upregulation was significantly associated with advanced tumour–node–metastasis stage, larger tumour size and presence of distant metastasis. Overall survival duration was significantly shorter in patients with relatively high miR-9 concentrations compared with those with relatively low miR-9 concentrations.

Conclusions

Serum miR-9 concentrations are significantly increased in patients with osteosarcoma compared with healthy controls. Upregulation of miR-9 is associated with tumour stage, size and metastasis. Serum miR-9 quantification may represent a useful diagnostic and prognostic marker of osteosarcoma.

Introduction

Osteosarcoma is the most common primary malignancy in children and young adolescents, accounting for ∼60% of malignant bone tumours in the first two decades of life. ¹ Despite of treatment combining chemotherapy, surgery and/or radiotherapy, the prognosis of patients with recurrent or metastatic osteosarcoma remains poor, with a 5-year survival rate of 50–60%.^2,3 Developments in molecular biology have provided insight into the pathogenesis of osteosarcoma, but the fundamental molecular mechanisms underlying the histological heterogeneity, drug resistance and development of metastasis remain largely unclear. Thus, the identification of novel drug targets is very important for improving the clinical outcome of these patients.

Micro RNAs (miRNAs) are short, highly conserved, small (22–25 nucleotides), noncoding RNA molecules that regulate gene expression via repression of translation and cleavage of mRNA by base pairing to the 3′ untranslated region of target genes.^4–6 Abnormal miRNA expression is found in various human tumours including osteosarcoma, ⁷ and is associated with proliferation, differentiation, apoptosis and invasion of tumour cells.^8–10 MiRNAs are present in a stable form in human serum and plasma, suggesting the potential use of circulating miRNAs as stable blood-based markers for the detection of cancer or other human diseases.^11–13

MicroRNA-9 is upregulated in many tumour types including colorectal, ¹⁴ breast ¹⁵ and non-small cell lung cancer, ¹⁶ but its role in the progression of osteosarcoma remains unknown. The aim of this study, therefore, was to analyse the association between serum miR-9 concentrations and clinicopathological features and prognosis, in patients with osteosarcoma.

Patients and methods

Study population

The study retrospectively enrolled patients with primary osteosarcoma who were surgically treated in the Department of Orthopaedics, China–Japan Union Hospital of Jilin University, Changchun, China, between January 2007 and September 2012. Tumours were classified according to the 6th edition of the tumour–node–metastasis (TNM) classification of the International Union Against Cancer (UICC). ¹⁷ Clinicopathological features were recorded at the time of serum sampling. Inclusion criteria were no metastasis at presentation and no history of previous treatment. Patients were excluded if they had a previous or secondary malignancy.

Healthy control subjects were recruited from individuals with no previous cancer diagnosis who were undergoing routine health checks at the Physical Health Examination Centre, China–Japan Union Hospital of Jilin University.

The study was approved by the Research Ethics Committee of China–Japan Union Hospital of Jilin University and all participants provided written informed consent prior to enrolment.

Quantitative RT–PCR

Peripheral venous blood (10 ml) was taken from all study participants using standard methods, after an overnight fast. Blood was kept at room temperature for 60 min then centrifuged at 1000  g for 10 min at 4℃. Serum was immediately separated, frozen and stored at −80℃ until analysis.

Total RNA was extracted from 400 µl serum (miRNeasy Mini Kit; QIAGEN, Hilden, Germany) according to the manufacturer’s instructions, and miR-9 was quantified by real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) using the TaqMan® miRNA assay system (Applied Biosystems, Foster City, CA, USA). RNA was reverse transcribed using the Universal cDNA synthesis kit (Exiqon; Woburn, MA, USA) according to the manufacturer’s instructions. Primer sequences were 5′-UCUUUGGUUAUCUAGCUGUAUGA-3′ (for miR-9) and 5′-CGTTCACGAATTTGCGTGTCAT-3′ (for RNA U6, internal control; both Invitrogen, Carlsbad, CA, USA). The PCR reaction mix comprised 4 µl cDNA, 10 µl LightCycler® 480 ProbesMaster (Roche Diagnostics, Mannheim, Germany), 4 µl water and 2 µl gene-specific TaqMan® primer/probe mix, in a final volume of 20 µl. Cycling conditions were an initial denaturation step at 95℃ for 5 min, followed by 40 cycles of 95℃ for 10 s and 60℃ for 1 min, then a final elongation step at 72℃ for 5 min. PCR was performed on a LightCycler® 480 System II (Roche Diagnostics). The concentration of miR-9 relative to RNA U6 was calculated according to the equation 2^{−ΔΔ CT} , where ΔΔCT = (CT_miR-21−CT_U6). ¹⁸ All samples were examined in triplicate.

Follow-up

For analysis of survival time, the date of surgery was used as the beginning of the follow-up period. Patients who died from other disease or from unexpected events were excluded from the study. Follow-up continued until July 2013.

Statistical analyses

Data were expressed as mean ± SD. Between-group comparisons were made using Student’s t-test or Pearson’s χ²-test, as appropriate. Patients were stratified into high or low miR-9 groups relative to the median miR-9 concentration. The association between miR-9 serum concentrations and overall survival was evaluated using Kaplan–Meier analysis. Statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA) and SPSS® version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows®. All tests were two-tailed, and P-values < 0.05 were considered statistically significant.

Results

The study included 118 patients with osteosarcoma (50 male/68 female; mean age 22.2 ± 2.2 years, range 8–68 years) and 60 control subjects (28 male/32 female; mean age 23.5 ± 2.1 years, range 7–59 years). There were no significant between-group differences in age or sex. Clinicopathological features of the patient group are summarized in Table 1. The median duration of follow-up was 48 months (range 10–81 months). During the follow-up period, 38 patients (32.2%) died of disease. The 5-year survival rate was 41.5% (49/118).

Table 1.

Relationship between demographic and clinicopathological features of osteosarcoma, and microRNA-9 (miR-9) concentration, in serum samples from patients with osteosarcoma (n = 118).

Parameter	n	Relative miR-9 concentration a	Statistical significance b
Age, years
<20	60	4.44 ± 0.65	NS
≥20	48	4.30 ± 0.79
Sex
Male	50	4.39 ± 0.81	NS
Female	68	4.41 ± 0.85
Clinical stage 17
I–IIA	64	3.83 ± 0.67	P < 0.01
IIB–III	54	4.68 ± 0.82
Tumour diameter
<8 cm	88	4.11 ± 0.71	P < 0.01
≥8 cm	30	4.88 ± 0.86
Distant metastasis
Absent	84	4.09 ± 0.69	P < 0.01
Present	34	5.09 ± 0.84
Tumour location
Tibia/femur	80	4.39 ± 0.69	NS
Other	38	4.51 ± 0.78

Data presented as mean ± SD.

a

Calculated using 2^−ΔΔCT method, where ΔΔC = (C _{t miR-9}–C _{t U6}) osteosarcoma group – (C _{t miR-9}–C _{t U6}) control group.

b

Student’s t-test.

NS, not statistically significant (P ≥ 0.05).

Serum miR-9 concentrations were significantly higher in patients than healthy control subjects (4.45 ± 0.74 and 2.53 ± 0.62, respectively; P < 0.01).

Data regarding the association between miR-9 concentration and clinicopathological features are shown in Table 1. MiR-9 was significantly upregulated in patients with advanced clinical stage (IIB–III vs I–IIA), large tumour diameter (≥8 cm vs < 8 cm) and distant metastasis (vs no distant metastasis) (P < 0.01 for each comparison). There was no significant association between miR-9 concentration and age, sex or tumour location.

Kaplan–Meier analysis (Figure 1) found that patients with relatively high miR-9 concentrations (expression) had significantly shorter survival times than those with relatively low miR-9 concentrations (P = 0.002).

Figure 1.

Kaplan–Meier analysis of overall survival in patients with osteosarcoma, stratified according to median serum microRNA-9 (miR-9) concentration (expression). The colour version of this figure is available at: http://imr.sagepub.com

Discussion

Serum miR-9 concentrations were increased in patients with osteosarcoma compared with healthy control subjects in the present study. In addition, miR-9 concentrations were significantly associated with TNM stage, tumour diameter and metastasis, and patients with relatively high serum miR-9 concentrations had a significantly poorer outcome than those with relatively low concentrations. Taken together, these data suggest that miR-9 could be a valuable prognostic indicator for osteosarcoma. To our knowledge, this is the first study to investigate the clinical significance of miR-9 in a large number of patients with osteosarcoma.

MicroRNA-9 was identified as a crucial regulator of nervous-system development, physiology and pathology in organisms including zebra fish, Drosophila and mammals.^19–21 Altered miR-9 expression is associated with the development and progression of cancers,^22–25 but the expression patterns and role of miR-9 are diverse. In some tumours (including neuroblastoma, ²⁶ medulloblastoma ²⁷ and ovarian cancer ²⁸ ) miR-9 is downregulated and functions as a tumour suppressor; in other cancers (including colorectal, ¹⁴ breast ¹⁵ and nonsmall-cell lung cancer ¹⁶ ) highly expressed miR-9 promotes cancer-cell growth and/or metastasis. MiR-9 concentrations were higher in patients with osteosarcoma than normal controls in the present study, and upregulation of miR-9 was associated with tumour aggressiveness and metastasis. Our data support the notion that miR-9 plays different roles in different tumours.

The current finding that miR-9 was upregulated in patients with osteosarcoma compared with healthy controls is consistent with others who found that miR-9 was highly expressed in osteosarcoma clinical samples and inversely correlated with the predicted target gene, transforming growth factor β receptor II (TGFBR2). ²⁹ The molecular mechanisms underlying miR-9 and the regulation of osteosarcoma carcinogenesis remain unclear and require further study.

In conclusion, the present study found that serum miR-9 levels are significantly increased in patients with osteosarcoma compared with healthy controls, and that the upregulation of miR-9 is associated with tumour stage, size and metastasis. Serum miR-9 may represent a useful diagnostic and prognostic marker of osteosarcoma.