Decreased microRNA-452 expression and its prognostic significance in human osteosarcoma.

BACKGROUND: MicroRNA-452 (miR-452) was previously reported to be dysregulated in several types of human cancers and involved in tumor progression. The aim of this study was to investigate the clinical significance and prognostic value of miR-452 expression in human osteosarcoma.
METHODS: The expression of miR-452 was detected in 95 pairs of osteosarcoma specimens and adjacent noncancerous bone tissues using quantitative RT-PCR (qRT-PCR) assay. Then, the association of miR-452 levels with clinicopathological features and prognosis was analyzed. The roles of miR-452 in regulating osteosarcoma cell proliferation, apoptosis, and invasion were evaluated in vitro.
RESULTS: miR-452 expression was significantly downregulated in osteosarcoma tissues than those in corresponding noncancerous bone tissues (P < 0.001). Decreased miR-452 expression was linked to larger tumor size, high tumor grade, advanced clinical stage, distant metastasis, and shorter overall survival. Multivariate Cox regression analysis confirmed that low level of miR-452 expression predicted poor prognosis independently. miR-452 overexpression in MG-63 cells suppressed cell proliferation, promoted cell apoptosis, inhibited cell invasion, and led to decreased BMI1 protein levels.
CONCLUSIONS: These findings suggest that miR-452 downregulation may be involved in osteosarcoma formation and progression and that miR-452 would serve as a novel prognostic biomarker for patients with this disease.

Background

Osteosarcoma is one of the most common primary bone tumors and occurs predominantly in children and young adults [1]. With the development of multiple therapeutic strategies including wide tumor excision, neoadjuvant or adjuvant chemotherapy, and radiotherapy, the 5-year survival of the non-metastatic patients has increased to 65 % [2]. However, osteosarcoma is very aggressive, and approximately 40–50 % of patients will eventually develop metastases, especially pulmonary metastases [3]. The prognosis of these patients is rather poor, and the long-term survival rate is only 10–30 % [4]. Although several genetic alterations have been reported to be linked with osteosarcoma, the complex molecular mechanisms in osteosarcoma pathogenesis remain largely unclear. Therefore, the identification of novel diagnostic and prognostic biomarkers and potential therapeutic targets is crucial for improving the prognosis of osteosarcoma patients.

MicroRNAs (miRs) are short (about 22 nucleotides in length), highly conserved small non-coding RNA molecules that negatively regulate gene expression by binding to target messenger RNAs (mRNAs) at their 3′-untranslated region, leading to mRNA degradation or translation suppression [5]. It is now clear that miRs play a vital role in diverse biological processes, and deregulation or dysfunction of miRs is involved in a variety of human diseases including cancer [6-8]. Emerging evidence demonstrates that miRs participate in the regulation of cancer cell growth, apoptosis, tumor angiogenesis, epithelial-mesenchymal transition, tumor invasion and metastasis, and chemotherapy resistance [9-11]. In osteosarcoma, aberrant expression of several miRs and their functions have been reported. For example, miR-124 showed decreased expression in osteosarcoma tissues, and its downregulation was correlated with advanced clinical stage, positive distant metastasis, and poor response to neoadjuvant chemotherapy [12]. Low miR-217 and miR-646 expression levels were unfavorable prognostic factors for overall survival of osteosarcoma patients [13]. Serum miR-191 and miR-221 may be used as diagnostic biomarkers with the ability to discriminate between patients with osteosarcoma and the healthy cohort [14, 15]. Restoration of miR-154 in osteosarcoma cells inhibited cell proliferation, colony formation, migration, and invasion, as well as induced cell cycle arrest at the G1 stage [16]. Furthermore, ectopic expression of miR-30a promoted chemotherapy-induced apoptosis and enhanced chemosensitivity in osteosarcoma [17]. Therefore, exploring the roles of miRs in osteosarcoma development and its underlying mechanisms may be helpful for its early diagnosis, accurate assessment, targeted therapy, and prognosis evaluation.

miR-452 is a member of miR-224/miR-452 cluster. Previous studies have proved that miR-224 is involved in tumor formation and progression of several human cancers [18-20]. Recently, aberrant miR-452 expression has been demonstrated in glioma [21], non-small cell lung cancer (NSCLC) [22], bladder cancer [23], prostate cancer [24], urothelial carcinoma [25], and hepatocellular carcinoma [26]. In these malignancies, miR-452 acts as a potential oncogene or a candidate tumor suppressor in different cellular contexts. However, very little is known about the expression and clinical significance of miR-452 in human osteosarcoma till now. Therefore, in the current study, we examined miR-452 expression in osteosarcoma specimens and paired adjacent nontumorous tissues and investigated the correlation between miR-452 expression and clinicopathological factors as well as patient’s survival. We also explored the role of miR-452 in the regulation of biological behaviors of osteosarcoma cells.

Methods

Patients and tissue specimens

Paired fresh osteosarcoma specimens and noncancerous bone tissues were collected from 95 patients at The First Affiliated Hospital of Zhengzhou University (Zhengzhou 450000, Henan Province, People’s Republic of China) between January 2007 and December 2010. All the samples were collected before any treatment. The specimens were immediately frozen in liquid nitrogen and stored at −80 °C until use. Table 1 shows the details of clinical and pathological characteristics of the patients. The diagnosis was confirmed by two senior pathologists. The clinical stage was classified according to Enneking’s staging system [27]. The follow-up data were available and complete for each patient. The overall survival was calculated from the day of primary surgery to death or last follow-up. The Research Ethics Committee of Zhengzhou University approved our study, and all patients provided written informed consent.

Table 1

Association of miR-452 expression with clinicopathological factors in osteosarcoma

Clinicopathological features	Number of cases	miR-452 expression		P
		High	Low
		n (%)	n (%)
Age
<20 years	70	33 (47.1 %)	37 (52.9 %)	0.352
≥20 years	25	15 (60.0 %)	10 (40.0 %)
Gender
Male	68	35 (51.5 %)	33 (48.5 %)	0.823
Female	27	13 (48.1 %)	14 (51.9 %)
Tumor size
>8 cm	39	14 (35.9 %)	25 (64.1 %)	0.012
≤8 cm	56	34 (60.7 %)	22 (39.3 %)
Anatomic location
Tibia/femur	66	31 (47.0 %)	35 (53.0 %)	0.374
Elsewhere	29	17 (58.6 %)	12 (41.4 %)
Tumor grade
Low	31	22 (71.0 %)	9 (29.0 %)	0.008
High	64	26 (40.6 %)	38 (59.4 %)
Histological type
Osteoblastic	33	19 (57.6 %)	14 (42.4 %)	0.830
Fibroblastic	21	10 (47.6 %)	11 (52.4 %)
Chondroblastic	14	6 (42.9 %)	8 (57.1 %)
Telangiectatic	15	8 (53.3 %)	7 (46.7 %)
Others	12	5 (41.7 %)	7 (58.3 %)
Enneking stage
I	24	20 (83.3 %)	4 (16.7 %)	<0.001
II	48	23 (47.9 %)	25 (52.1 %)
III	23	5 (21.7 %)	18 (78.3 %)
Distant metastasis
Absent	72	43 (59.7 %)	29 (40.3 %)	0.001
Present	23	5 (21.7 %)	18 (78.3 %)

Cell culture and transfection

Human osteosarcoma MG-63 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) at 37 °C in a humidified chamber with 5 % carbon dioxide.

miR-452 mimics and negative control (miR-NC) were obtained from GenePharma (China). MG-63 cells were seeded into six-well plates and transfected with 50-nM miR-452 mimics or miR-NC using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer protocol.

RNA extraction, reverse transcription, and real-time PCR

Total RNA isolation was performed using the miRNeasy Mini Kit (Qiagen, Shanghai, China). Total RNA concentration was assessed by measuring absorbance at 260 nm using a NanoDrop spectrophotometer (ND-1000, Thermo Scientific, Wilmington, USA). Complementary DNA (cDNA) was synthesized from isolated RNA using TaqMan MicroRNA Reverse Transcription Kit and miRNA specific stem-loop primers (both from Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed with a Taqman MicroRNA Assay Kit (Applied Biosystems, Foster City, CA, USA) on ABI7500 real-time PCR detection system. Quantitative PCR was conducted at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. All reactions were run in triplicate, and miR-452 expression was normalized to U6 small nuclear RNA. The reverse transcription primer for miRNA-452 was 5′-GTCGTATCCAGTGCGTGTCGTG GAGTCGGCAATTGCACTGGATACGACTCAGTT-3′. The PCR primers for miRNA-452 were 5′-GCGCAACTGTTTGCAGAG-3′ (forward) and 5′-GTGCAGGGTCCGAGG T-3′ (reverse) and those for U6 were 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). The relative amount of miR-452 to U6 was calculated using the equation 2−ΔCt, where ΔCT = (CTmiR-452−CTU6). The fold change of miR-452 in osteosarcoma relative to the matched noncancerous bone tissues was determined by the 2−ΔΔCt method.

Cell proliferation assay

Cell viability was evaluated using the MTT method. In brief, MG-63 cells were seeded in a 96-well plate, and 20 μl MTT solution (Sigma, USA) was added to the culture medium every 24 h, after transfection with miR-452 mimics or miR-NC. After 4 h, the medium was removed, and 150 μl DMSO was added to dissolve the formazan. Optical density was measured at 490 nm, using a plate reader (Molecular Devices, Sunnyvale, CA, USA).

Flow cytometry analysis

After transfection with miR-452 mimics or miR-NC, the MG-63 cells were washed and resuspended in phosphate-buffered saline and then doubly stained using the Annexin V-FITC/Propidium Iodide kit (BD Biosciences, USA). After incubation in the dark for 15 min, cells were analyzed with a FACScan flow cytometer (BD Biosciences, USA).

Cell invasion assay

Cell invasiveness was determined by using Transwell insert chambers (8 μm pore size; Corning, USA). After miRNA transfection, the MG-63 cells were collected and resuspended in serum-free medium at a concentration of 5 × 104 cells/ml and 200 μl cell suspensions were then added into the upper chamber. The bottom chamber was filled with 500 μl culture medium containing 10 % FBS. After the incubation for 24 h, the non-invaded cells were removed by a cotton swab, and cells on the lower surface of the membrane were stained with crystal violet and counted.

Western blot

Cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors. The protein extracts were loaded onto a 10 % sodium SDS-PAGE gel and transferred to a PVDF membrane. The blots were probed with primary antibodies (anti-BMI1, 1:1000, Abcam, Cambridge, UK) followed by the HRP-conjugated secondary antibody. Following three Tris-buffered saline containing 0.1 % Tween-20 (TBST) washes, the membranes were developed using ECL Plus (Millipore, MA, USA) and exposed to X-ray film. GAPDH served as the loading control.

Statistics

All statistical analyses were performed using the SPSS for Windows statistical software, version 16.0 (SPSS, Chicago, IL, USA). Data are presented as the mean ± standard deviation (SD). The differences between two groups were analyzed using the Student’s t test. The relationship between miR-452 levels and clinicopathologic parameters was analyzed by chi-square test. Survival curves were constructed with the Kaplan-Meier method and compared by log-rank tests. The multivariate analyses were evaluated with Cox proportional hazards models. P < 0.05 was considered statistically significant.

Results

Decreased miR-452 expression in osteosarcoma samples

miR-452 expression was detected by using quantitative real-time PCR (qRT-PCR) and normalized to U6. Figure 1a shows decreased miR-452 expression in osteosarcoma compared to matched adjacent noncancerous bone tissues. Figure 1b shows that the relative level of miR-452 in osteosarcoma specimens was significantly lower than that in corresponding noncancerous bone tissues (mean ± SD, 4.03 ± 0.96 vs. 10.07 ± 1.98, P < 0.001).

Fig. 1

The relative expression levels of miR-452 in osteosarcoma specimens and matched adjacent noncancerous bone tissues. a The fold change of miR-452 in osteosarcoma relative to the matched adjacent noncancerous bone tissues. b miR-452 expression was significantly lower in osteosarcoma specimens than in the corresponding adjacent noncancerous bone tissues. miR-452 levels were calculated by the 2−ΔCt method and normalized to U6 small nuclear RNA

Association between miR-452 expression and clinicopathological factors

The median miR-452 expression level was used as a cut-off value to divide all 95 patients into two groups: high miR-452 expression group (n = 48) and low miR-452 expression group (n = 47). As shown in Table 1, we found that low miR-452 expression was significantly associated with larger tumor size (P = 0.012), high tumor grade (P = 0.008), advanced Enneking stage (P < 0.001), and distant metastasis (P = 0.001). However, there were no significance between miR-452 expression and other clinicopathological factors, such as age, gender, anatomic location, and histological type.

Prognostic value of miR-452 in osteosarcoma

Then, we evaluated the prognostic significance of miR-452 expression in patients with osteosarcoma. As shown in Fig. 2, patients in high miR-452 expression group had longer overall survival than those in low miR-452 expression group (P < 0.001). Univariate Cox regression analysis demonstrated that larger tumor size (P = 0.026), high tumor grade (P = 0.015), positive distant metastasis (P = 0.002), and advanced Enneking stage (P < 0.001) were also significantly associated with shorter overall survival (Table 2). Multivariate analyses using the Cox proportional hazards regression model revealed that miR-452 expression (relative risk (RR) 3.263; P = 0.018), tumor grade (RR 3.013; P = 0.022), distant metastasis (RR 4.985; P = 0.003), and Enneking grade (RR 5.458; P = 0.001) were independent prognostic factors for overall survival of osteosarcoma.

Fig. 2

Osteosarcoma patients with low miR-452 expression had a significantly shorter overall survival than those with high miR-452 expression (P < 0.001, log-rank test)

Table 2

Cox regression analysis of factors associated with overall survival in 95 osteosarcoma patients

Variable	Univariate analysis		Multivariate analysis
	HR	P value	HR	P value
Age	1.638	0.223	–	–
Gender	1.126	0.772	–	–
Anatomic location	1.695	0.178	–	–
Tumor grade	3.572	0.015	3.013	0.022
Tumor size	3.108	0.026	1.165	0.082
Clinical stage	5.979	<0.001	5.458	0.001
Distant metastasis	4.392	0.002	4.985	0.003
miR-452	5.756	<0.001	3.263	0.018

The effects of miR-452 on the biological behavior of MG-63 cells

Further, we assessed the biological role of miR-452 in MG-63 cells. Transfection with miR-452 mimics resulted in increased miR-452 level in MG-63 cells (P < 0.001, Fig. 3a). The MTT assay showed that miR-452 overexpression obviously reduced MG-63 cell proliferation (Fig. 3b). Flow cytometry confirmed promoted apoptosis in miR-452-overexpression MG-63 cells (Fig. 3c). Transwell invasion assay showed that miR-452 mimic transfection impeded MG-63 cell invasion ability (Fig. 3d).

Fig. 3

Effects of miR-452 on the biological behaviors of MG-63 cells. a qRT-PCR analysis confirmed increased miR-452 expression in MG-63 cells after miR-452 mimics transfection. **P < 0.01. b MTT assay showed that miR-452 reduced cell proliferation in vitro. **P < 0.01. c Flow cytometric analysis indicated that miR-452 promoted cell apoptosis. d Upregulation of miR-452 impeded cell invasion in MG-63 cells. **P < 0.01

miR-452 could downregulate BMI1 expression in MG-63 cells

At last, we detected the changes in BMI1 protein levels after miR-452 mimic transfection by western blot. The results showed that miR-452 overexpression in MG-63 cells led to the downregulation of BMI1 protein levels (Fig. 4).

Fig. 4

miR-452 overexpression in MG-63 cells led to the downregulation of BMI1 protein levels

Discussion

Up to now, the exact mechanisms underlying osteosarcoma are not fully understood. Identification of genetic alterations would be important for the screening, diagnosis, and treatment of osteosarcoma. Accumulating evidence has suggested the important roles of miRs in tumor formation and progression. In the present study, we revealed that the expression of miR-452 was significantly reduced in osteosarcoma tissues, and reduced miR-452 expression was significantly associated with larger tumor size, high tumor grade, advanced clinical stage, and distant metastasis. Patients with low level of miR-452 showed shorter overall survival than those with high miR-452 level. miR-452 overexpression in MG-63 cells suppressed cell proliferation, promoted cell apoptosis, and inhibited cell invasion in vitro. Thus, loss of miR-452 expression might be involved in osteosarcoma development and serve as a novel biomarker for poor prognosis.

Previous research has demonstrated the tumor-suppressive function of miR-452 in several human malignancies. For example, Kristensen et al. reported that miR-452 inhibited proliferation, migration, and invasion of PC3 and DU145 prostate cancer cells [24]. He et al. showed miR-452 downregulation in NSCLC tissues and its association with positive tumor metastasis and advanced TNM stage [22]. Zhang et al. revealed that upregulated miR-452-suppressed NSCLC cell epithelial-mesenchymal transition and further disrupted migration and invasion [28]. In human gliomas, decreased miR-452 expression was correlated with shorter patient survival time [21]. Ectopic expression of miR-452 reduced tumorigenicity in a nude mouse xenograft model. Furthermore, overexpression of miR-452 in MCF-7 breast cancer cells could significantly enhance cell apoptotic and increase chemosensitivity to Adriamycin [29].

In contrast to the antitumor properties mentioned above, miR-452 may act as a potential oncogene in bladder cancer and hepatocellular carcinoma [23, 25, 26]. Overexpression of miR-452 in HepG2 and QGY-7703 hepatocellular carcinoma cells dramatically accelerated proliferation, induced cell cycle from G1 to S transition, blocked apoptosis, and promoted cell migration and invasion in vitro [26]. Increased miR-452 expression in bladder urothelial carcinomas was associated with high incidence of lymph nodes metastasis and poor prognosis [25]. Thus, the function of miR-452 may be complex in different cancer types, and the role of miR-452 should be tumor specific and possibly dependent on the nature of its targets.

Undoubtedly, miRs exert important functions in carcinogenesis by regulation of target gene expression [30]. In human cancers, some highly expressed miRs could function as oncogenes by repressing tumor suppressor genes, whereas low-expressed miRs could function as tumor suppressors by negatively regulating oncogenes [31]. Previous studies have reported several downstream genes of miR-452, including BMI1 [22], CDKN1B [26], IGF-1R [29], and KRAS [32]. BMI1 has been identified as an oncogene in several cancers [33-36]. Wu et al. revealed that overexpression of BMI1 could promote osteosarcoma cell growth and induce resistance to cisplatin [37]. He et al. identified BMI1 as a direct target of miR-452 in NSCLC [22]. Our study also observed decreased BMI1 protein levels in MG-63 cells after miR-452 overexpression. However, there is no “one-to-one” connection between miRs and target mRNAs. An average miR can have more than 100 targets [38]. Conversely, several miRs can converge on a single transcript target [39]. Therefore, the potential regulatory circuitry afforded by miR-452 is enormous, and the accurate mechanisms on how miR-452 influences osteosarcoma progression need further clarification.

Conclusions

In summary, our research showed miR-452 downregulation in osteosarcoma and its correlation with aggressive clinicopathological features. Decreased miR-452 expression might be an independent biomarker for poor prognosis. Large-scale prospective studies are needed to confirm our conclusion and clarify the mechanisms.