Down-regulation of microRNA-26a and up-regulation of microRNA-27a contributes to aggressive progression of human osteosarcoma.

BACKGROUND: Osteosarcoma is the most common primary bone malignancy with high local aggressiveness and rapid metastasizing potential, resulting in poor survival. Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression of miR-26a and miR-27a in osteosarcoma need further investigation in clinical samples. In our study, we evaluate the expression of these miRNAs in osteosarcoma tissues and compared with paired adjacent non-tumor bone tissues using RT-qPCR.
METHODS: Total RNA was purified from patients with osteosarcoma and noncancerous bone tissues. Real-time PCR was applied to quantify the expression level of miR-26a and miR-27a. Moreover, the correlation of these markers with clinicopathological characteristics was also evaluated in osteosarcoma patients. A cox proportional hazards model was performed to assess multivariate analyses of prognostic values.
RESULTS: Our result suggested that miR-26aexpression level in osteosarcoma bone tissue was significantly lower than that in the paired noncancerous bone tissues. MiR-27a expression was higher in osteosarcoma bone tissue in comparison with paired noncancerous bone tissues. The results indicated that low expression level of miR-26a and high expression of miR-27a were associated with high TNM stage (P = 0.001; P = 0.012), tumor grade (P = 0.007; P = 0.016), and distant metastasis (P = 0.004; P = 0.001). Kaplan-Meier analysis and log-rank test indicated that patients with low expression of miR-26a and high expression of miR-27a had shorter overall survival (log-rank test: P < 0.001). Multivariate Cox proportional hazards model analysis showed that low expression of miR-26a and high expression of miR-27a (P = 0.021; P = 0.011), high TNM stage (P = 0.001; P = 0.003), tumor grade (P = 0.005; P = 0.01), and distant metastasis.(P = 0.002; P = 0.005) were independent prognostic factors for overall survival patients with osteosarcoma cancer.
CONCLUSIONS: In conclusion, our findings suggested that expression level of miR-26a and miR-27a contributes to aggressive progression of this malignancy. Therefore, may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients.

Background

Osteosarcoma is one of the most frequent primary skeletal neoplasms, second only to plasma cell myeloma, in children, adolescents and young adults [1]. Despite the advances in multiple therapeutic strategies such as chemotherapy, surgery, and sometimes radiotherapy, overall clinical outcomes for osteosarcoma patients is still dissatisfactory, especially for patients with metastasis or recurrent osteosarcoma. It can be helpful to determine novel markers for osteosarcoma, which can accurately identify biological characteristics of cancers, improve therapeutic strategies, and predict clinical outcome. MicroRNAs (miRNAs) are small non-coding RNAs 18–25 nucleotides in length which regulate gene expression through repressing translation and cleaving their target mRNAs by binding to complementary sites in their 3′-untranslated region (3′-UTR)[2, 3]. Dys-regulation of miRNA expression has been reported in different kinds of human cancers, such as breast cancer, lung cancer, and colon cancer, chronic lymphocytic leukemia and malignant glioma [4-7]. Different human miRNAs have been shown to be dys-regulated in osteosarcoma [8-11].

MiR-26a was down-regulated and may serve as a potential cancer suppressor in various kinds of cancer such as nasopharyngeal carcinoma, hepatocellular carcinoma, thyroid anaplastic carcinomas and breast cancer [12-14]. Moreover, miR-26a was remarkably expressed in lymph node metastatic cancers when compared with primary cancers and enhanced lung cancer cell migration and invasion [15]. It has been reported that miR-26a can suppress cell differentiation, migration and invasion by targeting a number of genes [15, 16].

MiR-27a is located at chromosome 19p13.1, belongs to the miR-23a/24-2/27a cluster existing intergenically in the vertebrate genome [17]. It has been reported that aberrant expression of miR-27a occurred in different human cancer kinds, which show it act as a regulator in carcinogenesis. its oncogenic role has been verified in many kinds of malignancies such as hepatocellular carcinoma, renal cell carcinoma, breast cancer gastric adenocarcinoma, colon cancer, and cervical cancer; while, miR-27a is suggested to be a cancer suppressive miRNAs in non-small cell lung cancer, oral squamous cell carcinoma, and acute leukemia, oesophageal cancers [18-22]. Therefore, biological role of miR-27a in cancers is controversial. It indicated that it has context-dependent activities.

Some studies have indicated that miR-27a expression level is involved in promotion of proliferation, migration and invasion in osteosarcoma cells [23].

Therefore, in current study, the clinical importance of miR-27aand 26a in human osteosarcoma were evaluated also their relationship with clinicopathological factors was investigated.

Methods

Patients and tissue samples

We obtained 53 patients with osteosarcomas from Mashhad Hospitals between April 2008 and September 2014. None of the patients enrolled in this study had received and chemotherapy or radiotherapy before surgery. Clinical data was collected from the patients’ database. In our study, 53 pairs of osteosarcoma tissues and corresponding noncancerous bone tissues were collected from the same patients. The specimens were immediately snap-frozen in liquid nitrogen and stored at − 80 °C until use. Moreover, the diagnosis and the histological grading were approved by pathologists. The clinicopathological features were summarized in (Table 1). Ethical approval for the study was obtained according to the Declaration of Helsinki. All subjects were volunteers and informed consents were obtained.

Table 1

Correlation of miRNAs expression with clinicopathological features of osteosarcoma

Clinicopathological features	No. of cases	expression of miR-26a		expression of miR-27a		P value of miR-26a	P value of miR-27a
		Low	High	high	Low
Gender
Male	30	18	12	19	11	0.723	0.673
Female	23	15	8	10	13
Age
Children, Adolescents	20	14	6	11	9	0.634	0.694
Young adults	33	19	14	18	15
Tumor diameter (cm)
≤5	36	22	14	17	19	0.372	0.301
>5	17	11	6	12	5
Location
Distal	24	14	10	14	10	0.473	0.524
Proximal	29	19	10	15	14
Tumor grade
Low	24	13	11	11	13	0.007	0.016
High	29	20	9	11	9
Histological type
Osteoblastic	21	11	10	13	8	0.454	0.483
Chondroblastic	15	10	5	6	9
Telangiectatic	12	8	4	8	4
Fibroblastic	5	4	1	2	3
TNM stage
I + II	34	19	15	16	18	0.001	0.012
III + IV	19	14	5	13	6
Distant metastasis
Yes	16	13	3	16	0	0.004	0.001
No	37	20	17	13	24

Quantitative real-time PCR

The expression level of miRNAsin the osteosarcoma and corresponding non-cancer tissues was evaluated by qRT-PCR assay. The total RNA was purified from samples noncancerous bone tissue using TRI zolaccording to the manufacturer’s instructions. CDNA was reverse transcribed from total RNA samples using specific miR primers from the Taq Man MicroRNA Assays and reagents from the Taq Man MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA,USA. Real-time PCR was carried out using an invitrogen kit by system of Rotor-gene 6000 (Qiagen). The universal small nuclear RNA U6 (RNU6B) was used as an endogenous control for miRNAs. The ΔΔCt (ΔΔCt = ΔCt tumor samples – ΔCt control sample) to qualify the relative expression level of miRNAs.

Statistical analysis

All data were presented as the mean SD and were analysed using SPSS 16.0 software (SPSS Inc., USA). Associations between miRNAs expression level and clinicopathological features were determined using theχ2test. The Kaplan-Meier method was used to estimate survival rates, and the log-rank test was used to evaluate survival differences between groups. Cox proportional hazards multivariate survival analysis was applied to evaluate predictors correlated with overall survival. Differences were considered statistically significant when p was less than 0.05.

Results

Our result suggested that miR-26a expression level in osteosarcoma bone tissue was significantly lower than that in the paired noncancerous bone tissues (mean ± SD: 5.12 ± 2.53; 10.75 ± 3.23; P = 0.001; Fig. 1), Furthermore, miR-27a expression was higher in osteosarcoma bone tissue in comparison with paired noncancerous bone tissues (mean ± SD: 6.35 ± 1.23; 2.85 ± 0.64; P = 0.003; Fig. 1). We categorized the patients into a high expression group and a low expression group according to the median expression level of miRNAs, and the relationship of the miR-6a and miR-7a with various clinical features was analysed (Table 1).

Fig 1

MiRNAs expression levels in osteosarcoma and adjacent normal tissues

The results indicated that low expression level of miR-26a and high expression of miR-27a were associated with high TNM stage (P = 0.001; P = 0.012), tumor grade (P = 0.007; P = 0.016), and distant metastasis (P = 0.004; P = 0.001). However, there were no significant correlations of miR-26a and miR-27a expression levels with other clinical features. The correlation of miR-26a and miR27a expression with overall survival of osteosarcoma patients was investigated by Kaplan-Meier analysis and log-rank test. The results indicated that patients with low expression of miR-26a and high expression of miR-27a had shorter overall survival (log-rank test: P < 0.001; Fig. 2).

Fig 2

Relationship between miRNAs expression levels and survival time (log-rank test: P < 0.001)

Multivariate Cox proportional hazards model analysis showed that low expression of miR-26a and high expression of miR-27a (P = 0.021; P = 0.011), high TNM stage (P = 0.001;P = 0.003), tumor grade (P = 0.005;P = 0.01), and distant metastasis.(P = 0.002;P = 0.005) were independent prognostic factors for overall survival patients with osteosarcoma cancer (Table 2 and 3).

Table 2

Multivariate analyses of different prognostic parameters on osteosarcoma cancer survival (miR-26a)

Clinicopathological	HR	95 % CI	P-value
Characteristics
Gender	0.981	0.713–2.023	0.634
Age	1.017	0.794–3.643	0.731
Tumor grade	3.432	2. 456–10.032	0.005
Location	0.523	0.435–3.245	0.402
Distant metastasis	3.632	2.185–14.483	0.002
TNM stage	3.792	2.327–14.127	0.001
Histological type	2.843	1.543–3.953	0.402
miR-26a expression	2.643	1.743–9.167	0.021

Table 3

Multivariate analyses of different prognostic parameters on osteosarcoma cancer survival (miR-27a)

Clinicopathological	HR	95 % CI	P-value
Characteristics
Gender	0.925	1.275–2.128	0.773
Age	1.107	1.056–2.617	0.534
Tumor grade	3.456	2. 383–10.425	0.01
Location	0.656	0.738–2.820	0.512
Distant metastasis	3.743	2.573–14.132	0.005
TNM stage	3.223	2.843–13.435	0.003
Histological type	1.547	1.653–3.342	0.423
miR-27a	3.035	1.731–9.897	0.011

Discussion

Dys-regulation of miRNAs was reported to be associated with the development and progression of human malignancies [24]. Aberrant expression of circulating miRNAs has been demonstrated to be as potential sensitive and accurate biomarkers for cancer diagnosis and prognosis osteosarcoma [3]. Therefore, determination of functional and clinical importance of a specific miRNA may provide effective management of disease. In current study, the clinical importance of miR-27a and miR-26a in human osteosarcoma were evaluated also their relationship with clinicopathological factors was investigated.

Our result suggested that miR-26aexpression level in osteosarcoma bone tissue was significantly lower than that in the paired noncancerous bone tissues. The low expression level of miR-26a was associated with high TNM stage, tumor grade, and distant metastasis. Down-regulated miR-26a has been reported to play an important role in the progression of tumor, and miR-26a functions as a potential tumor suppressor in different types of cancer [12-14]. Song et al. suggested that down-regulation of miR-26a is associated with tumor aggressiveness and tumor metastasis, and miR-26a inhibits cell migration and invasion by targeting the EZH2 gene in osteosarcoma [25]. The decreased expression of miR-26a in osteosarcoma tissues has been reported to be significantly correlated with adverse clinicopathological features including adverse clinical stage and with the presence of distant metastasis. [25].

On the other hand, miR-26a was remarkably expressed in lymph node metastatic tumors when compared with primary tumors and enhanced lung cancer cell migration and invasion [15]. It has been reported that miR-26a can suppress cell differentiation, migration and invasion by targeting a number of genes including, SMAD1, MTDH, CDK6, CCNE1, CCNE2 CCND2, PTEN, PB1, MAP3K2 and enhancer of zeste homolog 2 (EZH2) [15, 16]. In current study, Kaplan-Meier analysis and log-rank test indicated that patients with low expression of miR-26a had shorter overall survival than those with high miRNAs expression.

Multivariate Cox proportional hazards model analysis showed that low expression of miR-26a high TNM stage, tumor grade, and distant metastasis were independent prognostic factors for overall survival patients with osteosarcoma cancer. It has been reported that down-regulation of miR-26a is associated with poor prognosis in patients with osteosarcoma, and the patients with low miR-26a expression tended to have a shorter overall and disease-free survival time. Also it was indicated that the expression of miR-26a may be a prognostic factor for overall and disease-free survival independent of these adjusted clinicopathologic characteristics [25]. Furthermore, in the present study, miR-27a expression was higher in osteosarcoma bone tissue in comparison with paired noncancerous bone tissues and high expression level of miR-27a was associated with high TNM stage, tumor grade, and distant metastasis. Tang et al. [26] showed that the serum levels of miR-27a expression increased in osteosarcoma patients and confirmed its significant associations with aggressive clinicopathological features including advanced clinical stage, positive distant metastasis and poor response to chemotherapy, indicating its oncogenic role in this malignancy. Aberrant expression of miR-27a has been suggested in various human cancer kinds, which indicates it act as a regulator in carcinogenesis. Furthermore, it has been shown that miR-27a might be as oncogenic mirRNA in many kinds of malignancies such as hepatocellular carcinoma, renal cell carcinoma, breast cancer gastric adenocarcinoma, colon cancer, and cervical cancer; but miR-27a is suggested to be a tumor suppressive miRNAs in non-smallcell lung cancer, oral squamous cell carcinoma, and acute leukemia, oesophageal cancers [18-22]. Nevertheless, biological role of miR-27a in cancers is controversial. These result suggested that it has context-dependent activities. Moreover, miR-27a expression level can promote proliferation, migration and invasion in osteosarcoma cells [23]. A study reported that miR-27a might have oncogenic in laryngeal squamous cell carcinoma via suppressing the expression of PLK2 and serve as a diagnostic and therapeutic biomarker in this malignancy [27]. Li et al. [28] indicated the miR-27a was up-regulated in lung adenocarcinoma patients treated with cisplatin-based chemotherapy and confirmed that it might be associated with low expression of RKIP, decreased sensitivity to cisplatin, and poor prognosis. It has been shown that oncogenic miR-27a had important role in ovarian cancer cell growth and metastasis [29]. Tang et al. [26] showed that the serum levels of miR-27a expression increased in osteosarcoma patients and confirmed its significant associations with aggressive clinicopathological features including advanced clinical stage, positive distant metastasis and poor response to chemotherapy, indicating its oncogenic role in this malignancy. Nowadays, further investigations performed on miRNAs in early detection of cancers by the other authors [30].

In the present study, the results indicated that patients with high expression of miR-27a had shorter overall survival than those with low miRNAs expression. Moreover, Multivariate Cox proportional hazards model analysis showed that high expression of miR-27a, high TNM stage, tumor grade, and distant metastasis were independent prognostic factors for overall survival patients with osteosarcoma cancer. Tang et al. [26] found that osteosarcoma patients with high miR-27a expression had both worse overall and disease free survival, and demonstrated that clinical stage, distant metastasis and serum miR-27a expression were all independent prognostic factors for both overall and disease-free survival. They confirmed the prognostic value of serum miR-27a expression in osteosarcoma patients.

Conclusions

In conclusion, our findings suggested that expression level of miR-26a and miR-27a contributes to aggressive progression of this malignancy. Therefore, may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients.