MicroRNA-99b predicts clinical outcome of osteosarcoma and suppresses tumor cell proliferation, migration and invasion

Osteosarcoma (OS) is a primary bone tumor and characterized by the bimodal distribution that mainly occurs in children and adolescents [1]. Statistics indicate that the incidence of OS is approximately 4.4 per 100,000, and this malignancy ranks the second common primary tumor in bone [2]. Some risk factors have been identified to be closely related with the occurrence of OS, including medical history of radiation or chemotherapy, benign bone lesions and some genetic conditions [3]. Surgical operation is the predominant chose for OS treatment, and the adjuvant therapy, such as chemotherapy, contributes to the reduction of mortality [4]. Currently, the 5-year survival rate of this malignancy has reached to 80% [5]. However, the prognosis remains dismal for the OS patients diagnosed with advanced tumors [6]. Thus, accurate prognosis and efficient therapy are urgently needed to improve the treatment of OS.

Targeted cancer therapy has received increasing attention in the cancer research field as a large number of deregulated key molecules have been identified in various tumors [7, 8]. Numerous microRNAs (miRNAs) with abnormal expression patterns have been detected in human cancer samples, and they are considered to be involved in the pathogenesis of various cancers [9, 10]. MiRNAs are a groups of small noncoding RNAs with the important regulatory function in the gene expression at post-transcriptional levels [11]. In addition, miRNAs serve critical roles in the regulation of some cell processes, such as cell proliferation, migration, invasion, differentiation and cell apoptosis [12]. In OS, some aberrant miRNAs have also been identified, such as miR-124 [13] and miR-491 [14], with close correlation with diagnosis, prognosis and tumorigenesis. Decreased expression of microRNA-99b (miR-99b) has been reported in several types of human cancer, including gastric cancer [15] and colorectal carcinoma [16]. The regulatory role of miR-99b in the chemotherapy of OS has been analyzed in a study by Gougelet et al. [17], but its expression pattern and clinical significance in OS remain elusive.

In this study, the expression of miR-99b in the patients with OS was investigated, and the potential clinical significance of miR-99b was evaluated. To further understand the functional role of miR-99b in OS progression, its effects on tumor cell processes were assessed.

Accumulated studies indicate that tumor progression involves the dysregulation of a large number of molecules, of which miRNAs are an important component [18]. Numerous ectopic miRNAs have been identified in various types of tumor and serve critical roles in the pathogenesis of malignancies [19]. For example, the expression of miR-144 was reported to be downregulated in gastric cancer cell lines compared with the normal cells, and the overexpression of miR-144 in tumor cells resulted in inhibited cell proliferation and invasion [20]. In NSCLC tissues, miR-34b was downregulated and associated with the degree of differentiation and pathological stage of the patients, indicating its potential to serve as a biomarker for this malignancy [21]. The aforementioned studies suggest the closely relationship between miRNAs and human malignancies. In OS, there are also some miRNAs with aberrant expression profiles. A study by Chu et al. shown that the expression of miR-136 was reduced in OS tissues and cells, and might be involved in the regulation of tumor cell proliferation, migration and invasion [22]. Another study by Xia et al. found that miR-377 served a tumor suppressor in the tumor progression, which evidenced by the inhibited effects of miR-377 on tumor growth and the enhanced effect on cell apoptosis [23]. Similarly, the decreased miR-139 in OS was also been identified as a suppressor in tumorigenesis, and its suppressive effects on tumor cell proliferation and invasion were exerted by targeting ROCK1 [24]. Taken together, it is important to identify more deregulated miRNAs in the development of OS.

In this study, we found a significantly decreased expression of miR-99b in both the tissue and cell samples of OS when compared to the corresponding normal controls. Thus, we considered that the aberrant miR-99b might play a tumor suppressor role in OS. The downregulated expression patterns of miR-99b have been determined in other human cancers, and it has been demonstrated to serve as a tumor suppressor. For instance, miR-99b expression was demonstrated to be reduced in gastric cancer tissues and cell lines, and it could suppress the tumor cell proliferation and cell cycle by targeting IGF-1R [15]. In colorectal carcinoma, the expression of miR-99b was also downregulated in tumor tissues, and tumor cell migration could be inhibited by the overexpression of miR-99b [16]. In our research cohort, we further investigate the relationship between miR-99b expression and the clinical features of the patients, and found that the decreased expression of miR-99b was associated with metastasis and TNM stage. Thus, we considered that the deregulated expression of miR-99b might be related with the development of OS.

Emerging studies highlight the clinical significance of miRNAs in the diagnosis and prognosis of human cancers [25]. The prognostic value of miR-99b has been analyzed in some cancers, such as clear cell renal cell carcinoma [26]. With the recorded survival information, we plotted the survival curves, which indicated that low miR-99b expression was related with shorter survival time. Moreover, the Cox regression data implied that the expression of miR-99b was an independent prognostic indicator. Thus, we considered that the decreased expression of miR-99b could predict poor prognosis of OS.

Considering the reduced expression of miR-99b in OS tissues and cell lines, its functional role in tumor progression was explored after the in vitro regulation of miR-99b. This study found that the OS cell proliferation, migration and invasion were all promoted by the knockdown of miR-99b, while were inhibited by the overexpression of miR-99b, which confirmed that miR-99b served a tumor suppressor in the progression of OS. Although we provided evidence for the inhibiting effects of miR-99b on OS progression, the underlying mechanisms remain unclear. Li and his colleagues have reported that miR-99b could suppress cervical cancer cell proliferation, invasion, migration and cell cycle by the inhibition of the PI3K/AKT/mTOR signaling pathway [27]. Liu et al. have demonstrated that the non-small cell lung cancer cell proliferation, migration and invasion abilities were downregulated by miR-99b through targeting Frizzled-8 (FZD8) [28]. Furthermore, FZD8 has been reported to serve as an oncogene in OS [29]. Therefore, we suspected that the effects of miR-99b in OS might exerted also through targeting FZD8 or via the PI3K/AKT signaling. Further studies need to be carried out to confirm our hypothesis on the molecular mechanisms. In addition to the limited understanding about the underlying mechanisms, another limitation of this study was the small sample size that might lead to limited accuracy of our results. Thus, future studies are needed with a large research cohort.

In conclusion, all the data in this study demonstrated that the decreased expression of miR-99b is a candidate biomarker in the prognosis of OS. OS tumor cell proliferation, migration and invasion can be suppressed by the overexpression of miR-99b, indicating the potential of miR-99b as a therapeutic target in the treatment of OS.