Role of MicroRNAs in Human Osteosarcoma: Future Perspectives

Osteosarcoma (OS) is a rare form of cancer with high death rate but is one of the most frequent forms of bone cancer in children and adolescents. MiRNAs are small endogenous RNAs that regulate gene expression post-transcriptionally. The discovery of miRNAs could allow us to obtain an earlier diagnosis, predict prognosis and chemoresistance, and lead to the discovery of new treatments in different types of tumors, including OS. Despite the fact that there is currently only one clinical trial being carried out on a single miRNA for solid tumors, it is very probable that the number of clinical trials including miRNAs as prognostic and diagnostic biomarkers, as well as potential therapeutic targets, will increase in the near future. This review summarizes the different miRNAs related to OS and their possible therapeutic application.


Introduction
Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Although advances made in chemotherapy, radiation therapy, and surgery have improved survival rates in different cancers, little to no progress has been seen in recent years for OS [1,2]. Unfortunately, in osteosarcoma, 15-20% of patients admitted for treatment, already have an advanced form of the disease with metastases, that are most commonly found in the lungs [3][4][5]. These lung metastases are what cause the death of the patient in 30-40% of cases [6]. In patients without lung metastases, the 5-year survival rate is 58-75% [7]. This prognosis is determined by the rate of occurrence of metastasis and the appearance of chemoresistance [6]. Given the aggressiveness and high mortality rates of OS, early detection is key to a successful treatment. Recent studies have shown that OS is becoming increasingly resistant to chemotherapeutic agents. Therefore, understanding the underlying molecular mechanisms causing the resistance, and the development of novel and more successful treatments, has become a necessity. In fact, Basile et al. (2020) published a meta-analysis in which they found different prognostic factors in patients under the age of 40, enabling an accurate prediction of the risk a patient has of developing lung metastasis. Optimizing the treatment of patients, based on these different prognostic factors, could play an important role in increasing OS survival rates. [8]. In recent years, microRNAs (miRNAs) have been presented as possible diagnostic and prognostic biomarkers, as modulators of chemoresistance and as a plausible new treatment option in the fight against different tumors, including OS.

Human Osteosarcoma
Osteosarcoma (OS) is a high-grade malignant bone tumor composed of mesenchymal stem cells (malignant osteoblasts). It is characterized by the deposition of an immature osteoid matrix resulting in the formation of immature woven bone. The incidence of this Biogenesis of miRNAs begins in the cell nucleus, where miRNAs are transcribed by RNA polymerase II or III as primary transcripts (pri-mRNA) to specific genes. Later, the enzyme Drosha and its cofactor DGCR8/Pasha cut this pri-miRNA to form a pre-miRNA (70-90 nucleotides long in the shape of a stem-loop). The nuclear transport receptor Exportin 5 (XPO5), that plays an important role in various cancers, then actively transports the pre-miRNA from the nucleus to the cytoplasm. In the cytoplasm, the pre-miRNA is cut by a Dicer enzyme (regulated by XPO5), to form a mature and short double-stranded miRNA molecule and, after the degradation of one of the two chains, a single strand of miRNA is incorporated into the RISC (RNA-induced silencing complex) protein complex. The RISC protein complex is responsible for directing the silencing of messenger RNA and regulating post transcriptional gene expression, via the inhibition of protein translation or destabilization of target transcripts [23][24][25][26][27][28][29][30][31] (Figure 1).

Figure 1.
Diagram of miRNA biogenesis. In the nucleus, miRNA that are transcribed by RNA polymerase II or III as primary transcripts (pri-miRNA). The enzyme Drosha and its cofactor DGCR8/Pasha then cut the pri-miRNA molecule to form a pre-miRNA. This pre-miRNA is actively transported from the nucleus to the cytoplasm by the nuclear transport receptor Exportin 5 (XPO5) in a Ran-GTP (guanosine triphosphate) protein dependent manner by XPO5. In the cytoplasm, the pre-miRNA is cut by a second enzyme called Dicer, to form a mature and short double-stranded miRNA molecule. The miRNA duplex unwinds and is incorporated into the RISC protein complex [17].

Biological Functions of miRNAs
MiRNAs are distributed in the intragenic and intergenic regions of the genome. miRNA genes are observed in defined units or clusters [32], containing two or more adjacent sequences with a common transcriptional triggering mechanism. These clusters are involved in different biological functions, as regulators of biological events such as cellular homeostasis, and their aberrant expression has been associated with pathophysiological events [33].
Regarding cancer, the main hallmarks are cell cycle control, apoptosis and senescence, autophagy, cell proliferation and growth, angiogenesis, and metastasis. miRNA clusters play a crucial role in these steps, and their role and type can differ depending on the type of cancer. More specifically, the miR-17/92 and miR106b/25 clusters are related to proliferation, tumor initiation, metastasis, senescence, cell cycle regulation, apoptosis, and autophagy [47][48][49][50][51][52][53][54][55][56][57]. Other miRNA clusters, their pathways, and related cancer hallmarks are shown in Figure 2. In the nucleus, miRNA that are transcribed by RNA polymerase II or III as primary transcripts (pri-miRNA). The enzyme Drosha and its cofactor DGCR8/Pasha then cut the pri-miRNA molecule to form a pre-miRNA. This pre-miRNA is actively transported from the nucleus to the cytoplasm by the nuclear transport receptor Exportin 5 (XPO5) in a Ran-GTP (guanosine triphosphate) protein dependent manner by XPO5. In the cytoplasm, the pre-miRNA is cut by a second enzyme called Dicer, to form a mature and short double-stranded miRNA molecule. The miRNA duplex unwinds and is incorporated into the RISC protein complex [17].

Biological Functions of miRNAs
MiRNAs are distributed in the intragenic and intergenic regions of the genome. miRNA genes are observed in defined units or clusters [32], containing two or more adjacent sequences with a common transcriptional triggering mechanism. These clusters are involved in different biological functions, as regulators of biological events such as cellular homeostasis, and their aberrant expression has been associated with pathophysiological events [33].
Indeed, some of these clusters are associated with cell cycle progression, migration, and metastasis in osteosarcomas. More specifically, miR-17/92 is found to be abnormally regulated in human OS cell lines and is linked to cell cycle progression [58] and activation of migration and metastasis via the downregulation of the Quaking (QKI) and β-catenin pathways [59]. In fact, patients with a high expression of miR-17/92 presented poor recurrence-free and overall survival rates [60].

MiRNAs and Human Osteosarcoma
Many studies reflect the important role that miRNAs play in the molecular regulation, the appearance and the progression of human OS. Some of these miRNAs act as tumor suppressors, whilst others act as oncogenes [61]. Different miRNAs will act on a diverse range of molecular targets and pathways. Different tumor suppressing miRNAs and their molecular targets in human OS are shown in Table 1.  Indeed, some of these clusters are associated with cell cycle progression, migration, and metastasis in osteosarcomas. More specifically, miR-17/92 is found to be abnormally regulated in human OS cell lines and is linked to cell cycle progression [58] and activation of migration and metastasis via the downregulation of the Quaking (QKI) and β-catenin pathways [59]. In fact, patients with a high expression of miR-17/92 presented poor recurrence-free and overall survival rates [60].

MiRNAs and Human Osteosarcoma
Many studies reflect the important role that miRNAs play in the molecular regulation, the appearance and the progression of human OS. Some of these miRNAs act as tumor suppressors, whilst others act as oncogenes [61]. Different miRNAs will act on a diverse range of molecular targets and pathways. Different tumor suppressing miRNAs and their molecular targets in human OS are shown in Table 1.
The presence of onco-miRNAs affecting different targets in human osteosarcoma is demonstrated in Table 2.     Interestingly, miRNAs can act as both tumor suppressors and as onco-miRNAs depending on the target that they act upon. For example, miR-23a can act as tumor suppressor in osteosarcomas, via the inhibition of the special AT-rich-binding protein 1 (SATB1), a gene related to chromatin structure regulation [62]. This finding is supported by Wang et al. who suggests that its role as a tumor suppressor could serve as a potential therapeutic target to fight osteosarcoma. MiR-23a is frequently found to be downregulated in osteosarcoma specimens and cell lines in vitro, which contributes to the aggressiveness of this cancer [63]. Indeed, an experiment carried out by He et al. in an in vivo mice model showed that the downregulation of miR-23a activates the transcription of its target mRNAs, RUNX2 and CXCL12, which play an essential role in OS cellular growth, migration, and invasion [64]. On the contrary, other studies have shown miR-23a acting as an onco-miRNA, and in this case, its downregulation could effectively reduce migration and invasion of osteosarcoma cell lines. As a matter of fact, miR-23a specifically targets the 3 -untranslational regions of PTEN (phosphatase and tensin homolog), lowering its expression. This enhances migration and invasion of osteosarcoma cell lines and might serve as a risk factor for cancer patients [65]. Similar results have been found for miR-20a and miR-107. Depending on which target they bind to, these miRNAs are able to act as both onco-genes or as tumor suppressors. Indeed, the increased expression of miR-20a can reduce colony formation and tumor growth in OS cells, by targeting and suppressing TAK1 expression [66], whereas when this miRNA binds to early growth factor 2 (EGF2) mRNA, it promotes OS cell proliferation and cell cycle [67]. Furthermore, miR-20a seems to participate in the regulation of OS chemoresistance by modulating the activities of the MAPK/ERK and cAMP/PKA signaling pathways [68]. Meanwhile, miR-107 is able to activate MEK/ERK and NF-κB signaling pathways via downregulation of tropomyosin 1 (TPM1). This promotes cell viability, migration, and invasion in human OS cells [69]. Conversely, in vitro studies have demonstrated that miR-107 can inhibit the development of OS via the Wnt/β-catenin signaling pathways, by downregulating Dickkopf-1 (Dkk-1) (Wnt inhibitory factor) [70,71].

Possible Applications of miRNAs in Human Osteosarcoma
As shown in the previous section, miRNAs have the capacity to act as tumor suppressors and as onco-miRNAs in human osteosarcomas, by acting on different signaling pathways. In fact, more than 177 different miRNAs are found to be differentially expressed in osteosarcoma cell lines [72], and could serve as prognostic, diagnostic, and chemoresistance biomarkers. MiRNAs could also act as possible therapeutic targets to treat human OS. In the following section, the possible uses of certain relevant miRNAs are detailed.

MiRNAs as Potential Biomarkers
One of the most studied miRNAs in osteosarcoma is miR-9. Recently, Wu et al. published a meta-analysis and concluded that a high expression of miR-9 was associated with poor prognosis [73]. Patients with highly expressed levels of miR-9 in osteosarcoma tissues present bigger tumor size, more advanced clinical stages, more frequent distant metastasis, and lower survival rates [74]. The effect that miR-9 has on OS progression seems to be related to it targeting p16 and mediating the activation of the ERK/p38/JNK pathways [75]. Interestingly, the high expression of miR-9, occurs not only in OS tissues, but also in the serum of patients [76]. Similar results have been obtained for the miR-29 family (miR-29a, miR-29b, and miR-29c) [77]. In this case, the underlying mechanism seems to be that miR-29 inhibits the expression of PTEN, thus promoting OS cell proliferation, migration, and metastasis formation [78]. With regards to metastasis occurrence, high circulating levels of miR-21 in patients' blood sera have been positively correlated with the presence of metastasis and lower 5-year survival rates [79,80]. MiR-95 promotes OS growth by targeting the sodium channel epithelial 1α subunit (SCNN1A) and hepatomaderived growth factor (HDGF) [81,82]. Other circulating miRNAs found in patients' sera include miR-95 and miR-194. The higher expression of miR-95 and the lower expression of miR-194 in patients' sera, contributes to higher metastasis formation and leads to a poorer prognosis in patients with OS [83,84]. These results suggest that altered serum levels of miR-9, miR-21, miR-29, miR-95, and miR-194 have great potential to serve as novel, non-invasive prognostic or diagnostic biomarkers.
Some miRNAs are related to p53, a known oncogene found in many cancers. One of these miRNAs is miR-34a, who's downregulation in human OS tissues is associated with an unfavorable prognosis [85]. In fact, Xi et al. demonstrated that the downregulation of miR-34 in the tissues of OS patients, inhibited apoptosis through targeting regulated transforming growth factor-β-induced factor homeobox 2s' (TGIF2) expression [86]. Another miRNA whose expression is lower in OS tissues is miR-126. This miRNA is able to downregulate metalloprotease 9 (ADAM-9), a protease that plays a role in promoting tumorigenesis, cell invasion, and metastasis [87]. Therefore, lower levels of miR-126 could potentially be associated with faster tumor progression, more advanced cancer stages, and shorter overall survival times [88].
MultimiRNA signature for prediction of prognosis of OS patients, has been studied, and three miRNAs (miR-153, miR-212, and miR-591) were used to generate a classified prognosis and prediction signature for OS patients [89]. It was found that miR-153 could potentially act as a tumor suppressor, as high levels of the latter decrease SMAD2, SMAD3, EGFR, and IGFBP-3 expressions, which in turn negatively regulates TGF-β [90]. On the contrary, miR-212 has been seen to inhibit apoptosis and promotes cell proliferation, by upregulating the hedgehog signaling pathway [91]. As for miR-591, although its presence in osteosarcoma has not yet been studied, its function as a tumor suppressor in breast cancer has been suggested, via the inhibition of the Hippo-YAP/TAZ signaling pathway [92]. In addition, miR-591 upregulation has been seen to confer resistance to paclitaxel, a medication used to treat a number of cancers including ovarian cancer. To date however, no research has been carried out on the effects of miR-591 on cisplatin or methotrexate resistance (the most common drugs used to treat OS) [93].

MiRNAs and OS Chemoresistance
One of the most important steps in the human OS is the patient's response to treatment. The presence of certain miRNAs seems to affect tumor cell chemoresistance. Curiously, high levels of the miR-29 family, which are related to a poor prognosis (see Section 4.1), sensitizes tumor cells against methotrexate and increases the effect of carbon ion radiotherapy [94,95]. The combined use of cisplatin in addition to methotrexate is common when trying to treat osteosarcoma. In a study carried out by Jiang L et al., these two chemotherapy drugs were seen to promote apoptosis in cells that overexpressed miR-126, but not in the silenced osteosarcoma cells [96]. This suggests that the differential expression of miRNAs may play an important role in chemotherapy resistance. Finally, high levels of miR-34a-5p were seen to affect the expression of the tyrosine kinase receptor CD117, thus promoting osteosarcoma's multidrug resistance via the mTOR pathway [97,98].

MiRNAs as Targets for OS Therapy
Different roles of miR-1 have been studied in diseases such as cardiac diseases, sclerosis, pulmonary arterial hypertension, non-alcoholic fatty liver disease, and cerebral palsy [99][100][101][102]. Furthermore, miR-1 has been related to different types of human cancers such as medulloblastoma, breast cancer, colorectal cancer, and lung cancer [103][104][105][106]. In osteosarcoma, some studies reveal its potential as a therapeutic target; for example, miR-1 is able to suppress growth, proliferation, migration, and invasion of OS cells. In an experiment carried out by Fujii et al., overexpression of miR-1 downregulated PAX3 (and increased p21 levels), and induced arrest in the G 0 /G 1 phase both in vitro and in vivo [107]. In addition, in vitro studies have shown that miR-1 inhibits the protein expression of vascular endothelial growth factor A (VEGFA), a necessary factor for the vascularization of tumors [108]. These results suggest that miR-1 could be a potential therapeutic target, via the p21 or VEGFA pathways. Regarding the latter pathway, miR-134 (a miRNA of the 14q32 locus) is capable of decreasing VEGFA and VEGFR1 expression in vivo [109], and inhibiting osteosarcoma angiogenesis and proliferation. Moreover, when this microRNA binds to the 3 -UTRs of metalloproteinases 1 and 3 (MMP1 and MMP3), it reduces tumor invasion and metastasis [110]. Therefore, miR-134 could also act as a possible therapeutic target. Other members of the 14q32 locus, such as miR-382, miR-369-3p and miR-544, suppress the expression of the c-MYC (oncogene) in human OS cells [111]. Studies in patient OS cells have demonstrated that another MMP, more precisely MMP16, is upregulated by miR-145 [112].
However, MMP16 is not miR-154 s only target. Other molecules, such as ROCK1, VEGF, and HDAC4 are also targets of this miRNA [113][114][115]. High levels of miR-145 are seen to inhibit cell proliferation, invasion, and metastasis in OS. A recent meta-analysis carried out by Xu et al. [116] showed that the level at which miR-145 was expressed in different tumors was correlated with patient prognosis, however this was not shown in patients with OS. Nevertheless, other studies related the downregulation of miR-145 with advanced tumor progression and poor prognosis in OS [117]. Another microRNA, miR-21, has been put forward as alternative possible therapeutic target. Indeed, miR-21 suppresses PTEN expression, and induces apoptosis in human osteosarcoma cells [118].
There are a large number of studies providing relevant information about the possible functions of microRNAs as potential biomarkers for prognosis, diagnosis and resistance to chemotherapy or as possible therapeutic targets in different cancers. Despite the good potential OS has, as a target of future clinical trials based on miRNA therapy, the truth is that there are actually only very few clinical trials based on miRNAs in this cancer to date. Possible explanations for this are that as a rare form cancer, there are only very few patients and therefore the recruitment potential for clinical trials is lower than it is in other cancer types. In addition, obtaining miRNAs from a bone microenvironment requires complicated procedures. Finally, the complexity of the human genome makes it difficult to interpret the results obtained from miRNA trials. We are still uncertain of the effects each miRNA may exert on the rest of the genome, and resistance mechanisms are likely. As a matter of fact, some clinical trials for solid tumors based on miRNA have been carried out. One of them was based on the therapeutic molecule MRX34, whose target is miR-34a. The results of MRX34 treatment with dexamethasone premedication did show some clinical activity without serious toxicity. However, some patients presented severe cytokine release syndrome, and the trial was terminated [119]. The final results of this clinical trial are not published yet. Another clinical trial, based on miR-16 (a tumor suppressor miRNA), has been carried out in lung cancer and mesothelioma, and included 27 patients. Although the safety of the trial was acceptable, twenty of patients died during the clinical trial due to tumor progression [120]. The results found in these different clinical trials indicate the amount of knowledge that still needs to be acquired on miRNAs before they can be used as biomarkers or as cancer therapies, including data on their safety and effectiveness.
Currently, there are a total of 334 clinical trials addressing miRNAs as possible biomarkers or therapeutic targets in different types of cancer. However, only one of them is for osteosarcoma (NCT01190943). This clinical trial is studying DNA biomarkers, including miRNAs, in tissue samples from patients with osteosarcoma. Other clinical trials, based on different miRNAs in the hope of treating further pathologies are currently being carried out. More specifically, currently, two clinical trials with therapeutic molecules targeting miRNAs in other pathologies are being carried out, and they are showing promising results. These molecules target miR-122 for the treatment of the hepatitis C viral infection and are in phases 1 and 2 of clinical trials [121,122].

Conclusions
OSs are a rare form of cancer with high death rates in humans, mainly children. The discovery of miRNAs marked a new era in molecular biology and their presence can give valuable information about the physiopathology of osteosarcoma. The relationship between different miRNAs and patient prognosis, cancer diagnosis, cancer progression, chemotherapy resistance, and their use as possible new drug targets have been demonstrated. However, there is currently only one clinical trial related to the inhibition of miRNA in solid tumors; this miRNA is miR-34a. It seems that one of the issues is the delivery system of the therapies. Another issue is the localized delivery of miRNA therapeutics. The hope is that in the near future these drawbacks will be overcome, and anti-cancer therapy based on miRNAs will be a reality, especially for solid tumors such as OS, with poor prognosis and high mortality rates.