Research Progress of Micro-RNAs in Apoptosis of Osteosarcoma

Osteosarcoma is a primary malignant bone tumor with no effective treatment. Apoptosis, one of the programmed cell death, is any pathological form of cell death mediated by intracellular processes. Under the pathological state, the unregulated regulation of apoptosis can disrupt the balance between cell proliferation and death, causing osteosarcoma proliferation and metastasis. As carcinogenic or tumor suppressor factors, microRNAs (miRNAs) regulate apoptosis of osteosarcoma cells by regulating apoptosis-related signaling pathways and apoptosis-related genes. This review provides the current knowledge of miRNAs and their target genes related to the apoptosis of osteosarcoma.


Introduction
Osteosarcoma is a malignant tumor originating from mesenchymal tissue, accounting for 20% of primary malignant bone tumors. It is the most common primary malignant bone tumor in children and adolescents, with 70-80% of patients aged 10 to 25 years and an annual incidence rate of 1 to 3 cases per million [1]. Osteosarcoma is a solid tumor that produces osteoid, which occurs in fast-growing epiphysis (such as proximal humerus, distal femur, and proximal tibia), but rarely in the spine, pelvis, and sacrum. It is often accompanied by amputation, lung metastasis, death, and other consequences, and the cure rate of simple operation is only 15-20% [2]. At present, the clinical treatment of osteosarcoma is unsatisfactory. Although some progress has been made in multi-drug chemotherapy and surgical resection, for patients with osteosarcoma who underwent radical amputation combined with chemotherapy, the ve-year survival rate can be increased to 50%-70%, 10% of the patients still have a local recurrence [3]. Therefore, it is essential for osteosarcoma research to nd new treatment methods to further improve the patients' survival rate.
MicroRNAs (miRNAs) is a kind of single-stranded RNAs, with long 19 ~ 25 nt, which does not have an open reading frame and does not encode any protein [4]. MiRNAs has been implicated in the occurrence and progression of cancer in a growing number of studies that miRNAs in malignant tumor cells present abnormal, which can positively or negatively regulate cancer progression, such as maintaining proliferative signal transduction, anti-apoptosis, inducing angiogenesis, and cancer cell invasion and metastasis [5]. Recent studies have shown that the expression pattern of miRNAs in osteosarcoma has changed. Lulla et al. showed that differential expressions of twenty-two kinds of miRNAs between osteosarcoma cells and osteoblasts [6], which can regulate the proliferation and apoptosis of osteosarcoma cells in a variety of ways, play a role in promoting or inhibiting cancer in the occurrence and development of osteosarcoma. Although the molecular regulatory mechanism of miRNAs on osteosarcoma cells has attracted widespread attention, its speci c biological effect is still not very clear (see in Table 1). The whole life process of the body is accompanied by cell death, which also occurs in the pathological process of tumor growth. Therefore, it is imperative to determine how to induce cell death in treating human osteosarcoma effectively [7,8]. Cell death can be divided into programmed cell death (PCD) and unprogrammed death according to the mode of death. Amongst these, PCD, an active cell death controlled by speci c genes and regulated by a unique signal transduction pathway, plays a vital role in the occurrence and development of osteosarcoma. As a polygene-controlled death process, apoptosis is the most typical and well-studied amongst the forms of PCD. The body can remove aging or abnormal cells through apoptosis so that the homeostasis of the internal environment is maintained in the normal physiological state, while in the pathological state, the disorder of apoptosis regulation will destroy the balance between cell proliferation and death, which has a destructive effect on the body and causes a series of diseases including osteosarcoma. Current studies have found that miRNAs can participate in the apoptosis process of osteosarcoma cells by regulating apoptosis-related proteins and pathways [9][10][11]. It has gradually become a hotspot to explore the mechanism of miRNAs that regulates apoptosis in osteosarcoma Based on the regulatory process of apoptosis and the biological roles of miRNAs, this review focuses on the regulation of apoptosis by miRNAs in recent years, and discuss remaining problems and likely prospects associated with curing osteosarcoma by promoting miRNAs-induced apoptosis, with a view of more comprehensively exploring the important role of miRNAs in the occurrence and development of osteosarcoma, and then provide new ideas for molecular targeted therapy of osteosarcoma.

Mirnas And Apoptosis-related Proteins
There are many proteins involved in the regulation of apoptosis, such as the Bcl-2 family, the Caspase family, the matrix metalloproteinase family (MMPS), and other apoptosis-related proteins. These factors can in uence the apoptosis process of osteosarcoma cells by regulating the three major apoptosis pathways: death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway (see in

Bcl-2 family
B-cell lymphoma/leukemia-2 (Bcl-2) family plays a vital role in the regulation of apoptosis in the signal transduction of apoptosis pathway, which is also one of the most noticed genes amongst many genes related to apoptosis. A study related to human follicular lymphoma has been rst discovered the Bcl-2 gene [12]. Bcl-2 protein, the rst discovered protein among many Bcl-2 family members, is also the rst proved anti-apoptotic protein and plays an essential role in the cell apoptotic pathway. Chen et al. found that miRNA449a may exert its pro-apoptotic function by inhibiting the expression of Bcl-2 [13]. The CCK-8 and apoptosis assays results suggested that cell proliferation could be inhibited while apoptosis was promoted by restoring of miRNA449a expression in osteosarcoma U2OS cell lines and Saos-2 cell lines.
In addition, Bcl-2 was identi ed as the target of miRNA449a by using TargetScan prediction technology and luciferase reporter gene detection technology, and the nal experimental results also showed that the expression of Bcl-2 was negatively correlated with miRNA449a. Studies have shown that both miRNA326 and miRNA143 promoted cell apoptosis by targeting the down-regulation of Bcl-2 [14,15]. Bax (Bcl-2 associated X protein), a pro-apoptotic protein closely related to the function of Bcl-2. Overexpressed Bax tends to follow increased apoptosis, on the contrary, Bcl-2 acts as an anti-apoptotic protein. Zhang et al. showed that transfection of miRNA144 analogs inhibited cell proliferation and signi cantly increased apoptosis in U2-OS cells. Furthermore, the detected results showed that the expression of Bax and caspase-3 was increased, while the expression of anti-apoptotic protein Bcl-2 was decreased [16]. The interaction relationship between Bax and Bcl-2 can be described: increased Bax can numerously and signi cantly lead to the production of Bax/Bax homodimers, while overpressed Bcl-2 can depart many Bax/Bax dimers so that generates a more stable Bcl-2/Bax heterodimer, and eventually play a role in inhibiting apoptosis. All of these demonstrate that the ratio of Bax/Bcl-2 can affect cell apoptosis.
The Mitochondrial pathway is one of the molecular regulatory pathways of apoptosis, which is also called the Bcl-2 regulatory pathway because it is regulated by Bcl-2 family [17]. It can be regulated by the interaction among Bcl-2 family members. As an effector, Bax can form pores in the outer mitochondrial membrane through oligomerization, eventually leading to MOMP, and then triggering the Caspase cascade Response to promote cell apoptosis because of the release of pro-apoptotic proteins such as cytochrome C and Smac located in the mitochondrial membrane space into the cytoplasm. Wang

Caspase family
Caspase family is a group of proteases that can regulate cell apoptosis and be activated sequentially Compared with normal osteoblast cell lines, miRNA221 and miRNA196a were increased in osteosarcoma cell lines. Overexpression of miRNA221 and miRNA196a inactivated caspase3. Transfection of the two inhibitors into osteosarcoma cell lines, respectively, showed a signi cant increase in caspase3 levels. It signi cantly inhibited cell proliferation, migration and invasion, and cell cycle stagnation in G0/G1 phase [31,32]. The expression of miRNA638 and miRNA190b was down-regulated in patients with osteosarcoma, and the overexpressed miRNA638 and miRNA190b could up-regulate caspase3 and induce apoptosis [33,34]. MiRNA34 increases the content of caspase3 by targeting the expression of TGIF2, thus promoting the apoptosis of osteosarcoma cells [35]. These ndings suggest that miRNA221, miRNA196a, miRNA638, miRNA190b, and miRNA34 can promote the apoptosis of osteosarcoma cells by acting on caspase3, a common downstream key factor of different apoptosis pathways. The role of MiRNAs in regulating caspase3 is also related to the mitochondrial pathway. Related studies have shown that miRNA143 can activate caspase3 by targeting Bcl-2 and induce apoptosis of osteosarcoma cells [15]. The activation of miRNA302b on caspase3 can regulate the expression of Bcl-2/Bim, inhibit the proliferation of osteosarcoma cells, and increase apoptosis [24]. Inhibition of miRNA421 can promote cell apoptosis rate, caspase3 activity and Bax/Bcl ratio [36]. Fan et al. showed that the overexpression of miRNA661 could activate caspase9, inhibit the growth of osteosarcoma cells, and promote apoptosis [37], indicating that miRNA661 can activate the signal transduction of death receptor pathway and lead to the apoptosis of osteosarcoma cells by promoting the activity of caspase9, the initiation effector of apoptosis.

MMPs
Because the progression of osteosarcoma is closely associated with matrix metalloproteinases ( [39]. Currently, in humans, there are 23 recognized MMPs that stimulate cancer survival and spread, and they represent the target group of anticancer drugs [40].
As one of the MMPs family, the degradation substrates of MMP9 mainly include gelatin, type IV, and V collagen, elastin, glass adhesive protein, etc. Because the basement membrane is mainly composed of type IV collagen, glycoprotein and proteoglycan, MMP9 is the most intensely studied and important amongst the MMP family. The overexpression of MiRNA495 can inhibit the proliferation and invasion of osteosarcoma cells and induce their apoptosis, which is related to the inhibition of the expressions of HMGN5, Cyclin B1, Bcl-2 and MMP9 [41]. In addition, both the overexpressed miRNA29b and miRNA181a can target down the expression of MMP9 and promote cell apoptosis [42,43]. MiRNA138 can reduce the invasion of osteosarcoma cells and promote apoptosis by down-regulating the expression of MMP2 and MMP9 [44]. In addition, miRNAs can also affect the apoptosis of osteosarcoma cells by acting on other MMPs. The expression of miRNA2682-3p was signi cantly decreased in osteosarcoma tissues and cell lines, and the overexpressed miRNA-2682-3p could inhibit cell proliferation and promote apoptosis by downregulating the expression levels of CCND2, MMP8 and Myd88 [45]. Transfection of miRNA192 mimics into osteosarcoma cell lines showed that overexpressed miRNA192 could down-regulate MMP11 content, reduce cell proliferation, migration and invasion, and promote apoptosis [46].

Other apoptosis-related proteins
Due to regulating cell apoptosis is a complex network, the simple way to study osteosarcoma cells apoptosis often cannot well explain the tumor biological behavior, so the related factors of osteosarcoma cells apoptosis in more in-depth research is likely to reveal preliminarily in its role in the development of osteosarcoma cells apoptosis mechanism, and to provide more broad prospects in the treatment of osteosarcoma. Survivin gene belongs to the inhibitor of apoptosis (IAP) family, and its expression can regulate cell cycle, inhibit apoptosis, promote cell proliferation and angiogenesis [47]. con rmed that mirNA-493-5p could target down KLF5 and block the PI3K/Akt signaling pathway, reduce the activity, migration and invasion of osteosarcoma U20S cells, and promote the apoptosis of osteosarcoma cells.
In addition to the regulation of cell growth and proliferation, SOX family can also participate in the regulation of cell apoptosis [52]. The three domains contained in Sox4 protein, the high mobility group (HMG) DNA-binding domain (DBD), and the glycine-rich domain (AA152-227) can all participate in the regulation of apoptosis [53]. Pan et al. [54] studied the function of pediatric osteosarcoma and found that the expression of miRNA188 was negatively correlated with SOX4, and restoring the expression of SOX4 could eliminate the pro-apoptotic effect of miRNA188 on OS cells. SOX5 is involved in chondrogenesis and promotes chondrocyte differentiation, and can directly bind DNA or regulate gene expression through binding with other proteins [55]. Literature has shown [56] that P53 inhibits tumorigenesis by regulating cell apoptosis, metabolic network, free radicals, and senescence. MiRNA34a can reduce FOXP1, known as a B-cell oncogene, through the p53 network [57]. By targeting FOXP2, the overexpression of miRNA139 can promote apoptosis of osteosarcoma cells [58].

Mirnas And Apoptosis-related Signal Pathway
MiRNAs affect downstream related factors and thus participate in regulating the proliferation, invasion, and metastasis of tumor cells and in uencing tumor cells' biological behavior through the activation status of related signaling pathways, thus affecting tumor development and outcome regression.
Currently, miRNAs have been found to regulate classical signaling pathways such as MAPK/ERK signaling pathway, PI3K/Akt signaling pathway, Wnt/β-catenin signaling pathway, Notch signaling pathway, and NF-кB signaling pathway, etc. In osteosarcoma, miRNAs can interact with signaling pathways to exert their oncogenic or oncogenic effects (see in Fig. 1 and Fig. 2) [59]. As a negative regulator of PI3K-dependent Akt signaling, gene of phosphates and tensin homolog deleted on chromosome 10 (PTEN) deletion can lead to PI3K/Akt hyperactivation. In a study of osteosarcoma, thicket (HNK) was found to decrease miRNA21 expression in a dose-dependent manner while upregulation of PTEN levels and PI3K/Akt signaling inhibition pathway were detected [70]. miRNA19a, miRNA21, and miRNA221 also exhibited similar effects [71][72][73].

Wnt/β-catenin signal pathway
Several studies have proposed that the Wnt signaling pathway is aberrantly activated in malignant bone tumors as well as tumor bone metastases, such as multiple myeloma, Ewing's sarcoma, osteosarcoma, and bone metastases from breast or prostate cancer [74]. A growing number of studies have shown that the Wnt/β-catenin signaling pathway is closely related to osteosarcoma development [75]. wnt1, wnt4, wnt5a, wnt7a, and wnt14 were expressed in osteosarcoma cell lines. wnt/β-catenin signaling can upregulate oncogenes (e.g., c-Myc, CCND1, c-MET), leading to osteosarcoma development [76].
Osteosarcoma contains tumor stem cells, and the Wnt/β-catenin signaling pathway also has an important role in osteosarcoma cancer stem cells [77].

Discussion
Osteosarcoma is a malignant bone tumor that is very common in adolescents or children, and its treatment is currently based on surgery and neoadjuvant chemotherapy. Although the current treatment protocols for osteosarcoma can improve patients' survival rate, most patients still suffer from local recurrence or pulmonary metastasis after treatment, which seriously affects the prognostic outcome of the patients involved. In recent years, with the continuous research on the molecular mechanism of osteosarcoma, studies have shown that miRNAs play a crucial role in the biological processes of proliferation, metastasis, invasion, and drug resistance of osteosarcoma. Amongst them, the regulation of miRNAs on apoptosis has become a research focus in recent years.
With the continuous improvement in the study of apoptosis mechanism and the elucidation of the relationship between apoptosis and osteosarcoma occurrence, it has been found that promoting apoptosis of tumor cells is gradually becoming a new strategy for osteosarcoma treatment. Through indepth research on the regulation of apoptosis by apoptosis-related proteins and apoptosis signaling pathway, inducing apoptosis in osteosarcoma cells become a new popular osteosarcoma treatment option. In radiotherapy, which triggers DNA damage in osteosarcoma cells by radiation, p53 gene expression initiates apoptosis and removes osteosarcoma cells, a process that is also mediated by the death receptor ligand Fas/FasL pathway of the apoptosis pathway and involves the Bcl-2 gene family.
Many antitumor drugs (e.g., DNA damaging agents, antimetabolites, topoisomerase inhibitors, etc.) can kill tumor cells by inducing apoptosis, and their mechanisms of action include (1) damaging the DNA of osteosarcoma cells and initiating the p53 gene; (2) regulating the Bcl-2/Bax ratio or inhibiting the activity of Bcl-2; and (3) regulating mitochondrial membrane permeability and promoting the release of cytochrome C. In contrast, heat therapy involves p53, Bcl-2, and Bax in addition to heat shock proteins and calcium ions. Apoptosis-related gene therapy involves introducing apoptosis-related genes into osteosarcoma cells to enhance the sensitivity of osteosarcoma cells to apoptosis or their resistance to apoptosis induction by radiotherapy. In addition, therapeutic measures targeting apoptosis-related receptor ligands, caspases family, and NF-κB are also available. Although comprehensive international therapeutic measures for osteosarcoma have improved survival rates, patients often die of complications, such as pulmonary metastases, shortly after surgery.
MiRNAs can regulate their target genes by degrading miRNAs or inducing miRNAs translation silencing.
Abnormal miRNAs expression can be detected in almost all human malignancies. Since miRNAs have been found to regulate the expression levels of more than 90% of protein-coding genes, indirectly in uencing the biological behavior of tumors by interfering with miRNAs expression, which is an extremely promising therapeutic approach. Mature miRNAs can play an essential role in the pathogenesis of osteosarcoma as tumor suppressors or promoters, as miRNAs expression is signi cantly associated with cell proliferation, adhesion, invasion, migration, metastasis, and apoptosis. MiRNAs are stable in circulation because they are resistant to degradation of endogenous circulating RNA enzymes thus quantitatively detected in plasma, serum and whole blood. It seems that circulating miRNAs re ect the pathological changes of miRNAs spectrum in tissues. Circulating miRNAs have great promise as diagnostic, prognostic or predictive biomarkers in the clinical treatment of patients with osteosarcoma.
Besides, compared with other bone malignant tumors insensitive to radiotherapy and chemotherapy, radiotherapy and chemotherapy play a vital role in the treatment of osteosarcoma. Many miRNAs regulate the sensitivity of osteosarcoma to chemotherapy and radiotherapy, affecting the therapeutic effect of osteosarcoma. Thus, reducing the expression of pro-oncogenic miRNAs, such as miRNAs silencing, antisense blocking, and miRNAs modi cations, may be regarded as a potential therapeutic option. However, there also exist many limitations for treating osteosarcoma, for instance, the sequence error of miRNAs sequence library, inferior RNA extraction methods, the variability of detection and analysis, the diversity of biological information data analysis, and the non-standard clinical testing of miRNAs. In this paper, we review that the miRNAs family plays a role in inhibiting or promoting osteosarcoma cell death by regulating apoptosis, suggesting that miRNAs regulation of programmed cell death in osteosarcoma may be an emerging therapeutic option for osteosarcoma treatment.
In recent years, exosomes have become the focus of extensive attention of scholars. Almost all cells in the human body can secrete exosomes, and exosomes can carry miRNAs and exist stably in peripheral blood and body uids. Since abnormal expression of the corresponding miRNAs can be detected in the early development stage of osteosarcoma, detecting the content of tumor cell-derived exosomes carrying miRNAs associated with osteosarcoma progression in early peripheral blood is expected to provide early diagnosis and disease progression monitoring for osteosarcoma. Previous studies have shown that the level of miRNA-25-3p in blood and secrete can be used as a prognostic indicator for patients with osteosarcoma. In addition, mesenchymal stem cell-derived exosomes (MSC-EXO) have attracted extensive attention due to their low immunogenicity, easy access, and storage and are considered to have good potential as targeted gene therapy vectors. Using MSC-EXO as vectors to transfect corresponding miRNAs analogs or inhibitors can regulate the apoptosis of osteosarcoma cells more precisely and stably.
In summary, given the key role of miRNAs in the regulation of apoptosis in osteosarcoma cells, future therapeutic measures targeting apoptosis-related miRNAs genes are expected to play a crucial role in treating osteosarcoma. In addition, the better utilization of exosomes and miRNAs will facilitate the early diagnosis of osteosarcoma and targeted therapy as drug carriers.

AUTHOR CONTRIBUTIONS
All authors contributed to the manuscript and approved of the nal version. Con ict of Interest: All author declare that they have no con ict of interest.