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 influence the apoptosis process of osteosarcoma cells by regulating the three major apoptosis pathways: death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway (see in Fig. 1, and Fig. 2).
2.1 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 first discovered the Bcl-2 gene . Bcl-2 protein, the first discovered protein among many Bcl-2 family members, is also the first 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 . 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 identified as the target of miRNA449a by using TargetScan prediction technology and luciferase reporter gene detection technology, and the final 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 significantly 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 . The interaction relationship between Bax and Bcl-2 can be described: increased Bax can numerously and significantly 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 . 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 et al. showed that knocking out the LncRNA SNHG20, which is the target of miRNA139, can up-regulate Bax’s expression and promote the apoptosis of osteosarcoma cells through the mitochondrial apoptotic pathway . Protectors such as Mcl-1 and Bcl-xL can inhibit the activity of effectors. MiRNA29 family can down-regulate the mRNAs and protein expression of Mcl-1 in osteosarcoma cells, promoting cell apoptosis and inhibiting their resistance to methotrexate . MiRNA133b and miRNA133a can also down-regulate the expression of Bcl-xl and Mcl-1 to promote osteosarcoma cell apoptosis [20, 21]. The initiator can promote the permeation of the mitochondrial outer membrane by regulating effectors or protectors. For example, Bim can directly activate the effector to promote apoptosis. Sun et al. observed that down-regulating the expression of miRNA24 can target up-regulating the expression of Bim, and promoting DOX-induced apoptosis through the mitochondrial pathway . In addition, miRNA340 can increase the expression of Bim to promote osteosarcoma cell apoptosis by inactivating CTNNB1 mediated Notch signaling pathway . MiRNA488 can target Bim to inhibit osteosarcoma cell apoptosis, miRNA302b can up-regulate the expression of Bim by regulating the AKT signaling pathway in osteosarcoma cells [24, 25].
2.2 Caspase family
Caspase family is a group of proteases that can regulate cell apoptosis and be activated sequentially through a protease cascade reaction . The occurrence of most cell apoptosis depends on caspases, which induced cell apoptosis by selectively cleaving substrate proteins, which leads to the structural change and the loss of function of the substrate protein. The occurrence and development of tumors can be induced by the dysfunction of cell apoptosis in the body, which is caused by the inhibition of caspase functionally. Caspase family consists of caspase-2, 3, 6, 7, 8, 9 and 10, among which caspase-9 is an upstream initiating factor in the caspase factor family, which is the initiating factor of apoptosis and is important for the regulation of cell apoptosis process . Caspase-3 is the most critical factor in the apoptotic cascade, located downstream of the apoptotic cascade. It can be activated by activating of caspase-9 in the endogenous apoptosis pathway, inducing the repair of related genes and the inactivation of cell cycle-related enzymes, then leading to cell apoptosis . Related studies have shown that the high expression of caspase3 and caspase9 can be detected in both miRNA130 and miRNA34a transfected osteosarcoma cells, while cell invasion and metastasis are inhibited, and apoptosis is promoted [18, 29]. Wang et al. confirmed that miRNA139 increased the expression of caspase3 and caspase9 through targeting the regulation of SNHG20. MiRNA381, on the other hand, increases caspase3/9 activity by inhibiting the expression of LRH-1.
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 significant increase in caspase3 levels. It significantly 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 . These findings 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 . The activation of miRNA302b on caspase3 can regulate the expression of Bcl-2/Bim, inhibit the proliferation of osteosarcoma cells, and increase apoptosis . Inhibition of miRNA421 can promote cell apoptosis rate, caspase3 activity and Bax/Bcl ratio . Fan et al. showed that the overexpression of miRNA661 could activate caspase9, inhibit the growth of osteosarcoma cells, and promote apoptosis , 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.
Because the progression of osteosarcoma is closely associated with matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM), MMPs play an important role in cancer survival and invasion and the development of tumor vascular networks . MMPs as zinc-dependent endopeptidase is secreted mainly by fibroblasts, leukocytes, vascular smooth muscle cells, and rapidly proliferating tumor cells. MMPs biological properties are present in various physiological and pathological processes, such as the degradation of collagen and elastin, the formation of endothelial cells during angiogenesis, migration of vascular smooth muscle, and proliferation of tumor cells migration . Currently, in humans, there are 23 recognized MMPs that stimulate cancer survival and spread, and they represent the target group of anticancer drugs .
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 . 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 . In addition, miRNAs can also affect the apoptosis of osteosarcoma cells by acting on other MMPs. The expression of miRNA2682-3p was significantly 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 . 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 .
2.4 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 . Studies have shown that overexpressed miRNA206 can inhibit cell  Krüppel like factors (KLFs) play an important regulatory role in cell proliferation, cell differentiation, cell apoptosis and other aspects by regulating the expression of genes rich in GC and CACCC promoters . Wang et al.  revealed that miRNA10b promotes the development of osteosarcoma cells by downregulating KLF4 expression, while inhibiting miRNA10b in osteosarcoma cells can promote cell apoptosis. KLF5 is a transcription factor closely related to KLF4 in the KLF family. Several studies have shown that overexpression of KLF5 can promote tumor formation and lead to tumor deterioration . In the study of osteosarcoma, Zhang et al.  confirmed 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 . 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 . Pan et al.  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 . Literature has shown  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 . By targeting FOXP2, the overexpression of miRNA139 can promote apoptosis of osteosarcoma cells .