Drug resistance‐related microRNAs in osteosarcoma: Translating basic evidence into therapeutic strategies

Abstract Although the application of multiple chemotherapy brought revolutionary changes to improve overall survival of osteosarcoma patients, the existence of multidrug resistance (MDR) has become a great challenge for successful osteosarcoma treatment in recent decades. Substantial studies have revealed various underlying mechanisms of MDR in cancers. As for osteosarcoma, evidence has highlighted that microRNAs (miRNAs) can mediate in the processes of DNA damage response, apoptosis avoidance, autophagy induction, activation of cancer stem cells, and signal transduction. Besides, these drug resistance‐related miRNAs showed much promise for serving as candidates for predictive biomarkers of poor outcomes and shorter survival time, and therapeutic targets to reverse drug resistance and overcome treatment refractoriness. This review aims to demonstrate the potential molecular mechanisms of miRNAs‐regulated drug resistance in osteosarcoma, and provide insight in translating basic evidence into therapeutic strategies.

skeletal growth rate. 2 Because of its strong tendency to extensive metastasis and tumour relapse, OS consequently causes high mortality and poses a great threat of life to children and adolescents. With the emergence of next-generation sequencing, OS was gradually discovered to have a rather complicated genetic background. 3 The inactivation of tumour suppressor genes TP53 and/or RB1 was corroborated to remarkably induce OS tumourigenesis. 4 The congenital mutations of TP53 and/or RB1 are enough for developing tumour, but the occurrence rate of these congenital mutations was underestimated before. 5 Currently, the combination of surgical resection and multiple chemotherapy including neoadjuvant therapy, has been standardized for OS clinical remedy since 1970s. This regimen tremendously ameliorated symptoms and extended overall survival time of OS patients. 6 However, there exists a low response to therapeutic drugs in many OS patients, which is responsible for their subsequent aggressive progression and unfavourable outcomes. It is noted that the 5-year survival rate has remained at the level of 65%-75% in recent three decades, even with substantial research progress in OS clinical treatment approaches. [7][8][9] Obviously, distant metastasis, tumour recurrence and drug resistance are three pivotal reasons for the treatment refractoriness of OS. Much attention should be paid to better decipher and understand the underlying molecular mechanisms. Hence, it would be conceivable that molecules implicated in these mechanisms can serve as therapeutic Ruiling Chen, Gangyang Wang and Ying Zheng contributed equally to this work. targets for extending survival time of OS patients. Noticeably, extensive evidence has supported the involvement of miRNAs in OS pathogenesis.
In recent years, miRNAs have been explored to have a close connection to the mechanisms of pathogenesis and drug resistance in different cancer types, and establish a competitive endogenous RNA regulatory network that remains to be investigated. [10][11][12][13] Inspiringly, miRNAs seem to play an emerging role in OS drug resistance. 14,15 This might provide a brand-new insight in seeking for promising prognostic biomarkers and therapeutic targets for successful treatment in OS. To our knowledge, a single miRNA can target at least 200 genes involved in one signalling pathway or diverse signalling pathways. 16 Therefore, miRNAs might be valuable and effective for treating cancers with inherent heterogeneity and abnormality of multiple genes, among which OS can be taken as a good example.
In this review, we will elaborate on the emerging role of miRNAs in OS drug resistance under the mechanisms of DNA damage response, apoptosis avoidance, autophagy induction, activation of cancer stem cells (CSCs), and alteration in signal pathways. Also, we will provide insight in the potential clinical utility of these miRNAs as promising biomarkers and therapeutic targets to reverse chemoresistance.

FUNCTION OF MIRNAS
microRNAs (miRNAs) were first discovered by Victor Ambros et al in 1993, and perceived as endogenous small RNA molecules with biologically regulatory functions. 17 They are broadly conserved sequences among species with only 18-25 nucleotides in length for their mature forms, and have regulatory roles in gene expression at the post-transcriptional level. 18,19 Through binding to the 3′-untranslated region (3′-UTR) perfectly or imperfectly, they consequently contribute to the translational suppression or the degradation of diverse target mRNAs. 20 The detailed biogenesis process and functional mechanism of miRNAs have been well-elucidated ( Figure 1). It has been estimated that over 70% of human genome DNA has transcripts. Among them, about 2% transcripts code for protein synthesis and 3% can transcribe endogenous miRNAs. Of note, over 30% of human genes are under the regulation of miRNAs. 19,21 Intriguingly, a single small miRNA can interact with several regions of one or multiple target mRNAs. Conversely, a mRNA can be modulated by a multitude of miRNAs simultaneously, which is a unique advantage of miRNAs for cancer treatment.
Evidence has accumulated that miRNAs participate in various biological processes, 22,23 such as development, proliferation, differentiation, apoptosis, cell cycle, and metabolism, together with some human diseases 24 including cancer. 25 These deregulated miRNAs can be categorized as oncogenic ones and tumour suppressor ones. They play a regulatory function in tumourigenesis, progression, or chemosensitivity of different cancers. Besides, some miRNAs were reported to possess clinical values as predictive factors or therapeutic targets. [26][27][28] Noticeably, the emerging role of miRNAs in OS chemoresistance has been reported in recent studies, holding promise for improving the quality of life in OS patients. 15

RESISTANCE IN CANCER
Although the application of chemotherapeutic agents contributes to effective cancer treatment to a large extent, the occurrence of acquired multidrug resistance (MDR) remains a tough issue that ought to be solved. Substantial studies have discovered several universal mechanisms underlying acquired MDR, 29 including drug transport, drug metabolism, aberrant drug targets, DNA damage response, apoptosis evasion, autophagy, epithelial-to-mesenchymal transition (EMT), and activation of CSCs.
Drug transport mechanism has been well-studied in cancer MDR, which is closely associated with up-regulated drug transport proteins presenting on the surface of cytoplasmic membrane, that is, ATPbinding cassette (ABC) transporters. 30 Drug metabolism is a complicated process of xenobiotics detoxification with the participation of drug metabolism enzymes (DMEs) and the consequent metabolites are transported by ABC transporters. 31 As we can see, the concerted efforts of DMEs and ABC transporters finally lead to the decreased drug accumulation in the cytoplasm to reduce drug toxicity. DNA damage response (DDR) is a cellular stress response to DNA damage caused by cytotoxic drugs endogenously or exogenously. It aims to repair existing DNA lesions by arresting cell cycle temporarily, and prevent further or irretrievable damage such as cell senescence and apoptosis. 32 Therefore, the enhanced DNA repair can promote cell viability and resistance to cytotoxicity. Programmed cell death, an integrated concept of apoptosis, autophagy, and programmed necrosis, is an intracellular program triggered in the context of adverse conditions to determine the ultimate fate of cells, namely, survival or death. Interestingly, in malignant cells, apoptosis and programmed necrosis are invariably associated with death, while autophagy executes a dual role. 33 Furthermore, mechanisms modulated by apoptosis or autophagy have been confirmed to contribute to enhanced drug resistance.
Recently, ever-growing evidence has shown that exosomes and miRNAs can also play a significant role in drug responsiveness of cancers including OS. 15

| DNA damage response
Cytotoxic agents can cause cellular DNA damage and initiate a cellular stress response called DDR, which aims to repair existing DNA lesions through temporary cell cycle arrest and protect cells from irreversible damage. 32 To our knowledge, the DDR process contains DNA tolerance mechanisms, base excision repair, nucleotide excision repair, mismatch repair, and DNA double-strand break repair. 41 It has been reported that there exists an interplay between DDR genes and noncoding RNAs (ncRNAs) including miRNAs in cancer. 42,43 Several recent studies have shown that miRNAs can be a regulator of OS drug resistance via involving in DDR mechanism (Table 1).
MiR-124 was previously reported to regulate glucocorticoid resistance in haematological malignancies, for which glucocorticoid is common therapeutic drug. 44,45 Up-regulated miR-124 was newly shown to enhance cell response to diverse DNA-damaging drugs by binding to the 3′-UTR of ATMIN and PARP1 mRNAs in U2OS cells. 46 Protein PARP1, an abbreviation of poly (ADP-ribose) polymerase 1, is well-known to attract DNA repair proteins for repair through binding to DNA breaks. 47 Its inhibitors have been validated to sensitize cancer cells and have an anticancer effect in various cancers. 48,49 Protein ATMIN (ATM interactor) interacts with a significant DNA damage checkpoint kinase, ataxia telangiectasia mutated (ATM), and regulates ATM activity for DNA repair. 50 The role of miR-15b in cancer drug resistance has been reported in the last decade. 51 A recent study 52 first pointed out a significant decrease in miR-15b in OS MDR cell lines and identified WEE1 mRNA as its direct target. WEE1 gene codes for a protein kinase to modulate the G2 checkpoint in response to DNA damage. Besides, a restoration of miR-15b was observed to suppress WEE1 and partially reverse drug resistance in vitro. By establishing a MDR models of OS, Zhenfeng Duan et al F I G U R E 1 Biogenesis and biological function of miRNAs. First, a specific miRNA gene transcribes into pri-miRNA through polymerase II or III in the nucleus. Next, Drosha cleaved the hairpin structure of pri-miRNA to produce pre-miRNA which is subsequently exported to the cytoplasm by Exportin5. Then, the miRNA duplex is released after the excision of Dicer. After that, a combination of miRNA duplex and Argonaute protein forms a RNA-induced silencing complex (RISC), in which the passenger strand of miRNA is degraded. Finally, RISC causes mRNA degradation or translational suppression by targeting the 3′-UTR of mRNA F I G U R E 2 MiRNAs modulate OS drug resistance through several mechanisms discovered an attenuate resistance to DOX after systemic administration of miR-15b mimics.
As we all know, intracellular genomic instability is an intrinsic hallmark of tumourigenesis and tumour progression. Some cancer cells rely on a limited set of repair mechanisms for survival. Studies have found that disruption of DNA damage repair pathways can be utilized for current anticancer therapies. 53,54 However, it is still obscure in OS chemotherapy and requires deeper exploration of potential mechanisms of miRNAs-regulated DDR in OS.  and TDX, accompanied by overexpression of p21 in a p53-dependent manner. 63 Elevated miR-301a enhanced drug resistance because of apoptosis avoidance by directly targeting AMPKa1. 64 It has been identified that miR-21 mostly exerts oncogenic roles in cancers including OS. 65 A study revealed that Bcl-2 expression had a positive connection with miR-21 which inhibited apoptosis and induced a resistance to CDDP, while Bcl-2 siRNA ameliorated miR-21-induced resistance. 66 Another recent study identified Spry2 as a direct target of miR-21, and confirmed the positive role of miR-21in OS drug resistance. 67  Collectively, these oncogenic or tumour suppressor miRNAs contribute to OS drug resistance by regulating expression of apoptosisrelated genes to avoid cell apoptosis, such as CCND1 and BCL2.

| Apoptosis avoidance
Considering that BCL2 is a classic anti-apoptotic protein that promotes cell survival by inhibiting activation of a caspase cascade, and is associated with several miRNAs in OS chemoresistance, it's presumable that BCL2 might be critical for the reversal of MDR in OS.
However, further identification and confirmation of the above F I G U R E 3 Aberrant expression of apoptosis-related miRNAs miRNAs is needed and great efforts should be invested to translate these findings into clinical applications.

| Autophagy induction
On one hand, autophagy refers to a lysosomal degradation pathway by secluding damaged or excess cellular molecules and organelles within autophagosomes and clearing them to keep cellular homeostasis. 76 On the other hand, it's a protective prosurvival pathway by sustaining a balance among the synthesis, degradation, and succeeding recycling of essential molecules in the condition of nutrient deprivation. 77 Conversely, autophagy will trigger cell death in the context of excessive loss of proteins, indicating that autophagy can exert paradoxical roles. 78 Accumulated evidence has highlighted the participation of autophagy regulation in cancer diseases including OS, [79][80][81] and revealed the promoted activity of this degradative pathway after administration of cytotoxic drugs to acquire drug resistance. [82][83][84] Recently, the involvement of autophagy modulated by miRNAs in OS drug resistance has been explored (Table 1).

MiR-101 is viewed as an important regulator in fibrotic diseases
and is used as therapeutic agents. 85 But except for that, it is also newly reported in cancer drug resistance. MiR-101 significantly blocked the expression of autophagy-related gene in U2OS cells and promoted cell sensitivity to DOX treatment. 86 MiR-22 was reported to couple with high-mobility group box 1 (HMGB1) and suppress HMGB1-modulated autophagy in OS cells treated with DOX and CDDP. 87,88 Previous studies imply that HMGB1, a chromatin-binding nuclear protein, can regulate the balance of autophagy and apoptosis, and promote drug resistance by facilitating autophagy in OS cells with administration of agents. 89,90 It was confirmed that miR-30a targeting Beclin-1 reduced chemoresistance to DOX via inhibition of Beclin-1regulated autophagy in vitro. 91 MiR-199a-5p also bound to Beclin-1 contributing to blockage of autophagy and CDDP-induced cytotoxicity in MG63 cells. 92 Multifunctional miR-155 is enriched and important in cellular immune system, and its overexpression is well-known to result in cancer development and drug resistance. The miR-155-based therapy has been commonly considered in cancer treatment. 93 A recent study revealed that elevated expression of miR-155 promoted autophagy induced by anti-cancer drugs and increased cell viability to modulate drug resistance in OS cells. 94 MiR-140-5p played a positive role in OS drug resistance through induction of autophagy with a direct interaction with inositol 1,4,5-trisphosphate kinase 2 (IP3k2). 95 Since autophagy is a double-edged sword in the process of biological degradation, and tight control of autophagy is beneficial for the survival of normal or cancer cells, it would be a considerable notion that manipulation of autophagy can be applied in cancer therapy by inhibiting its protective function and inducing cell death instead. 96,97 This has been studied preclinically in its infancy in OS treatment. 98 was the first lncRNA reported to play a role in OS-CSCs with expression of CD133. 105 Remarkably, several current studies shed light on the involvement of miRNAs in OS-derived CSCs, which needs much more investigations to have a good understanding of potential mechanisms for their future applications in OS treatment (Table 1).
MiR-143 is viewed as a novel regulator in type II diabetes, which can specially suppresses insulin-AKT pathway and causes insulin resistance. 106 Besides, chemically modified miR-143 has been considered as a RNA medicine for treating colorectal tumours. 107 A study reported a reduced level of miR-143 in OS patients with drug treatments, which contributed to enhanced chemoresistance by apoptosis avoidance and activation of autophagy and ALDH1 + CD133 + cells. 108 It is acknowledged that ALDH1 and CD133 are common cancer stem cell markers for identifying and selecting CSCs. 109

| Alteration in signal pathways
Abnormal signal transduction pathways seem to regulate initiation, progression, and chemotherapy sensitivity to anticancer drugs in various cancers. There are several common OS-associated signal pathways which include Wnt/β-catenin, PI3K/Akt, IGFIR, Notch, TGF-β, and so on. Wnt/β-catenin pathway plays a role in osteoblast differentiation and was reported to be the most important one for OS tumourigenesis. 113 PI3K/Akt pathway is another crucial pathway participating in OS pathogenesis, and has been recently confirmed as a key vulnerability for OS treatment. 114 Some recent studies have demonstrated that miRNAs could elicit aberrant activities of OS-associated pathways to affect chemosensitivity (Table 1).
Decreased miR-34c resulted in OS metastasis and chemoresistance by directly targeting the 3′-UTR of Notch1 and LEF1. 115 Sirolimus was reported to induce cell apoptosis and increase cell sensitivity to therapeutic drugs with an up-regulation of miR-34b targeting p21-activated protein kinase 1 (PAK1) and ABCB1. 116 The inactivating Wnt/β-catenin signalling pathway. 119 Generally, these results provide an appealing strategy to target miRNAs implicated in signal pathways to improve OS therapeutic effectiveness.

OSTE OSARCOMA DRUG RESISTAN CE
According to the above preclinical studies, these drug resistance-related miRNAs are expected to supplement or replace existing biomarkers of diagnosis or prognosis, and serve as promising candidates for therapeutic targets to overcome drug resistance in the coming future. 40 Several drug resistance-related miRNAs were mentioned to have a predictive role in clinical prognosis and survival time of OS patients. Clinically, reduced miR-382 was correlated with unfavourable prognosis in OS patients, due to its potent effect on chemoresistance to anticancer drugs. 62 OS patients with low expression level of miR-15b had obviously poor prognosis and shorter survival times because of chemotherapy resistance. 52 A low expression level of miR-143 was observed in OS samples, which had a significant connection with poor outcomes and shorter survival of OS patients with chemotherapy. 108 Reduced miR-34b level was perceived as a predictor of unfavourable outcomes of OS patients and associated with MDR, which was reversed by administration of sirolimus in vitro. 116 Besides, OS patients had a markedly higher level of serum miR-21, which was associated with advanced Enneking stage and chemoresistance, and served as an independent prognostic factor for OS patients. 120 Noticeably, some miRNAs have been reported to be rather promising therapeutic targets in preclinical or clinical studies in recent years (Table 3). The miR-34 family including miR-34a, miR-34b, and miR-34c, has been known as a tumour suppressor in cancers including OS and gained extensive attention. [121][122][123][124][125][126] In substantial preclinical studies, treatment with miR-34 mimics was viewed as a novel miRNA-target therapy in cancers. 121,127,128 Besides, replenishment of miR-34 encapsulated in lipid nanoparticles was demonstrated to exhibit an anticancer effect in several malignancies in a phase I clinical trail (NCT01829971). 129 A recent study revealed that miR-34 mimics could trigger the perturbation of microtubule network and cell death in OS cells, implicating its possibility as a therapeutic agent in OS. 130 Recently, it is noted that miR-34 mimic brought significant benefits for treatment of metastasis in OS mouse models. 131 However, the optimal drug doses require further identification for application. The drug toxicity mentioned in this study was not associated with drug resistance.
The loss of let-7 is a prevalent phenomenon in various cancers, and its restoration obviously suppressed tumour growth and extended survival time in vivo. 132 It is indicated that replenishing let-7 might be a beneficial method in OS treatment, which remains to be investigated.
MiR-155 plays a critical and positive role in diverse cancer types. 133 Recently, a study has reported a successful delivery of antimiR-155 conjugated with a small peptide called pHLIP, and its therapeutic benefits without toxicity in a lymphoma mouse model, 134  To sum up, this review focuses on drug resistance-related miR-NAs in OS through several molecular mechanisms, and provides insight in creating promising therapeutic strategies by targeting these miRNAs to reverse OS chemoresistance.

CONFLI CT OF INTEREST
The authors declare no conflict of interest.