MicroRNAs in osteosarcoma: From biological players to clinical contributors,a review

Abstract

Osteosarcoma is a primary malignant bone tumour with high morbidity that occurs mainly in children and adolescents. While the molecular basis of osteosarcoma has received considerable attention, the cellular and molecular mechanisms underlying pre- and postoperative metastasis formation and the development of chemoresistance remain unclear. MicroRNAs (miRNAs), a class of 22-nucleotide noncoding RNAs, have emerged as critical components of gene-regulatory networks controlling numerous important pathophysiological processes, including the initiation and progression of cancers. Studies on miRNAs have opened new avenues for both the diagnosis and treatment of cancer. This review discusses the roles of miRNAs in osteosarcoma and their potential applications for the diagnosis, prognosis and treatment of this malignancy. As a rapidly evolving field of basic and biomedical science, miRNA research will have a revolutionary impact on the management of osteosarcoma.

Keywords

Diagnosis microRNA osteosarcoma prognosis treatment tumourigenesis

Introduction

Osteosarcoma is the most common primary bone malignancy, mainly occurring in children and adolescents.^1–3 Despite advances in therapeutic strategies (including wide tumour excision, adjuvant chemotherapy and radiotherapy) the prognosis for patients with osteosarcoma remains poor. Of all children diagnosed with osteosarcoma, 30% will not survive for more than 5 years; <50% will live beyond 10 years.^1–3 The development of new strategies to treat osteosarcoma remains an important but unmet clinical need.

Studies have demonstrated diverse genetic alterations in osteosarcoma cells including structural abnormalities, gain and/or loss of chromosomes, mutation in tumour suppressor genes and epigenetic modifications,^4,5 but the molecular mechanisms underlying the initiation, development and metastasis of this disease remain poorly understood. An in-depth understanding of osteosarcoma biology is required in order to optimize treatment strategies, identify biomarkers and develop new chemotherapeutic agents.

MicroRNAs (miRNAs) have been implicated in tumourigenesis and cancer progression.^6,7 They form an abundant class of small (22-nucleotide) noncoding RNAs that regulate gene expression at the post-transcriptional level, through binding to the 3′ untranslated region (3′ UTR) of target mRNAs.^8–10 Studies have shown that miRNAs are frequently deregulated in human cancers, and this has provided a new opportunity to elucidate the aberrantly expressed cellular pathways underlying neoplastic transformation.^6,7 However, in contrast to some other types of malignancy (such as breast,¹¹ lung,¹² pancreatic¹³ and colorectal cancer¹⁴) little is known about the role of miRNAs in the pathogenesis of osteosarcoma. The current review examines the significance of aberrantly expressed miRNAs in human osteosarcoma and discusses how these miRNAs may be involved in osteosarcoma tumourigenesis. In addition, this review provides an overview of findings that reveal the potential use of miRNAs as diagnostic and prognostic biomarkers and osteosarcoma treatment strategies.

Biogenesis and function of miRNA

MicroRNAs were initially identified in Caenorhabditis elegans in 1993,¹⁵ but were not found in mammals until 2000.¹⁶ To date, over 1000 human miRNAs have been registered, each of which can regulate hundreds of mRNA targets. This has led to the hypothesis that miRNAs regulate the expression of ≥30% of human gene transcripts.¹⁷

The biogenesis of miRNA involves several steps. First, a nascent miRNA transcript is transcribed by RNA polymerase II to yield primary miRNA. This is then cleaved within the nucleus by Drosha, a RNase III enzyme, creating a stem–loop hairpin structure of ∼70 nucleotides in length (precursor miRNA).^8–10 The precursor miRNA is transported to the cytoplasm by a Ras-related nuclear protein GTP-dependent exportin-5-mediated mechanism, where an RNase III enzyme, dicer and transactivation response RNA-binding protein cleave away the double-stranded portion of the hairpin, generating an asymmetric duplex miRNA/miRNA* intermediate of ∼22 nucleotides in length.^8–10 Of the two strands, one becomes active as mature miRNA (the guided strand) and the other is usually released and degraded (the passenger strand).^8–10 The mature miRNA is incorporated into the RNA-induced silencing complex, which binds imperfectly to the partially complementary binding sites in the 3′ UTR of target mRNAs, dependent mainly on the ‘seed’ sequences (comprising bases 2^–8 of the mature miRNA). Binding leads to inhibition of mRNA translation or mRNA destabilization by deadenylation.^8–10

Numerous studies have documented the importance of miRNAs as essential cornerstones of the genetic system. They play critical roles in a broad range of biological processes including development timing, cell differentiation, proliferation, apoptosis, cell signalling and the immune response.^8–10 The discovery of miRNA as an additional regulatory mechanism has been revolutionary in molecular biology and medicine.

Cancer and miRNA

Aberrant miRNA expression has been detected in almost all human cancers. The first evidence of miRNA involvement in cancer was obtained from studies in chronic lymphocytic leukaemia (CLL), where miR-15a and miR-16 were found to be silenced in ∼69% of patients.¹⁸ Following these initial observations, extensive mapping revealed that human miRNA genes are often located in cancer-associated regions or at fragile sites of chromosomes which are prone to deletion, amplification or mutation in cancer cells.¹⁹ Furthermore, experimental evidence suggests that abnormalities in certain miRNAs have causative roles in tumourigenesis, as miRNAs can act either as tumour suppressors or oncogenes, according to their function in cellular transformation and expression in tumours.^6,7 Overexpression of oncogenic miRNAs appears to be associated with transformation, metastatic progression, increased cell viability and proliferation in many solid tumours.^6,7 Examples include the mediation of cell survival and proliferation by miR-21-targeting of the tumour suppressor genes phosphatase and tensin homologue (PTEN) and programmed cell death protein 4 (PDC4).^20,21 Some miRNAs have been shown to possess tumour suppressor activity, since their loss of function promotes tumourigenesis.^6,7 For example, loss of miR-15a/16-1 in CLL is closely correlated with uncontrolled expression of the target antiapoptotic gene B-cell lymphoma 2 (BCL2) in CLL cells.²² The exact roles of most miRNAs in tumourigenesis are still largely unknown, however.

Because miRNAs are highly related to cancer progression (including growth, apoptosis, invasion and metastasis) and are responsible for cancer-related inflammation, anticancer drug resistance and regulation of cancer stem cells,²³ elucidation of the genetic networks regulated by abnormally expressed miRNAs is extremely useful for understanding their role in the induction of phenotypic changes. In addition to their causal involvement in tumour formation, miRNAs are being investigated as novel biomarkers for disease detection and prognosis, and potential therapeutic targets for human cancers.²⁴

miRNA dysregulation in osteosarcoma

A number of expression profiling studies have demonstrated miRNA dysregulation in osteosarcoma (Table 1). Lulla et al.²⁵ identified 22 miRNAs that were differentially expressed in osteosarcoma compared with normal osteoblasts, and further validated four of these miRNAs (miR-135b, 150, 542-5p and 652). Another study revealed downregulation of miR-199a-3p, 127-3p and 376c, and upregulation of miR-151-3p and 191, in osteosarcoma cell lines and osteosarcoma tissues when compared with osteoblast cell lines.²⁶ A study utilizing microarray probes for 723 human miRNAs identified 28 underexpressed and 10 overexpressed miRNAs in osteosarcoma.²⁷ Other research has identified decreased expression of several miRNAs from the 14q32 region (miR-382, 134, 369-3p, 127-3p, 154, 544 and 656) and increased expression of miR-17–92 cluster miRNAs (miR-17-5p, 17-3p, 18a, 19a, 19b-1, 20a and 92a-1) in osteosarcoma samples (normalized to normal bone) and osteosarcoma cell lines (normalized to osteoblasts).²⁸

Table 1.

Details of studies investigating dysregulation of microRNAs (miRNAs) in osteosarcoma.

Reference	Study design	Technique	miRNA	Trend	Putative function
Lulla et al.²⁵	Osteosarcoma cell line and FFPE osteosarcoma vs osteoblast cell line	TLDA; qRT–PCR	135b, 150, 542-5p, 652	UR	–
Duan et al.²⁶	Osteosarcoma cell line and osteosarcoma vs osteoblast cell line	Microarray; qRT–PCR	151-3p, 191	UR	–
			127-3p, 376c	DR	–
			199a-3p	DR	Inhibit cell growth/migration, regulate cell cycle
Maire et al.²⁷	Osteosarcoma vs normal osteoblast	Microarray	126, 142-3p, 148a, 181a*, 195, 199b-5p, 218, 223, 451, 497	UR	–
			31, 31, 100, 127-3p, 137, 154, 154, 221, 221, 299-5p, 329, 335, 335, 337-3p, 376a, 376a, 376c, 377, 382, 409-3p, 409-5p, 410, 432, 493, 495, 543, 654-3p, 758	DR	–
Thayanithy et al.²⁸	Osteosarcoma vs normal bone; osteosarcoma cell line vs osteoblast	Microarray; qRT–PCR	17-5p, 17-3p, 18a, 19a, 19b-1, 20a, 92a-1	UR	–
				UR
			127-3p, 154, 656, 382, 134, 369-3p, 544	DR

FFPE, formalin fixed, paraffin-wax embedded; TLDA, TaqMan® low-density array (Applied Biosystems, Foster City, CA, USA); qRT–PCR, quantitative reverse transcription–polymerase chain reaction; UR, upregulated; DR, downregulated.

Tumourigenesis

The molecular basis of osteosarcoma has received considerable attention. Although the biological roles of miRNAs in osteosarcoma remain largely uncharacterized, several targets through which miRNA effects are mediated in osteosarcoma have been identified (Table 2).

Table 2.

Details of studies investigating the roles of dysregulated microRNAs (miRNAs) in osteosarcoma tumourigenesis, metastasis and treatment resistance

Reference	Study design	Technique	miRNA	Trend	Target	Putative function
Tumourigenesis
Thayanithy et al.²⁸	Osteosarcoma vs normal bone; osteosarcoma cell line vs osteoblast	Microarray; qRT–PCR	382, 134, 369-3p, 544	DR	MYC	DR miR-17–92 cluster, induce apoptosis
Ziyan et al.²⁹	Osteosarcoma vs normal bone	qRT–PCR	21	UR	RECK	Induce invasion/migration
Liu et al.³⁰	Osteosarcoma vs corresponding noncancerous tissue	qRT–PCR	125b	DR	STAT3	Inhibit proliferation/migration
Zhang et al.³¹	Osteosarcoma vs adjacent normal tissue; osteosarcoma cell line vs osteoblastic cell line	qRT–PCR	143	DR	BCL2	Reduce cell viability, promote apoptosis in vitro, suppress tumourigenicity in vivo
Metastasis
Osaki et al.³⁴	Metastatic cell line vs nonmetastatic cell line	Microarray; qRT–PCR	143	DR	MMP13	Suppress lung metastasis
Huang et al.³⁷	Metastatic low FAS-expressing cells vs nonmetastatic high FAS-expressing cells	Microarray;qRT–PCR	20a	UR	FAS	Decrease FASLG sensitivity, promote lung metastasis
Montanini et al.³⁸	Osteosarcoma cell line and primary osteosarcoma culture vs normal osteoblasts; pulmonary metastatic sample vs primary osteosarcoma sample	Sequencing; qRT–PCR	93	UR		Increase cell proliferation/invasivity
Treatment resistance
Song et al.³⁹	Doxorubicin-, cisplatin- and ifosfamide-treated osteosarcoma xenograft vs untreated xenograft	qRT–PCR	140	UR	HDAC4	Suppress cell proliferation in cells with wild-type TP53
He et al.⁴⁰	Adriamycin treatment and irradiation vs untreated	qRT–PCR	34a, 34b, 34c	UR		Induce G₁ arrest and apoptosis in a TP53-dependent manner
Gougelet et al.⁴⁴	Ifosfamide good responder vs bad responder	TLDA	92a, 99b, 193a-5p, 422a	UR
Gougelet et al.⁴⁴	Ifosfamide good responder vs bad responder	TLDA	132	DR

RT–PCR, quantitative reverse transcription–polymerase chain reaction; DR, downregulated; MYC, v-myc myelocytomatosis viral oncogene homologue (avian); UR, upregulated; RECK, reversion-inducing-cysteine-rich protein with kazal motifs; STAT3, signal transducer and activator of transcription 3 (acute-phase response factor); BCL2, B-cell lymphoma 2; MMP13, matrix metallopeptidase 13 (collagenase 3); FAS, Fas (tumour necrosis factor receptor superfamily, member 6); FASLG, FAS ligand (TNF superfamily, member 6); HDAC4, histone deacetylase 4; TP53, tumour protein p53; TLDA, TaqMan® low-density array (Applied Biosystems, Foster City, CA, USA).

MicroRNAs are known to function as oncogenes during osteosarcoma tumourigenesis.²⁹ The oncogenic miRNA miR-21 is aberrantly overexpressed in all types of tumour examined thus far, and induces cancer cell growth, migration, invasion and metastasis by negatively regulating tumour suppressor genes such as PTEN and PDCD4.^20,21 The function and pathway of miR-21 in osteosarcoma carcinogenesis remains unknown, however. Research has indicated that miR-21 is significantly overexpressed in osteosarcoma tissues,²⁹ and induces invasion and migration of the osteosarcoma cell-line MG-63 by negatively regulating the tumour suppressor gene, reversion-inducing-cysteine-rich protein with kazal motifs (RECK).²⁹ In addition, there is an inverse correlation between miR-21 and RECK protein in human osteosarcoma tissues.²⁹ These results suggest that miR-21 is implicated in mediating the phenotypic characteristics of osteosarcoma cells, probably via regulation of RECK.

MicroRNAs are also known to influence carcinogenesis by acting as tumour suppressors.^26,28,30,31 Levels of miR-199a-3p were significantly lower in osteosarcoma cells than normal cells, in vivo and in vitro.²⁶ MiR-199a-3p may inhibit cell growth and migration, increasing the number of cells in G₁-phase, and directly or indirectly targeting oncogenes such as met proto-oncogene (hepatocyte growth factor receptor), mechanistic target of rapamycin, and signal transducer and activator of transcription 3 (acute-phase response factor) (STAT3).²⁶ Since miR-199a-3p acts as a tumour suppressor, it is possible that restoring its function may provide therapeutic benefits in osteosarcoma.

Ectopic overexpression of miR-125b suppressed cell proliferation and migration in MG-63 and SAOS-2 osteosarcoma cells, and inhibited tumour formation in nude mice.³⁰ Use of a combined bioinformatics and experimental approach identified STAT3 as the direct downstream target of miR-125b.³⁰ In addition, STAT3 could bind to the promoter region of miR-125b-1 and act as a transactivator to promote miR-125b transcription,³⁰ indicating feedback-loop regulation between STAT3 and miR-125b in osteosarcoma. These findings suggest that aberrant expression of miR-125b is critical for the development and progression of human osteosarcoma.

Osteosarcoma cell lines and primary tumour samples were shown to have significantly lower levels of miR-143 than osteoblastic cell lines and adjacent normal tissues, respectively;³¹ restoration of miR-143 expression reduced cell viability and promoted apoptosis in vitro, and suppressed tumourigenicity in vivo.³¹ In addition, the important antiapoptotic protein BCL2 was identified as a direct target of miR-143, and the proapoptotic function of miR-143 is mediated mainly via targeting of BCL2 expression.³¹ In combination with reports revealing the roles of miR-143 in other types of cancer,^32,33 these results confirmed that miR-143 may function as a tumour suppressor in carcinogenesis and cancer progression, indicating its potential application in cancer therapy.

It has been shown that miRNAs at the 14q32 locus are significantly downregulated in osteosarcoma, compared with normal bone tissue.²⁸ A subset of these 14q32 miRNAs (miR-382, 369-3p, 544 and 134) act co-operatively to regulate negatively the v-myc myelocytomatosis viral oncogene homologue (avian) gene (MYC) and in turn, downregulate miR-17–92 cluster, a direct transcriptional target of MYC.²⁸ Restoring expression of these four 14q32 miRNAs induced apoptosis in SAOS-2 cells: an effect that was attenuated by overexpression of MYC lacking the 3′ UTR or by introduction of the miR-17–92 cluster.²⁸ These data suggest the presence of a regulatory network involving 14q32 miRNAs, MYC and miR-17–92 in osteosarcoma, such that decreases in 14q32 miRNAs lead to increases in MYC protein and subsequent increases in miR-17–92, thus contributing to osteosarcoma pathogenesis. Further characterization of these networks may provide suitable targets for the more effective treatment of osteosarcoma.

In summary, miRNAs including miR-21, 199a-3p, 125b, 143 and 14q32 miRNAs have been shown to participate in osteosarcoma tumourigenesis. The role of dysregulated miRNAs in osteosarcoma carcinogenesis and progression remain to be fully elucidated.

Metastasis

Osteosarcoma is characterized by a high propensity for pulmonary metastasis, which is a major cause of death.^1–3 The 5-year survival rate is 60^–70% for patients without metastatic disease at diagnosis, but clinical outcomes are far worse for patients who present with pulmonary metastatis, who have a 5-year survival of <30%.^1–3 Novel therapeutic targets and approaches are required to suppress pulmonary metastasis of osteosarcoma and improve prognosis.

Studies have revealed antimetastatic effects of some miRNAs in osteosarcoma (Table 2). These include miR-143, which was shown to be downregulated in a metastatic osteoscarcoma cell line.³⁴ In addition, exogenous application of miR-143 significantly decreased invasiveness (but not cell proliferation) in vitro, and significantly suppressed lung metastasis in a spontaneous lung metastatic mouse model, but not in a primary lesion.³⁴ Matrix metallopeptidase 13 (collagenase 3; MMP13) was identified as a probable miR-143 target gene, suggesting that miR-143 downregulation may promote cellular invasion via MMP13 upregulation.³⁴

Other miRNAs may function as pro-metastatic factors in osteosarcoma (Table 2). FAS ligand (tumour necrosis factor [TNF] superfamily, member 6; FASLG) is constitutively expressed in lung tissue,³⁵ and it is therefore expected that FAS-positive osteosarcoma cells would be eliminated upon entrance into the lung. The ability of osteosarcoma cells to form lung metastases is therefore inversely correlated to cell-surface FAS levels, and FAS downregulation allows osteosarcoma cells to circumvent FASLG-mediated apoptosis in the lung.³⁶ It has been shown that miR-20a contributes to the metastatic potential of osteosarcoma cells by inhibiting FAS, with an inverse correlation shown between FAS and miR-20a in cell lines and patient samples.³⁷ MicroRNA libraries have been built and sequenced for the osteosarcoma cell lines MG-63 (tumour protein 53 [TP53]⁻/⁻) and 143B (TP53⁺/⁺), which differ in proliferation and transmigration rate.³⁸ The miRNA miR-93 was present at higher levels in 143B cells than MG-63 cells, and in primary osteosarcoma cultures compared with normal osteoblasts.³⁸ Transfection with miR-93 resulted in significantly increased cell proliferation and invasiveness in 143B cells, compared with MG-63 cells.³⁸ These data suggest that miR-93 confers a highly malignant, metastatic phenotype.

Such study findings indicate that miRNAs (including miR-143, miR-20a and miR-93) participate in osteosarcoma metastasis, but the molecular mechanisms involved remain largely unknown.

Treatment response

Osteosarcoma is characterized by complex resistance to conventional treatments, and survival has not improved since 1990, despite preoperative administration of chemotherapeutic agents.^1–3 The ability to predict treatment response before chemotherapy would be extremely useful. Some studies have suggested associations between miRNAs and chemotherapy response in osteosarcoma (Table 2).

Screening of osteosarcoma tumour xenografts treated with chemotherapeutic agents (including doxorubicin, cisplatin and ifosfamide) identified high expression of miR-140 in all three groups, suggesting that this miRNA may contribute to broad-spectrum chemoresistance.³⁹ Overexpression of miR-140 suppressed cell proliferation in U-2 OS osteosarcoma cells with wild-type TP53, but had less effect on MG-63 cells with mutant TP53.³⁹ In addition, miR-140 induced TP53/cyclin-dependent kinase [CDK] inhibitor-1A expression and G₁/G₂ arrest in U-2 OS cells only, indicating that the effect of miR-140 is dependent on the presence of functional TP53.³⁹ Histone deacetylase 4 was identified as an important target of miR-140.³⁹ miR-140 may therefore represent a potential target for the development of novel therapeutic strategies to overcome drug resistance.

Another miRNA, miR-34, has also been shown to affect gene expression in a TP53-dependent manner, inducing chemosensitivity in wild-type TP53 human osteosarcoma.⁴⁰ Members of the miR-34 family (miR-34a, miR-34b and miR-34c) are direct targets of TP53, and function as tumour suppressors by inhibiting proliferation, and inducing apoptosis and cell-cycle arrest, in several types of cancer.^41–43 Irradiation and adriamycin have been shown to induce miR-34 expression in U-2 OS cells (TP53⁺/⁺) but not in SAOS-2 cells (TP53⁻/⁻).⁴⁰ MiR-34 was found to affect the expression of CDK6, E2F transcription factor 3 (E2F3), cyclin E2 and BCL2, as well as to induce G₁ arrest and apoptosis in a TP53-dependent manner.⁴⁰ In addition, the miR-34 family underwent genetic and epigenetic alterations in osteosarcoma.⁴⁰

A signature comprising five miRNAs (miR-92a, 99b, 132, 193a-5p and 422a) is able to differentiate between good and bad response to ifosfamide treatment in both rat and human osteosarcoma.⁴⁴ The miRNAs 92a, 99b, 193a-5p and 422a were overexpressed in good responders and miR-132 was reduced.⁴⁴ It is possible that miRNA profiling may be a valuable tool in predicting treatment response, and may allow the development of more individualized preoperative chemotherapy regimens, thereby improving patient survival rates.

Circulating miRNAs as minimally invasive biomarkers

Studies have demonstrated that miRNAs are not only present inside cells but are also detectable in body fluids including serum, plasma, saliva, urine and milk.^45–49 Alterations in the levels and compositions of these extracellular circulating miRNAs have been shown to be tightly correlated with various diseases, including cancers.^45–49 The quantification of circulating miRNAs may be a valuable noninvasive biomarker to assess and monitor osteosarcoma patients, but this has not yet been investigated.

Therapeutic potential

The functional involvement of miRNAs in the development and progression of cancer has led to the development of new therapeutic strategies. Approaches include blocking upregulated oncogenic miRNAs by antisense oligonucleotides, or rescuing downregulated tumour suppressor miRNAs using miRNA mimics.⁵⁰ The miRNA could be introduced into the systemic circulation or injected into the body (such as into the peritoneal cavity or a limb) or directly into a tumour.⁵¹ Alternatively, the therapeutic agent could be introduced into stem or progenitor cells that would subsequently be transplanted.⁵² In an in vivo model, miR-143 delayed tumour formation and resulted in significantly smaller tumours when transfected into MG-63 or U-2 OS cells, compared with nontransfected cells.³¹ In addition, systemic injection of miR-143/atelocollagen complexes prevented spontaneous lung metastases in osteosarcoma.³⁴ These results suggest a potential for miRNAs to be used as targets for osteosarcoma therapy. As the understanding of the role of miRNAs in osteosarcoma remains limited, it is not known whether miRNAs could be used directly for the treatment of patients with osteosarcoma. In addition, extensive preclinical safety and toxicity studies would be necessary before a miRNA-based treatment could be considered for use in humans.

Conclusions and perspectives

The diagnosis of cancer has undergone major changes, from being based purely on morphology to now incorporating immunological, cytogenetic and molecular methods. Gene expression profiling has led to further refinement of the classification and diagnosis of cancer. The understanding of the molecular basis of osteosarcoma has advanced considerably, and miRNAs have now been found to be involved in tumour development, progression and metastasis. In addition, miRNAs have complex regulatory roles in human osteosarcoma and are closely linked to the clinical outcome of patients. These small molecules may become important contributors to the diagnosis and treatment of osteosarcoma, although their precise molecular mechanisms remain unclear. Additional studies are needed to define miRNA-mediated molecular pathways further, in osteosarcoma. The identification of dysregulated miRNAs in the tissue and circulation of patients with osteosarcoma would assist in the development of biomarkers for tumour progression and treatment response. Moreover, since therapeutic targeting of miRNAs holds significant promise towards improving the clinical management of patients with osteosarcoma, future studies should develop miRNA-based treatments with high delivery sufficiency, therapeutic effect and good safety profiles in animal models.

Footnotes

Declaration of Conflicting Interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by a grant from the National Natural Science Foundation of China (No. 81102039).

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